Hyperion Imaging System USER GUIDE

Transcription

1 PN A2 Hyperion Imaging System USER GUIDE

2 For Research Use Only. Not for use in diagnostic procedures. Information in this publication is subject to change without notice. It is Fluidigm policy to improve products as new techniques and components become available. Therefore, Fluidigm reserves the right to change specifications at any time. Every effort has been made to avoid errors in the text, diagrams, illustrations, figures, and screen captures. However, Fluidigm assumes no responsibility for any errors or omissions. In no event shall Fluidigm be liable for any damages in connection with or arising from the use of this publication. Patent and Limited License Information Fluidigm products are covered by issued and pending patents in the United States and other countries. Patent and limited license information is available at fluidigm.com/legalnotices. Trademarks Fluidigm, the Fluidigm logo, CyTOF, Helios, Hyperion, and Maxpar are trademarks or registered trademarks of Fluidigm Corporation in the United States and/or other countries. All other trademarks are the sole property of their respective owners. For EU's WEEE directive information, go to fluidigm.com/compliance Fluidigm Corporation. All rights reserved. 06/2017 IMC-QSG-02 rev 2 SW v6.7 Complies with 21 CFR and except for deviations pursuant to Laser Notice No. 50, dated June 24, Fluidigm Canada Inc Rodick Rd. Suite 400 Markham, ON L3R 4G5 CANADA For technical support visit fluidigm.com/support. North America Toll-free (US/CAN): support.northamerica@fluidigm.com Latin America techsupportlatam@fluidigm.com Europe/Middle East/Africa/Russia support.europe@fluidigm.com Japan techsupportjapan@fluidigm.com China (excluding Hong Kong) techsupportchina@fluidigm.com All other Asian countries/india/australia techsupportasia@fluidigm.com 2 Hyperion Imaging System: User Guide

3 Contents About This Guide Safety Alert Conventions Safety Alerts for Chemicals Log in to the software Add new accounts Define user settings Configure notifications (Administrator only) Safety Data Sheets Chapter : Operation Operator Safety Chapter : Hyperion Imaging System Introduction and Specifications Introduction Hyperion Imaging System Technology Solid-State Laser Specifications Hyperion Imaging System Specifications Standard Equipment Hyperion Tissue Imager to Helios Coupling Procedure Hyperion Imaging System Startup Procedure Software Provided System Requirements Software Startup Switching from Solution Mode to HTI Mode Turning on the System Loading the Tuning Slide Create a Panorama Recommended Slide Dimensions Consumables and Reagents Hyperion Imaging System Helios Instrument Tuning Materials Required Determine Laser Energy Monitor Background Signal Auto Tuning Chapter : Instrument Overview Hyperion Tissue Imager Sample Window and Coupling Window Hyperion Tissue Imager LED Panel Helios Status Panel Other Components Chapter : Software Overview Introduction HTI Acquisition (Batch Mode) Load a New Slide Upload an Image of the Slide Create an MCD File Create a Panorama Creating a Region of Interest Determine the Ablation Energy for the Sample Slide Hyperion Imaging System Shutdown Disconnecting the Hyperion Tissue Imager from the Helios Instrument Toolbar Switching to Helios Solution Mode Control Buttons Chapter : Maintenance Hyperion Imaging System Status Panel User management User management for CyTOF software administrators Cleaning Instructions Exterior Surfaces Injector Cleaning Torch Cleaning Hyperion Imaging System: User Guide 3

4 Coupling Tubing Periodic Maintenance Vacuum Interface Cones Replace the Load Coil Replace the Injector Sealer Cap Change the Interface Pump Oil Change the Oil on the Backing Pump Troubleshooting Appendix A: Safety General Safety Laser Safety Environmental Conditions Electrical Safety Chemical Safety Sample Handling and Preparation Safe Handling of Gas Cylinders Safety Interlocks Other Safety Considerations Disposal of Products Appendix B: Manual Tuning Manual Tuning (HTI Optimization) XY Parameter Optimization Makeup Gas Optimization Current Optimization Helium Flow Optimization Appendix C: Decontamination Decontamination of the Helios Instrument Biological Agents Hazardous Chemicals Radioactive Materials Pressurized Gas Safety 4 Hyperion Imaging System: User Guide

5 About This Guide CAUTION ABBREVIATED SAFETY ALERTS. Hazard symbols and hazard types specified in procedures may be abbreviated in this document. Refer to Appendix A: Safety for further information. Safety Alert Conventions This guide uses specific conventions for presenting information that may require your attention. Refer to the following safety alert conventions. Safety Alerts for Chemicals Fluidigm follows the United Nations Globally Harmonized System of Classification and Labelling of Chemicals (GHS) for communicating chemical hazard information. GHS provides a common means of classifying chemical hazards and a standardized approach to chemical label elements and safety data sheets (SDSs). Key elements include: Pictograms that consist of a symbol on a white background within a red diamond-shaped frame. Refer to the individual SDS for the applicable pictograms and warnings pertaining to the chemicals being used. Signal words that alert the user to a potential hazard and indicate the severity level. The signal words used for chemical hazards under GHS: DANGER Indicates more severe hazards. WARNING Indicates less severe hazards. Safety Alerts for Instruments For hazards associated with instruments, this guide uses the following indicators: Pictograms that consist of a symbol on a white background within a black triangleshaped frame. Hyperion Imaging System: User Guide 5

6 About This Guide Safety Data Sheets Signal words that alert the user to a potential hazard and indicate the severity level. The signal words used for instrument hazards: DANGER Indicates an imminent hazard that will result in severe injury or death if not avoided. WARNING Indicates a potentially hazardous situation that could result in serious injury or death. CAUTION Indicates a potentially hazardous situation that could result in minor or moderate personal injury. IMPORTANT Indicates information necessary for proper use of products or successful outcome of experiments. Safety Data Sheets Read and understand the SDSs before handling chemicals. To obtain SDSs for chemicals ordered from Fluidigm Corporation, either alone or as part of this system, go to fluidigm.com/sds and search for the SDS using either the product name or the part number. Some chemicals referred to in this user guide may not have been provided with your system. Obtain the SDSs for chemicals provided by other manufacturers from those manufacturers. Operator Safety The Hyperion Tissue Imager has been classified as a Class 1 laser device. 6 Hyperion Imaging System: User Guide

7 Chapter 1: Hyperion Imaging System Introduction and Specifications Introduction The Hyperion Imaging System is a mass cytometry-based high-resolution laser ablation system that allows the highly multiplexed imaging with 135 channels available. The system is designed to detect metal-tagged antibodies bound to the cell surface and intracellular proteins in tissue sections using immunohistochemical methods. This allows researchers to investigate cellular subpopulations and cell-to-cell interactions in various tissue microenvironments with greater resolution. The system allows for high-resolution cellular profiling in spatial proximity, enabling detection of disease cells and immune cells populations within the context of the tissue structure. Figure 1. The Hyperion Imaging System, left, coupled to Helios, a CyTOF system Hyperion Imaging System: User Guide 7

8 Chapter 1: Hyperion Imaging System Introduction and Specifications Introduction Hyperion Imaging System Technology The Hyperion Imaging System technology is an innovative system based on laser ablation technology coupled with mass cytometry time-of-flight (TOF) of the resulting ablation plume (see Figures 2 and 3). The Hyperion Tissue Imager uses a solid-state laser with a laser beam directed at the slide through the Sampler Cone. The sample on the slide or the tuning film on the slide is ablated and aerosolized. The ablation chamber, which houses the glass slide, is pressurized with helium and the resulting aerosol plume is delivered through the coupling tubing to the inductively coupled plasma (ICP) torch of the Helios through the argon and helium flow. Figure 2. A glass slide containing tuning film or a tissue section is loaded into the Hyperion Imaging System. The beam from the solid-state laser is directed through the cone to the sample on the slide. The resulting ablation plume is directed through the coupling tube in a stream of argon gas towards the ICP torch in the Helios instrument. 8 Hyperion Imaging System: User Guide

9 Chapter 1: Hyperion Imaging System Introduction and Specifications Introduction The system directs a pulsed laser beam through the optical components of the optics chamber through the attenuator, which functions to moderate the energy of the laser. The camera captures the image from the slide that has been loaded onto the stage of the ablation chamber. The laser beam ablates spots on the slide, resulting in plumes of aerosol particles (ablated material). The plumes are directed to the Helios ICP torch, where they are vaporized, atomized, and ionized in the plasma. The high-pass optic removes the low-mass ions, resulting in an ion cloud that enters the TOF mass analyzer. The ions are separated based on their mass and are accelerated to the detector. The detector measures the quantity of each isotope for each plume, corresponding to a single laser shot, from the ablated sample. Acquired data are stored in an MCD file format. In a typical workflow one MCD file is used for one sample slide which in turn can contain multiple regions of interest (ROI). The MCD files can be viewed in the MCDViewer software. MCDViewer can also export acquired data as separate images in a TIFF file format. Figure 3. Schematic of the Hyperion Imaging System coupled to the Helios instrument Ablation Plume Transients The material resulting from the laser shot that is directed to the sample slide is referred to as the plume. The time it takes for a single plume generated as a result of a single laser shot to be transferred to the Helios system, ionized, and then detected by the detector is defined as the plume transient time. The plume start is a parameter in the CyTOF software that indicates the time when the Helios system begins to detect the ions from a plume. The duration of the single plume integration is defined as the plume width. The default value for plume width at a frequency of 100 Hz or 200 Hz is 384 pushes (see Figure 4), which is approximately 5 milliseconds in duration. During manual tuning, the software can generate a transient curve, which is displayed by opening the Transient window of the software. Helium flow ramping can be setup and the software graphs the dual counts (number of ions) against time to determine the optimal helium flow required for effective transient delivery to the Hyperion Imaging System: User Guide 9

10 Chapter 1: Hyperion Imaging System Introduction and Specifications Solid-State Laser Specifications Helios instrument with minimal crosstalk (or overlap of plumes). Autotuning is recommended for optimizing Helium flow. Figure 4. Graph of 175 Lu intensity. The plume width of a single plume is represented as 384 pushes during 20 Hz operation. A 20 Hz frequency is used to record and optimize transients during tuning. Solid-State Laser Specifications Table 1. Performance specifications for the solid-state laser Performance Specifications Laser type Wavelength Nd:YAG nm Energy output µj Repetition rate (frequency) / / Hz 10 Hyperion Imaging System:

11 Chapter 1: Hyperion Imaging System Introduction and Specifications Hyperion Imaging System Specifications Hyperion Imaging System Specifications Table 2. System specifications for the Hyperion Imaging System System Specification Tissue thickness (full ablation) Addressable sample size Scan area µm thickness mm x mm mm / Hr (@ Hz) Ablation spot size % Recommended operating temperature C Optimal operating temperature ± C Table 3. Electrical specifications for the Hyperion Imaging System Electrical Specifications Power Operating voltage Operating frequency Operating mode Power dissipation, full load VA V AC / Hz Single-phase operation < W Hyperion Imaging System: 11

12 Chapter 1: Hyperion Imaging System Introduction and Specifications Hyperion Imaging System Specifications Table 4: Gas Specifications for the Hyperion Imaging System. Gas Specifications Purity Pressure (psi) Flow Rate (L/min) Impurities (ppm) Argon. % ± Oxygen < Nitrogen < Hydrogen < Water < Helium. % Grade. ± NA Oxygen <. Nitrogen <. Hydrogen <. Water <. Table 5. Instrument dimensions for the Hyperion Tissue Imager Instrument Dimensions Height Width Depth Weight cm ( in) cm ( in) cm ( in) kg ( lb) Table 6. Instrument dimensions for the Helios System Instrument Dimensions Height Width Depth Weight cm ( in) cm ( in) cm ( in) kg ( lb) 12 Hyperion Imaging System:

13 Chapter 1: Hyperion Imaging System Introduction and Specifications Hyperion Imaging System Specifications Table 7. Input and output connections for the Hyperion Imaging System Input and Output Connections Power Gases Sample Digital I/O AC power Argon gas, Helium gas Sample slide, ablated sample USB. communication cable, USB. camera, trigger (data acquisition card) Table 8. Workstation specifications for the Hyperion Imaging System Workstation Specifications CPU Memory. GHz Intel Core i GB Monitor Hard drive Ultrawide LG monitor GB x SSD, RAID Data storage TB x HDD, RAID Hyperion Imaging System: 13

14 Chapter 1: Hyperion Imaging System Introduction and Specifications Standard Equipment Standard Equipment Table 9. Standard parts included in the Hyperion Imaging System shipment Part Number Standard Equipment Hyperion Tissue Imager Helios System Universal computer, keyboard and mouse Ultrawide LG monitor Recommended Slide Dimensions Table 10. Recommended slide dimensions for the Hyperion Imaging System Dimensions (mm)* Width Length.... Thickness * Recommendations are based on the size of the Hyperion Tissue Imager slide stage. 14 Hyperion Imaging System:

15 Chapter 1: Hyperion Imaging System Introduction and Specifications Consumables and Reagents Consumables and Reagents Hyperion Imaging System Table 11. Consumables available for the Hyperion Imaging System Part Number Standard Equipment O-Ring, Coupling Tube 4 Pack Coupling Tube Kit Injector Hyperion Tissue Imager Nut Lock Gas Connection Kit Hyperion Tissue Imager Front and Back Ferrule Set Hyperion Tissue Imager 5 Pack Table 12. Reagents available for the Hyperion Imaging System Part Number Standard Equipment 3-Element Full Coverage Tuning Slide Helios Refer to the Helios User Guide (PN ). Consumables Ordering North America Customers in the US and Canada who have a Fluidigm account are already registered for online ordering. Go to fluidigm.com/catalog. New customers can set up an online account to place orders, view past order history, and see current order confirmations. Phone: Toll-free (US/CAN) salesadmin@fluidigm.com Hyperion Imaging System: 15

16 Chapter 1: Hyperion Imaging System Introduction and Specifications Consumables and Reagents Outside North America To reorder parts and reagents, contact your regional Fluidigm sales representative or distributor. Go to fluidigm.com/contacts. 16 Hyperion Imaging System:

17 Chapter 2: Instrument Overview Hyperion Tissue Imager WARNING The top cover of the Hyperion Tissue Imager functions as a safety interlock. When the cover is off, the DC power connection to the laser and the laser interlock line will be disengaged. WARNING If the equipment is used in a manner not specified by the manufacturer, the protection provided by the equipment may be impaired. Top cover Coupling window Coupling tubing Sample window Wheel retraction switch Interlock LED panel Lower cover AC and communication panel Figure 5. Schematic of the Hyperion Tissue Imager side view Hyperion Imaging System: 17

18 Chapter 2: Instrument Overview Hyperion Tissue Imager Coupling window Coupling attachment Ablation chamber Figure 6. Schematic of the Hyperion Tissue Imager back view 18 Hyperion Imaging System:

19 Chapter 2: Instrument Overview Hyperion Tissue Imager Sample Window and Coupling Window Closed Position When the sample window or the coupling window is in the closed position, the light corresponding to that window on the LED panel on the right side of the instrument is GREEN. Hyperion Imaging System: 19

20 Chapter 2: Instrument Overview Hyperion Tissue Imager Open Position When the sample window or the coupling window is in the open position, the light corresponding to that window on the LED panel on the right side of the instrument is NOT LIT and the user cannot perform tuning or acquisition on the system. 20 Hyperion Imaging System:

21 Chapter 2: Instrument Overview Hyperion Tissue Imager LED Panel Hyperion Tissue Imager LED Panel Hyperion Imaging System: 21

22 Chapter 2: Instrument Overview Hyperion Tissue Imager LED Panel Table 13. Instrument Interlock LEDs on the LED panel of the Hyperion Tissue Imager Instrument Interlock LED POWER Description When the light is CONTINUOUSLY GREEN, the system power supplies are on. When light is NOT LIT, the system power supplies are off. SAMPLE WINDOW COUPLING WINDOW COUPLING TUBE CHAMBER PRESSURE LASER ENABLED UPPER COVERS HELIUM SUPPLY LASER TEMP. INTERLOCK BYPASS When the light is GREEN, the sample window is closed. When the light is NOT LIT, the sample window is open and the interlock has been triggered. When the light is GREEN, the coupling window is closed. When the light is NOT LIT, the coupling window is open and the interlock has been triggered. When the light is LIT, this indicates that the coupling tubing has been connected to the ablation chamber. The wheel retraction system is not activated. When the light is LIT, this indicates that the chamber is pressurized. When the light is AMBER, the laser is firing and ready to ablate, or ablation is in progress. When the light is NOT LIT, this indicates that the upper cover housing the optics and the laser has been removed. When the light is GREEN, the pressure in the Helium supply line is within operating range. When the light is RED, this indicates that the laser has exceeded safe operating temperature. The laser will be disabled. When the light is FLASHING GREEN, all safety interlocks have been bypassed. (The interlock bypass is for the Field Service Engineering team.) 22 Hyperion Imaging System:

23 Chapter 2: Instrument Overview Helios Status Panel Helios Status Panel Table 14. Function and location of status panel LEDs. Sensor Location Interlock Status/Function T1 Backing pump Temperature sensor LED is RED if temperature is out of range. T2 Interface pump T3 Torch box T4 Vacuum interface VG1 TOF TOF pressure sensor LED is GREEN if the vacuum in TOF is <10-3. VG2 TOF LED is GREEN if the vacuum in TOF is <10-5. TP1 Turbo pump Three-stage Hyperion Imaging System: 23

24 Chapter 2: Instrument Overview Helios Status Panel Sensor Location Interlock Status/Function TP2 Turbo pump Turbo pump speed sensor Single-stage GATE OPEN/CLOSED Proximity sensor Indicates the status of the gate SHIELD Proximity sensor LED is GREEN if the shield is mounted. TORCH Torch LED is GREEN if the torch is mounted. ARGON Argon gas supply Pressure sensor LED is GREEN if argon pressure is > psi. RFG Radio frequency generator Proximity sensor LED is GREEN if the door is closed. AIR System exhaust Flow sensor LED is GREEN if the exhaust flow is > L/s ( cfm). CHILL Chiller Flow sensor LED is GREEN if chiller flow rate is above specified value. PLASMA OFF Plasma Press the button to manually stop plasma if required. Torch box Sampler cone Vacuum interface Figure 7. Interior view of the Helios instrument with front access door open 24 Hyperion Imaging System:

25 Chapter 2: Instrument Overview Helios Status Panel Front shield RF fingers Load coil Torch body Figure 8. Torch box Hyperion Imaging System: 25

26 Chapter 2: Instrument Overview Other Components Other Components Table 15. Other Helios components Parts Image Location AC distribution box Right side of Helios system Sampler cone In the vacuum interface 26 Hyperion Imaging System:

27 Chapter 2: Instrument Overview Other Components Parts Image Location Cooling fans Inside lower front door Hyperion Imaging System: 27

28 Chapter 3: Software Overview Introduction The Hyperion Imaging System software is designed to be user-friendly and intuitive and to provide continuous instrument feedback and diagnostic capabilities. This gives users the ability to troubleshoot quickly and effectively as well as to provide the highest quality of data output. The software interface has three sections: Toolbar, Workspace, and System Logs. The Toolbar, at the top of the start page, allows users to set up tuning protocols, create panoramas and regions of interest (ROIs), set up acquisition of slides, preview samples, acquire samples, and record results. The Control Tool Bar allows users to control various aspects of the software including plasma, laser, ablation chamber flushing and loading and unloading samples. The Workspace contains the windows involved in instrument tuning and sample acquisition, including the data acquisition settings, camera window, HTI acquisition window, rain plot, TOF plot, masses per reading (MPR), and sample panels. Users can visualize the tuning slides and sample slides that are run on the instrument. The System Logs on the bottom of the screen displays real-time instrument status and processes with a date and time stamp. 28 Hyperion Imaging System:

29 Chapter 3: Software Overview Toolbar The software has two modes: user mode, and administrator mode displayed on the right side of the toolbar. Administrator mode is available to Hyperion Imaging System operators and provides access to a subset of instrument parameters and settings for hardware configuration, performance optimization, and data acquisition. User mode provides nonoperators with basic instrument tuning and data acquisition functionality. Toolbar Table 16. Toolbar of the Hyperion Imaging System Button Description Administrator only. System settings for hardware configuration. Automated tuning procedures may be performed in this window. The Helios instrument is optimized for detector voltage, current, XY, and makeup gases to produce the optimal signal. The system uses 131 Xenon ions to optimize detector voltage each time the instrument is tuned. HTI (Hyperion Tissue Imager) acquisition of the sample slide is completed in this window. Users can create several panoramas and can select and ablate several user-defined regions of interest in this window. Users can open the window to obtain a two-dimensional plot from the text files generated by the software. Users may cluster their data and filter out particular pixels. Hyperion Imaging System: 29

30 Chapter 3: Software Overview Toolbar Button Description Provides users with a live or static view of the slide inserted into the ablation chamber. Create panoramas for instrument tuning or for single sample acquisition, navigate the XY stage to move the slide to the correct position, adjust the LED, auto focus the slide, and define the region of interest (single sample acquisition). Opens the HTI Status Panel and provides a view of the most recent status of the interlocks and sensors on the tissue imager, including laser state, chamber pressure, laser temperature, and helium pressure Opens the System Logs at the bottom of the window. Provides feedback on the status of the instrument and the ongoing processes with timestamps. Opens the User Management window and provides administrators the ability to add and remove users, verify instrument usage using timestamp provided. Users can update their personal information and change their password. The high-dimensional datasets generated by the Helios are analyzed with preferred thirdparty data management and analysis and software solution, Cytobank. Allows users to verify the software version number as well as to log out of one user mode to switch to another. Allows users to switch between windows in the workspace area. 30 Hyperion Imaging System:

31 Chapter 3: Software Overview Control Buttons Control Buttons Table 17. Control Buttons of the CyTOF software for Hyperion Imaging System Button Description Opens the Data Acquisition Settings window and allows users to manually ramp parameters between specified ranges. Parameters such as XY Parameters, Current, and Makeup Gas can be used to manually tune the instrument. Users can click the Run button to begin acquisition during manual instrument tuning. Stop allows users to stop the run before it reaches the selected endpoint. Provides the Masses per Reading graph of the parameters that were selected in the Data Acquisition Settings window Provides the time-of-flight plot of the data collected during tuning Plots the parameters that are selected in the Data Acquisition Settings window. Users can determine the optimal Helium flow during tuning. Start and Stop allow users to start or stop the plasma on the Hyperion System. Cancel halts the plasma start-up sequence. Start and Stop allow users to turn the laser on or off. Users can flush the chamber with helium after loading a new slide. Click Cancel to stop the flush before the recommended time. Note: 5 minutes is the optimal recommended time for the ablation chamber flush. The default flush duration is 5 minutes and can changed by administrators in HTI Settings > Chamber/Gases. Hyperion Imaging System: 31

32 Chapter 3: Software Overview Hyperion Imaging System Status Panel Hyperion Imaging System Status Panel The HTI Status Panel provides users the ability to monitor the current state of the Hyperion Imaging System, including the status of the instrument safety interlocks. A blue button indicates that the interlock or instrument state is OK. Amber indicates that an instrument interlock has been tripped or instrument state is not stable. The current values for Helium Mass Flow, Chamber Pressure, Helium Pressure and Chamber Temperature are displayed in the Values box. LED1, LED2, and LED3 are laser diagnostic indicators. 32 Hyperion Imaging System:

33 Chapter 3: Software Overview Hyperion Imaging System Status Panel Graph Table 19. The graph section of the Toolbar allows users to visualize tuning slides or samples. Graph Type Example Description TOF The TOF graph shows the time-of-flight values for the elements used in the experiment. Masses Per Reading The Masses Per Reading window plots the values of all the masses that are recorded. Transients The Transients graph shows the transients that are transferred through the coupling as the helium gas is ramped. This allows users to select the helium flow that provides the optimal 175 Lu signal with minimal cross talk. Hyperion Imaging System: 33

34 Chapter 3: Software Overview User management Graph Type Example Description Plotviewer Plotviewer allows users to cluster data and to view data in bivariate plots. User management User accounts are created and managed by the CyTOF software administrator. Two types of accounts are available: User accounts and Administrator accounts. User accounts have access to the basic user functionality. Administrator accounts have access to additional software and instrument configuration settings. User management for CyTOF software administrators CyTOF software administrators have the ability to: Create user accounts Manage, control, and monitor user attributes Track the instrument and software usage for each user account with time and date stamps and export this information to a comma separated values file (.csv) Configure notifications for user accounts Log in to the software 1 On the Windows desktop, double-click to the CyTOF software icon. 2 In the Username box, enter your user name. 3 In the Password box, enter your password. 34 Hyperion Imaging System:

35 Chapter 3: Software Overview User management 4 Click Login. NOTE Your user name and password are provided by the CyTOF software administrator. Add new accounts 1 On the toolbar, click User Settings. 2 In the User Management window type a new user name into the Username box and click the green plus symbol (or press Enter) Define user settings 1 On the toolbar, click User Settings. 2 To manage attributes for and account, click the user name in the Username column. Under User Attributes, the selected account name is displayed in the Username box. Hyperion Imaging System: 35

36 Chapter 3: Software Overview User management The following attributes can be modified (or remain empty): First Name, Last Name, Middle Name: Enter the names of the user that the account is assigned to Enter the valid address of the user; a valid address must be provided to enable notifications Phone: Enter the contact phone number of the user Additional administrator controls Locked checkbox: If checked, the locked account cannot be used to log into the software Reset Activity button: If clicked, the event history for the selected account is cleared 36 Hyperion Imaging System:

37 Chapter 3: Software Overview User management For Users Users can update a subset of user attributes for their account only. The changes will apply to the active account. The following attributes can be modified: First Name, Last Name, Middle Name: Enter the names of the user the account belongs to Enter a valid address of the user; a valid address must be provided to enable notifications Phone: Enter the contact phone number of the user Change Password button: Click to display the Change Password dialog box; type the old password, type new password, and then click Change Password to change the password for the active account Configure notifications (Administrator only) The CyTOF software can be configured to send notifications to users when batch acquisition stops. Data is encrypted and transmitted by Secure Sockets Layer (SSL) connection. NOTE Contact your site IT administrator for assistance with SMTP settings. Click User Settings. In the User Management window, click a user name in the table. Under User Attributes: Hyperion Imaging System: 37

38 Chapter 3: Software Overview User management a In the box, enter the recipient s address b In the SMTP Client Host, SMTP Client Port, SMTP Username, and SMTP Password boxes, type the credentials for the sender s address. NOTE To receive notifications by webmail accounts, configure webmail settings to SMTP. Open Authentication (OAuth) 2.0 protocol is not supported. 38 Hyperion Imaging System:

39 Chapter 4: Operation Hyperion Tissue Imager to Helios Coupling Procedure IMPORTANT The Hyperion Imaging System instrument should be placed in a vibrationfree environment. The following procedure outlines the coupling of the Hyperion Tissue Imager to the Helios instrument. This tubing connection allows the plume of the ablated material to be transported to the Helios for mass cytometry analysis. IMPORTANT The Hyperion Tissue Imager should be connected to the AUXILIARY power connection on the right panel of the Helios instrument to allow for raising and lowering of the instrument on its wheels and for efficient coupling to Helios. WARNING Do not replace detachable main supply cords with inadequately rated cords. 1 Inspect both ends of the Hyperion Tissue Imager Injector to check for debris or material prior to beginning this procedure. You may inspect under a microscope (if available). NOTE Use compressed air to remove any debris from the injector. Hyperion Imaging System: 39

40 Chapter 4: Operation Hyperion Tissue Imager to Helios Coupling Procedure 2 Insert the injector into the Torch Assembly. Gently rotate the injector to push it past the O-rings until it is fully seated into the torch assembly. WARNING Handle the injector carefully. The injector may break and cause injury if not inserted correctly on the Hyperion Imaging System. The instrument should not be moved while the coupling is still connected to Helios. The coupling tubing is provided fully assembled. There are two nuts on the coupling tubing. The larger-diameter nut is installed on the injector. 3 Inspect the coupling tubing to ensure that there is no debris or dust. You may use compressed air to remove any dirt from the coupling. Ablation chamber nut Injector fitting Back nut Coupling tubing 40 Hyperion Imaging System:

41 Chapter 4: Operation Hyperion Tissue Imager to Helios Coupling Procedure 4 Press the wheel retraction switch on the right side of the instrument to lift the system so that the castors are exposed for movement of the system. Wheel retraction switch Hyperion Imaging System: 41

42 Chapter 4: Operation Hyperion Tissue Imager to Helios Coupling Procedure 5 Loosen the knob of the alignment tool and push the arm in all the way. 6 Carefully slide the alignment tool onto the heater guide pins of the Helios, ensuring that the back of the tool is flush against the torch assembly. Secure the tool in position with one hand and tighten the right-side thumbscrew. 7 With the first thumbscrew secure, reach down from above with your other hand and tighten the thumbscrew on the left. Once both screws are secure, ensure that the alignment tool is level on the heater guide pins and that the back of the tool is against the torch assembly. 42 Hyperion Imaging System:

43 Chapter 4: Operation Hyperion Tissue Imager to Helios Coupling Procedure 8 Loosen the black thumbscrew on the top of the alignment tool and horizontally line up the arrow of the alignment tool with the center of the injector. Tighten the black thumbscrew to lock the position of the alignment tool. 9 Loosen the knob and extend the arm of the alignment tool fully. Tighten the knob to lock the arm in place. 10 Raise the Hyperion Tissue Imager completely by pressing the up arrow on the wheel retraction switch until the tissue imager is fully in the raised position. Lift the coupling window using the gray tab. 11 Carefully move the Hyperion Tissue Imager towards the tool so that the ablation chamber gently touches the two extended pads of the alignment tool arm. This will help ensure that the tissue imager is parallel with the Helios. The arrow on the alignment tool Hyperion Imaging System: 43

44 Chapter 4: Operation Hyperion Tissue Imager to Helios Coupling Procedure (pink box in the photo) should point towards the center of the coupling insertion on the Hyperion Tissue Imager. 12 Loosen the knob on the alignment tool and push in the arm of the alignment tool all the way back toward the Helios instrument. 13 With an arm over the top of the Hyperion Tissue Imager, press down on the wheel retraction switch to lower the module until it is seated on the ground. Watch for any shifting of the Hyperion Tissue Imager as it settles. 14 If the Hyperion Tissue Imager settles significantly to the left or right of the Helios instrument, loosen the small black thumbscrew at the end of the arm and offset the alignment arrow right or left to adjust for the settling. Repeat steps 10 through 14 as necessary. 15 Carefully remove the alignment tool from the heater guide pins. Lift the tool off the guide pins and then slide it out and away from the torch assembly horizontally below the pins. 44 Hyperion Imaging System:

45 Chapter 4: Operation Hyperion Tissue Imager to Helios Coupling Procedure 16 Position the Hyperion Tissue Imager in front of the Helios so that the Helios injector aligns with the connection opening in the ablation chamber on the Hyperion Tissue Imager. 17 Slide the injector fitting over the back end of the injector in the Helios. Grip the back nut with one hand while tightening the Injector fitting in the clockwise direction. Hyperion Imaging System: 45

46 Chapter 4: Operation Hyperion Tissue Imager to Helios Coupling Procedure 18 Lower the instrument to the ground using the wheel retraction switch so that it is ready for operation. The alignment should be both vertical and horizontal so that the output of the ablation chamber lines up with the coupling tubing and injector. 19 Insert the coupling tubing end into the coupling attachment in the ablation chamber. 46 Hyperion Imaging System:

47 Chapter 4: Operation Hyperion Tissue Imager to Helios Coupling Procedure IMPORTANT If the tubing is not straight then it will require adjustment. In this case, raise the instrument up using the wheel retraction switch so that the castors are exposed. And move the instrument. Check that the tubing is fully inserted into the hole. Lower the instrument back to the ground for operation. Check that the tubing is correctly positioned as straight as possible. 20 Tighten the ablation chamber nut to the ablation chamber in the clockwise direction. Hyperion Imaging System: 47

48 Chapter 4: Operation Hyperion Tissue Imager to Helios Coupling Procedure 21 Close the coupling window. 22 Ensure that all the communication cables have been connected. The USB 2.0 cable connects to the computer for software control. The USB 3.0 connects to the computer for the camera. The BNC trigger line connects to the data acquisition board. 23 Check that the gas supply lines are connected. The helium gas line should be connected to the external helium supply and the argon gas line should be connected to the makeup gas supply on the front cover of the Helios instrument. PC (USB 2.0) Camera (USB 3.0) Helium supply Argon supply Power supply 48 Hyperion Imaging System:

49 Chapter 4: Operation Hyperion Imaging System Startup Procedure 24 Open the helium and argon gas valves. 25 Turn on the instrument by clicking the power switch on the communication panel and begin tuning. (See Instrument Tuning section.) If you notice issues with the transient signal, you may need to go back and check the coupling between the Hyperion Tissue Imager and the Helios. Small adjustment to reduce bending of the coupling tubing may be necessary. WARNING TRIP HAZARD. The electrical cables and communications can cause a potential trip hazard. Be careful when walking close to the Hyperion Tissue Imager. Hyperion Imaging System Startup Procedure Software Provided CyTOF Software v6.7 MCD Viewer 1.0 System Requirements Windows 7 Pro 64-bit Software Startup Double-click the desktop icon to start CyTOF Software 6.7. Hyperion Imaging System: 49

50 Chapter 4: Operation Hyperion Imaging System Startup Procedure In the Toolbar, click About. Click Login. IMPORTANT If this is the first login you will be required to accept the License Agreement. Click I accept the terms of the license agreement. Check Do not show the message again. 50 Hyperion Imaging System:

51 Chapter 4: Operation Hyperion Imaging System Startup Procedure For the Username enter administrator. Click Login. Click Control Panel. Switching from Solution Mode to HTI Mode Establishing Communication with the Hyperion Tissue Imager NOTE If the instrument was run in solution mode previously, switch the settings to HTI mode. 1 Click the Devices tab and click HTI as Introduction. In the HTI box select HTI from the Type drop-down and click Auto Detect. Hyperion Imaging System: 51

52 Chapter 4: Operation Hyperion Imaging System Startup Procedure Click Setup. 52 Hyperion Imaging System:

53 Chapter 4: Operation Hyperion Imaging System Startup Procedure In the Serial Port Settings tab, verify that each hardware module has been assigned a COM port. Close the window. Select Control Panel > Devices and click Set Configuration Hyperion Imaging System: 53

54 Chapter 4: Operation Hyperion Imaging System Startup Procedure Turning on the System Turn on the power switch located on the communication panel of the Tissue Imager. NOTE Ensure that the helium has been turned on from the source. If the helium flow is on, the helium light on the LED panel is green. On the Toolbar, click the Plasma Start button. It will take approximately minutes for the plasma to start. The chamber will automatically flush for approximately 5 minutes. Check the HTI Status Panel > Chamber Flushing State to confirm that the chamber has been flushed (blue circle). Click HTI Status to open the HTI Status Panel window. When the Laser Warmup State indicator is blue, the warmup is complete. The separate Laser State indicator is blue and ON. This panel also provides information on the status of the Hyperion Imaging System interlocks and real-time readings of the helium flow, chamber pressure, helium pressure, and optical chamber temperature. When the helium pressure reaches 30 psi the chamber is pressurized. Loading the Tuning Slide WARNING Use care when loading and unloading the sample slide to avoid injury from broken glass. Obtain the 3-Element Full Coverage Tuning Slide (PN ). WARNING It is recommended that you store the tuning slide and the sample slide in a slide holder between uses to prevent dust buildup. 54 Hyperion Imaging System:

55 Chapter 4: Operation Hyperion Imaging System Startup Procedure On the Toolbar, click the HTI Sample Unload button. The sample stage will extend from the ablation chamber. Open the instrument sample window. When the stage is fully extended, carefully insert the slide (facing up) into the ablation chamber stage. IMPORTANT Gently grip the slide with your thumb and index finger to ensure that you are not pushing down on the slide. Place the slide in the grooves on the stage and insert. Push the slide until it is firmly seated. A loud noise will occur if the slide has been inserted incorrectly. Hyperion Imaging System: 55

56 Chapter 4: Operation Hyperion Imaging System Startup Procedure Click Load. If the instrument sample window is open, a dialog box confirms that a Cover Door is Closed. Close the sample window. Click Yes. NOTE The slide moves to the home position immediately after it is loaded. The home position is set to 14,714 µm from the left side of the inserted microscope slide and 13,300 µm from the bottom of the slide, which is considered the midpoint of the slide. The slide coordinate system starts on the bottom left hand corner of the slide, which represents X=0 and Y=0. 56 Hyperion Imaging System:

57 Chapter 4: Operation Hyperion Imaging System Startup Procedure NOTE If the slide does not go to the home position, select HTI Camera > Advanced Actions tab > XY Home. NOTE The area available for ablation is approximately 16,000 µm in the Y direction and 60,000 µm in the X direction. The chamber will automatically flush for approximately 5 minutes. (Unless the default flushing time has been changed in the HTI Settings). Check the HTI Status Panel to confirm that the chamber has been flushed (blue circle). In the Toolbar click Laser Start to turn on the laser power. Check the HTI Status Panel to confirm that the laser is on (blue circle). Create a Panorama The Panorama mode allows the software to scan the slide and create a high-level image of a slide section from which you can select a region of interest (ROI) for tuning. The panorama does not get ablated. The ROI is a small section of the panorama that users can define for ablation. IMPORTANT The panorama is created in order to find an unablated region with a uniform tuning film. 1 Click Camera in the Toolbar to open the HTI Camera software and visualize the tuning slide. Hyperion Imaging System: 57

58 Chapter 4: Operation Hyperion Imaging System Startup Procedure 2 In the Toolbar at the top of the HTI Camera window, click Start Video. This displays live video of the tuning slide. 3 Click Auto Focus to focus the camera and visualize the tuning slide. 58 Hyperion Imaging System:

59 Chapter 4: Operation Hyperion Imaging System Startup Procedure 4 Select the Actions tab on the right side of the window. In the Slide Move (µm) box enter a slide move value and scan the sample using the left, right, up, and down controls to find an area on the slide for your panorama. 5 Alternatively, enter the start position in the Slide X, µm and Slide Y, µm fields of the Slide Move box. Click Move to Slide Pos. The stage moves so that these coordinates are in the middle of the field of view, indicated by the red cross hairs. The coordinates are found in the status bar at the bottom of the Camera window. Hyperion Imaging System: 59

60 Chapter 4: Operation Hyperion Imaging System Startup Procedure 6 Enter these values in the Panorama X1 and Panorama Y1 fields to make this the top left coordinates of the panorama. 7 In the Width, µm and Height, µm fields, enter the desired values for your panorama. For example, enter Width 1000 µm and Height 1000 µm to create a 1 mm x 1 mm panorama. Enter the dimensions that correspond to the top-left X1, Y1 of the panorama to create. Click the button to save the panorama and enter a filename. 8 Click Create Panorama. The Auto Focus will begin focusing on the slide. View a realtime image of the slide as the panorama is generated in the camera window. 60 Hyperion Imaging System:

61 Chapter 4: Operation Instrument Tuning 9 Once the panorama is generated click the Panorama tab to view the image. NOTE A 1,000 µm x 1,000 µm panorama will take approximately 1 minute to generate. Instrument Tuning Materials Required 3-Element Full Coverage Tuning Slide (PN ) The Hyperion Tissue Imager is tuned using the 3-Element Full Coverage Tuning Slide. The helium flow is adjusted at specified increments to ensure optimal transport of transients to the Helios sample introduction system. The signal for 175 Lu is collected at the optimal helium flow, and the transient peak is recorded. The transient peak corresponds to the plume of ablated material that is transferred via the coupling tubing to Helios. Determine Laser Energy The laser energy (in db) represents the setting of the attenuator that controls the energy of the laser delivered to the sample. For optimal performance, the laser energy must be set to a value that will result in complete ablation of the sample from the slide. At installation, your Fluidigm Field Service Engineer will set laser energy to 0 for tuning on the Tissue Imager. Hyperion Imaging System: 61

62 Chapter 4: Operation Instrument Tuning IMPORTANT Over time, the energy required for full ablation of tuning film will deviate from 0 db. To restore the energy level to 0 db, the reference energy must be re-optimized. Contact your Field Application Specialist for assistance when you need to adjust the ablation energy to 0 db for tuning (approximately every three months). You can check the laser energy required for the tuning by acquiring a small region of interest and checking that you get a very clean line of ablation on the tuning film. Monitor Background Signal It is recommended that users create a background template to monitor the metals on the Imaging System in order to confirm metal contamination before tuning the instrument. 1 Click Acquisition Settings to open the Data Acquisition window. 2 Right-click on the Parameter table and select Apply template. Select the Tuning Slide template. Click Periodic Table and select Os(189). Click OK. 3 Click Select Template. 4 In the Parameter window click Parameter Name and then select Time from the drop-down. 5 In the Parameter window enter the following: Parameter Start Value: 0 Parameter End Value: 300 Parameter Step Value: 1 Ablation Energy: 0 Ablation Frequency: 20 6 Click Run. 7 Click the Graph Mass button in the Control Bar to open the Masses Per Reading window. Click Dual Count. IMPORTANT The Os(189) value should be <3 to ensure minimal background. 62 Hyperion Imaging System:

63 Chapter 4: Operation Instrument Tuning 8 Save this template to compare to subsequent acquisitions to compare the background. Auto Tuning 1 In the Toolbar click Tuning to open the Tuning window. 2 Click New. At the top of the Tuning Protocol table, a new Calibration will appear in blue with a date and time stamp for reference. Hyperion Imaging System: 63

64 Chapter 4: Operation Instrument Tuning 3 Click the General tab on the right side of the Parameters tab. Check the checkboxes for Enable QC Report, and Enable Transients Calibration. NOTE When switching from solution mode to HTI Acquisition mode, Enable Pre Calibration XY Optimization and Enable Fine XY Optimization are also checked. 4 Click Camera in the Toolbar to open the HTI Camera window and select a region for a new panorama. Enter 1000 µm x 1000 µm for the width and height. Click Create Panorama. Refer to Create a Panorama for more details. 64 Hyperion Imaging System:

65 Chapter 4: Operation Instrument Tuning 5 Click the Panorama tab and select a ROI by dragging and dropping the red translucent box on the desired area. Select a 100 um x 500 um area for tuning. Check the Log tab on the right side of the window to confirm the correct dimensions have been set. NOTE A 100 µm x 500 µm area or larger is recommended for tuning. In subsequent runs, it is recommended that you start at the coordinates where the last tuning was completed. Hyperion Imaging System: 65

66 Chapter 4: Operation Instrument Tuning IMPORTANT The ablation frequency is set to 20 Hz to allow each plume of transients to arrive at the detector as distinct peaks with minimal signal overlap. IMPORTANT If switching from solution mode to HTI mode or when the injector and coupling have been removed and replaced, perform the Pre Calibration XY Optimization and Fine XY Optimization. Check the Enable Pre Calibration XY Optimization and Enable Fine XY Optimization checkboxes. These tuning sub-calibrations may also be required if you see consistently low signal that may be related to the Helios XY alignment. 6 Open the HTI Status Panel and check that the Chamber Flushing State is flushed and the Laser State is on. 7 Click Run. 8 The tuning window is minimized after Run is clicked and restored when tuning is complete. The tuning is complete when the Tuning Protocol status changes to Completed and the row turns green. The entire Auto Tuning (with the two selected Sub calibrations) takes approximately minutes. 9 Close the Tuning window. NOTE The most recent successful Calibration is highlighted in green. 66 Hyperion Imaging System:

67 Chapter 4: Operation Instrument Tuning 10 Click the Results tab to view the results of the tuning. The pass criteria for Auto Tuning are as follows: Resolution (Mass 1): >400 Transients Cross Talk 1: < 0.15 Transients Cross Talk 2: < 0.05 Mean Duals for Lu(175): >500 (per laser shot) NOTE When the instrument has been switched from Solution mode to Imaging mode, the mean dual count for Lu(175) may be up to 80% of what they were prior to switching to solution mode. Verify that this value is not below the 500 specification indicated. IMPORTANT If Auto Tuning fails, follow the Manual Tuning HTI Optimization (Appendix B) procedure. Hyperion Imaging System: 67

68 Chapter 4: Operation HTI Acquisition (Batch Mode) HTI Acquisition (Batch Mode) Load a New Slide In the Toolbar, click HTI Sample Unload. NOTE Do not click the Unload button more than once. It will take a few seconds for the stage to extend. Open the instrument sample window. When the stage is fully extended insert the sample slide into the ablation chamber stage. Close the instrument sample window. Click HTI Sample Load. If you forget to close the sample window, a Warning dialog box alerts you that the Cover Door may be open. Close the sample window and click OK. 68 Hyperion Imaging System:

69 Chapter 4: Operation HTI Acquisition (Batch Mode) The chamber flushes in approximately 5 minutes. The flushing status is indicated on the Log tab on the right side of the HTI Acquisition window. The laser will turn on automatically. Click HTI Status Panel to open the Status Panel and confirm that the Laser State is ON (blue). The System Logs read Checking interlocks followed by Laser warmup. NOTE You may begin taking your panoramas in HTI acquisition mode during this time. Hyperion Imaging System: 69

70 Chapter 4: Operation HTI Acquisition (Batch Mode) Upload an Image of the Slide It is recommended that you upload a JPG image of the slide which can help you navigate the slide to select ROIs for ablation on a panorama. 1 Use a phone camera or a digital camera to take an image of the slide. The frosted end should be on the right side of the image. Ensure that the slide is on a dark background so that the tissue is clearly visible and the image is as straight as possible. Save the image on the E: drive of the instrument computer. 2 Crop the image as close to the edges of the slide as possible. NOTE Use Microsoft Paint to create a box to outline the image so that it s easy to locate the sample. 3 In the Toolbar click HTI Acquisition to open the batch acquisition window. 4 To open the Slide Selection window, click SETTINGS and select Slide from the dropdown. NOTE You may also right-click on the Slide space to set the slide image. 5 Double-click on an empty row to set up a new slide template. Enter a name (description), Height, and Width of your slide. 70 Hyperion Imaging System:

71 Chapter 4: Operation HTI Acquisition (Batch Mode) NOTE Most slides have a Height of 26,000 µm and Width 75,000 µm. 6 To insert the image in the Slide box in the HTI Acquisition window, click on the empty box under Image File. When the Open dialog box opens, browse to locate the image file. Double-click the image file to select it. On the Slide Selection window, click Apply Selection. Add appropriate Image file by clicking in this area 7 Use the arrow key to expand the Slide menu. NOTE Drag the mouse to resize the borders between the boxes in the HTI Acquisition window. Create an MCD File 1 In the menu of the HTI Acquisition window, click FILE and select New. 2 In the New File dialog box: a Click Browse to find the file folder and enter a filename. b In the Slide drop-down menu, select the slide that you have created (for example, TEST). c Click Save. NOTE If you have not created a new slide select Default. Hyperion Imaging System: 71

72 Chapter 4: Operation HTI Acquisition (Batch Mode) NOTE The area available for ablation is approximately 16,000 µm in the Y direction and 60,000 µm in the X direction. 3 Click Save Library and close the Slide Selection window. Create a Panorama The Panorama allows the software to scan the slide and create an optical image from which you can select a region of interest (ROI) for sample ablation. 1 Drag your mouse over the slide image to select an area for the panorama. The XY coordinates will auto populate. Click Create. 2 Alternatively, enter the coordinates and dimensions (width and height) of the panorama you would like to create in the left corner of the window. X1, Y1 represents the top-left corner of the panorama. 72 Hyperion Imaging System:

73 Chapter 4: Operation HTI Acquisition (Batch Mode) Hyperion Imaging System: 73

74 Chapter 4: Operation HTI Acquisition (Batch Mode) 3 In the main Toolbar click Camera. In the Camera window click Start Video. 4 Define the Width, (µm) and Height, (µm) of your panorama. Click the Actions tab. In the Slide Move (um) box, scan the sample using the up and down arrow keys to select an area for your panorama. Right-click and select Move to Here. Look at the Slide coordinates at the bottom of the camera window. Enter these values into the X1 µm and Y1 µm fields in Add Panorama in the HTI Acquisition window. 5 Click Add to add a panorama with these coordinates. A pink box will appears on the slide image to the right of the Add Panorama panel to represent the area of the panorama and a description will appear on the list in the panel. Enter a description for the panorama in the box. To delete a panorama, click the appropriate row and click the Delete key. NOTE You may also select more than one region for the panorama(s). These are added to the list and displayed as blue boxes in the slide image. 6 Click Create to begin acquiring the panorama(s). As the panoramas are created they are checked on the table. 74 Hyperion Imaging System:

75 Chapter 4: Operation HTI Acquisition (Batch Mode) 7 A dialog box confirms that each panorama is generated, and that panorama appears under Panorama in the center of the HTI Acquisition window. To view the image of each panorama click on the description in the table. NOTE Use a mouse with a scroll wheel to zoom into or out of the panorama. Once the panoramas have been created the boxes are unchecked and the Status is Done. Hyperion Imaging System: 75

76 Chapter 4: Operation HTI Acquisition (Batch Mode) Creating a Region of Interest 1 To select a region of interest from the panorama, right-click on the panorama and select ROI Tool (selected by default) from the drop-down. Click and drag to select a ROI. A light purple box appears on the panorama image indicating the selected ROI. The selection appears in the Acquisition table at the bottom of the window. 2 Alternatively, to select ROIs on the slide, click the Add ROI arrow in the HTI Acquisition window to open the Add ROI box. Enter the coordinates for the start position (top-left corner of the desired ROI) in the X (µm) and Y (µm) fields, and enter the dimensions in the Width (µm) and Height (µm) fields. IMPORTANT To delete a ROI, check the Select checkbox of that ROI in the table and press the Delete button on your keyboard. Click Control and select several ROIs to delete a group of ROIs. 76 Hyperion Imaging System:

77 Chapter 4: Operation HTI Acquisition (Batch Mode) 3 To apply the appropriate elements to each ROI selected, right-click on the Acquisition table and select Apply Template. The Acquisition Templates window opens. 4 Select the appropriate template or click Periodic Table to choose the elements from the Element Table. Click Export. Hyperion Imaging System: 77

78 Chapter 4: Operation HTI Acquisition (Batch Mode) 78 Hyperion Imaging System:

79 Chapter 4: Operation HTI Acquisition (Batch Mode) NOTE Alternatively, right-click on Apply Template to access the quick access drop-down menu. 5 Right-click on the Acquisitions table to enter the desired values for the ablation parameters in the table for each ROI selection including: Ablation Energy (db) Step X: 1 Step Y: 1 Ablation Frequency (Hz): 200. NOTE To select and apply values to multiple ROIs, hold down Ctrl and click each row. Hyperion Imaging System: 79

80 Chapter 4: Operation HTI Acquisition (Batch Mode) A purple box will appear and the dimensions of the ROI, with related information are recorded in red text. 80 Hyperion Imaging System:

81 Chapter 4: Operation HTI Acquisition (Batch Mode) After the template is applied, the channels appear to the right of the window in the Channels table. Check the boxes for the channels that you would like to see in your acquisition files and enter a label in the Label column. 6 Under the Acquisition Commands, in the lower right-hand corner of the HTI Acquisition window, check Generate Text Files. IMPORTANT You may also check Shutdown which will shutdown the plasma and Hyperion Tissue Imager when the HTI acquisition is complete. NOTE By checking ROI Images the software will generate an image before ablation and after ablation and save this to the MCD file. These images can later be viewed with MCD Viewer To create additional ROIs, click and drag on the panorama image to create a box with the desired dimensions and location. The dimensions will appear on the box and in the Log on the right side of the page. 8 The Status bar turns green and indicates the percent completion of the ablation. NOTE To reorder the list for a parameter, click that parameter. 9 The gray bar at the bottom of the window indicates the Total Ablation Time required to acquire the selected ROIs and the number of acquisitions to be run. Hyperion Imaging System: 81

82 Chapter 4: Operation HTI Acquisition (Batch Mode) 82 Hyperion Imaging System:

83 Chapter 4: Operation HTI Acquisition (Batch Mode) 10 From the Filter drop-down select ROIs to Acquire to view the acquisitions that are selected. 11 Select All ROIs to view all the acquisitions that are created. 12 Select Current Panorama ROIs to view the selected ROIs associated with the panorama selected in the Panorama table. Hyperion Imaging System: 83

84 Chapter 4: Operation HTI Acquisition (Batch Mode) Determine the Ablation Energy for the Sample Slide In order to determine the optimal ablation energy for ablating a tissue sample you should ramp up the ablation energy to find the optimal setting. 1 In the Standalone Ablation box enter the following: Ablation X Step, µm: 1 Ablation Y Step, µm: 1 Ablation Frequency, Hz: 100 Hz Ablation Energy: 5 db 2 Click Ablate Selected ROI. Ramp the energy up 1 db and ablate again. Continue until you determine the ablation energy that is sufficient to fully ablate the sample. 1 Check the HTI Status Panel to ensure that the laser is on and the chamber has been flushed. 2 Click Start at the bottom-right corner of the window to begin ablation. NOTE The Status bar in the Acquisition table indicates the progress of the ablation of each ROI. The ROIs are ablated in the order in which they appear in the table. 84 Hyperion Imaging System:

85 Chapter 4: Operation HTI Acquisition (Batch Mode) 3 Click Camera in the toolbar and then click the Camera tab to see the live camera image of the ablation region to confirm that all parameters have been correctly set and the ablation is progressing with clean lines of sample clearance. Click the Zoom In or Zoom Out buttons on the top Toolbar to adjust the level of visual detail. NOTE If Start Video is grayed out, this indicates that the live camera video is on. IMPORTANT Click the Stop button at the bottom of the HTI Acquisition window at any time during the acquisition to stop and adjust your settings or to select a new region. 4 A rain plot showing the signal for each of the elements in your sample appears on the right side of the HTI Acquisition window. Click Detach to move the Rain Plot (Mass) panel within the window and allows you to resize the panel. Hyperion Imaging System: 85

86 Chapter 4: Operation Hyperion Imaging System Shutdown Hyperion Imaging System Shutdown To remove the sample slide from the ablation chamber, open the sample window and click HTI Sample Unload. The laser stops automatically. Click HTI Sample Load to retract the stage and close the chamber. Click Plasma Stop. The plasma stop sequence will be initiated. Click HTI Gases Stop. Close the software. Disconnecting the Hyperion Tissue Imager from the Helios Instrument 1 Disconnect communication cables and gas lines including USB 2.0 connection, USB 3.0 connection, argon, and helium gas lines. 86 Hyperion Imaging System:

87 Chapter 4: Operation Disconnecting the Hyperion Tissue Imager from the Helios Instrument 2 On the Hyperion Tissue Imager ablation chamber, loosen the black nut on the coupling tubing. Disconnect the coupling interlock. 3 Using the wheel retraction switch on the right side of the instrument, raise the instrument. Hyperion Imaging System: 87

88 Chapter 4: Operation Disconnecting the Hyperion Tissue Imager from the Helios Instrument 88 Hyperion Imaging System:

89 Chapter 4: Operation Disconnecting the Hyperion Tissue Imager from the Helios Instrument 4 Turn off the RF Generator circuit breaker on the on the right side of the Helios instrument. Disconnect the AC power cord from the AUXILIARY connection panel. 5 Carefully roll the Hyperion Tissue Imager to a safe area in the laboratory using the instrument castors. 6 Re-connect the instrument power to a wall receptacle and use the instrument wheel retraction switch to lower the instrument and secure it in place. Hyperion Imaging System: 89

90 Chapter 4: Operation Switching to Helios Solution Mode Switching to Helios Solution Mode IMPORTANT Ensure that the nebulizer, torch, injector, spray chamber, cones and load coil on the Helios system are clean and correctly installed and aligned on the instrument. Check the condition of the interface pump oil in the visual inspection window of the Helios instrument. Refer to the Helios, a CyTOF System User Guide, Chapter 6: Maintenance (PN ) for more details. Disconnect the coupling tubing and move the Hyperion Tissue Imager out of the way to access the injector. Gently twist and pull the injector from the torch assembly. Insert the HT Injector (PN ) into the torch body and push in until it can go no farther. 90 Hyperion Imaging System:

91 Chapter 4: Operation Switching to Helios Solution Mode Reconnect the heater box to the torch assembly. Slide the heater box onto the support pins towards the torch assembly so that the spray chamber ball joint connection meets the injector. Connect the ball joint clamp to secure the spray chamber to the injector. Place the heat shield onto the heater and tighten the four thumbscrews, two on each side of the shield. Hyperion Imaging System: 91

92 Chapter 4: Operation Switching to Helios Solution Mode Figure 1. The heat shield on the Helios heater box. There are four screws, two on each side of the shield, which must be tightened before replacing the shield. Connect the heater connection to the front face of the instrument. Open the CyTOF Software 6.7. Click Control Panel in the Toolbar and select the Devices tab. In the HTI/PSI click PSI as Introduction. In the Introduction System drop-down menu select PSI. Click Set Configuration. 92 Hyperion Imaging System:

93 Chapter 4: Operation Switching to Helios Solution Mode Helios is now ready to run in solution mode for mass cytometry analysis. Refer to the Helios User Guide (PN ), Chapter 5: Operation. Run a Full Tuning Protocol before running the instrument in solution mode. Hyperion Imaging System: 93

94 Chapter 5: Maintenance The Hyperion Imaging System is very simple to maintain. You may be required to clean the outside of the instrument, but all internal components including the laser ablation chamber and the cover glass are addressed during the Preventive Maintenance every 6 months. Cleaning Instructions Table 19. The cleaning frequency and reagents required for correct maintenance and cleaning of the Hyperion Imaging System Parts Frequency Performed by Agents/Equipment Company and Part Number Injector As required Operator % Contrad in DIW Decon Labs Glassware brushes Torch As required Operator % Contrad in DIW Decon Labs Glassware brushes Skimmer- Reducer Sampler Cone Preventive Maintenance Preventive Maintenance Field Service Engineer Field Service Engineer NOTE For cleaning frequency in solution mode refer to the Helios User Guide (PN ), Chapter 6: Maintenance. It is recommended that you clean glassware and instrument cones weekly if you are running the Helios instrument in solution mode. Table 20. The equipment required for correct maintenance and cleaning of the Hyperion Imaging System Parts Equipment Company Product Name Part Number Torch Glassware brushes Restek Nylon Tube Brushes and Pipe Cleaner Injector Glassware brush Gordon Brush Nylon. Miniature Single- Spiral brushes with a stainless steel stem and a cut end PN PN 94 Hyperion Imaging System:

95 Chapter 5: Maintenance Switching to Helios Solution Mode Exterior Surfaces NOTE This procedure may only be done when the instrument has been disconnected from the Helios instrument and moved out of the way. Disconnect the Hyperion Imaging System from the power supply. Wipe the instrument exterior surfaces only using a towel dampened with a lab-grade cleaning agent (for example, isopropanol). Repeat Step 2, using a towel dampened with DIW. Dry the instrument exterior using a dry towel. Injector Cleaning 1 Soak in 10% Contrad for up to 1 hr. 2 Scrub the injector with the recommended glassware brush (PN 01023, Gordon Brush). 3 Insert the brush into the tip of the injector and pull in and out a few times to dislodge any buildup around the tip. 4 Rinse thoroughly with DIW. 5 Spray isopropanol on the injector and air-dry. 6 Alternatively, completely dry the injector with a blow dryer or heat gun. NOTE Ensure that the parts are completely dry before reinstalling. Torch Cleaning See the Helios User Guide (PN ) for the procedure Torch Assembly Removal, Cleaning, and Reassembly of the Injector and Torch. Soak in 10% Contrad for up to 1 hour. Scrub the parts with the recommended glassware brush (PN 20108, Restek). Rinse thoroughly with DIW. Spray isopropanol on the torch and air dry. Alternatively, completely dry the glassware with a blow dryer or heat gun. NOTE Ensure that the parts are completely dry before reinstalling. Hyperion Imaging System: 95

96 Chapter 5: Maintenance Periodic Maintenance Coupling Tubing IMPORTANT We do not recommend cleaning of the coupling tubing. Inspect the tubing periodically to ensure that there are no breaks or cracks. Replace the Coupling Tubing Assembly (PN ) if necessary. Periodic Maintenance Vacuum Interface Cones The vacuum interface cones are inspected and cleaned if necessary during the preventive maintenance of the instrument. If running solution mode the skimmer-reducer and sampler cone should be cleaned on a weekly basis. See the Helios User Guide (PN ) for the procedure to Removal, Cleaning, and Reinstallation of the Vacuum Interface Cones. Replace the Load Coil If the load coil (PN ) is damaged or misshapen it must be replaced. Inspect the load coil regularly to ensure that it is in good condition. Excessive deposits and discoloration Side arms are bent WARNING Before beginning the following procedure, the Helios instrument should be off and sufficiently cooled. Perform prior to starting plasma. 96 Hyperion Imaging System:

97 Chapter 5: Maintenance Periodic Maintenance 1 Remove the nebulizer line. Disconnect the Sample Line from the grounding nut and connect to the Sample Loader. 2 Remove the injector ball joint clamp. Slide the heater (with heat shield) off of the heater guide pins and rest it on the support pins. Remove the torch assembly from the front face of the instrument. 3 Open the front access door. Undo the four clips on the top and both sides of the front shield and lift off (magenta boxes below). 4 Using a 7/16 wrench loosen the nut holding the load coil. Use a 9/16 wrench simultaneously to apply counterforce on the larger nut. Repeat on the opposite side. Remove the old load coil. 5 Carefully remove the zip ties on the new load coil using a wire cutter. Remove the base of the load coil holder but keep the load coil core in place. 6 Install the new load coil using the 7/16 wrench to tighten the nuts and washers while applying counterforce on the larger nuts with the 9/16 wrench. Hyperion Imaging System: 97

98 Chapter 5: Maintenance Periodic Maintenance Nuts to apply counterforce Load coil core Nuts to loosen 7 Remove the load coil core. Examine the new load coil and make sure that the coils are equally spaced. Also ensure that the arms of the coil are straight and aligned. 8 Replace the front shield and fasten the four clips. 9 Reinstall the torch assembly. Check the alignment of the torch with the load coil (refer to the appropriate steps in the cleaning and maintenance section if necessary). See the Helios User Guide (PN ) for the procedure Z-alignment Adjustment. 98 Hyperion Imaging System:

99 Chapter 5: Maintenance Periodic Maintenance Replace the Injector Sealer Cap Replace the Injector Sealer Cap if required (PN ). You will also need an O-ring, which is included in the O-Ring Kit, Torch Body (PN ). Injector sealer cap 1 Insert the O-ring into the cap. Ensure that the O-ring is firmly seated in the inner cap and the center opening is unobstructed. Injector sealer cap O-ring 2 Loosely screw on the new injector sealer cap (with the O-ring) to the injector holder. Hyperion Imaging System: 99

100 Chapter 5: Maintenance Periodic Maintenance 3 Carefully insert the injector into the injector holder. Pull the injector in and out to confirm that there is sufficient tension on the injector but that you are still able to insert and remove. WARNING The injector may break if excessive pressure is used to insert the injector into the injector holder. 4 Ensure that the cap is finger tight. Do not overtighten. 5 Remove the injector and place aside. 100 Hyperion Imaging System:

101 Chapter 5: Maintenance Periodic Maintenance Change the Interface Pump Oil Before beginning the procedure have on hand: Funnel with extension tubing Flexible drain funnel Vacuum Pump Oil (PN ) 1 Switch off the RF generator power using the RFG circuit breaker on the right side of the instrument 2 Open the front access door using the door handle. Pull the spring pin and open the lower instrument door. 3 Open the lower right door of the instrument. The Interface Pump is on the right side of the instrument. Valve Figure 2. The interface pump in the lower right compartment of the Helios system. The visual inspection window is on the side of the interface pump (inset). 4 The oil level in the interface pump should be approximately ¾ full according to the Min and Max lines on the visual inspection window. Hyperion Imaging System: 101

102 Chapter 5: Maintenance Periodic Maintenance 5 Verify the condition of the oil using the oil inspection chart. The oil should be below Level 4 as indicated in the pump oil condition chart. IMPORTANT The interface oil condition should be checked weekly. Figure 3. Pump oil condition chart. When the oil color is above level 4 (as indicated by the black arrow) the oil should be replaced in the interface pump. 6 Remove the two white caps on each end of the Drain Kit (PN ). 102 Hyperion Imaging System:

103 Chapter 5: Maintenance Periodic Maintenance 7 Unscrew the gold valve on the interface pump, and connect the drain kit provided. 8 Drain the oil into a tray or plastic container. 9 Remove the drainage tube and reconnect the gold valve. Hyperion Imaging System: 103

104 Chapter 5: Maintenance Periodic Maintenance 10 Unscrew the top cap, using the Allen key provided. 11 If the oil is very dirty, add 100 ml of fresh Vacuum Pump Oil (PN ) to allow the oil to drain freely, and then drain using the drainage tubing before proceeding to the next step. 12 Using a funnel and drain tube provided, fill the oil slowly until it reaches ¾ full by viewing the oil inspection window. 13 Replace the top cap and tighten with the Allen key. Do not overtighten. 14 Close the lower left and right instrument doors and the front access door of the Helios system. NOTE Dispose of the oil in accordance with applicable local, regional, national and regulations. Change the Oil on the Backing Pump NOTE The backing pump oil is changed on an annual basis by the Fluidigm Field Service Engineer (FSE) as part of the preventive maintenance of the instrument. Contact Fluidigm technical support if you believe the oil in the backing pump requires changing. 104 Hyperion Imaging System:

105 Troubleshooting Table 21. Troubleshooting tips for the Hyperion Imaging System. Symptom Possible Cause Recommended Solution Sample Introduction Errors Laser not turning on in software. Instrument not connected to power source. Check the power cords. Cannot focus the image. Objective too far of/close to slide In the HTI Camera window click Auto Focus. Ablation is occurring, no or low ion signal. Gas lines not connected. Check helium and argon gas line connections. Ablation is occurring, no or low ion signal. Coupling tubing not connected or leaking. Check coupling tubing, and check that the connections are tight. Ablation is occurring, no or low ion signal. Leak in ablation chamber. Contact a Fluidigm Service. Ablation is occurring, no or low ion signal. Incorrect Ablation Power settings. Determine the most efficient ablation energy for the sample. Change the Ablation Power settings. Ablation quality poor, blurry image, no ions detected Slide may be inserted incorrectly. Ensure that the sample is facing up. Hyperion Imaging System: 105

106 Chapter 5: Maintenance Troubleshooting Symptom Possible Cause Recommended Solution Coupling Tubing Connection Errors Possible coupling tubing leak Coupling tubing not well-seated into the ablation chamber or into the injector. Verify coupling tubing connections to the injector and the ablation chamber. Poor transients Coupling tubing fitting and nuts are too tight. Check that coupling tubing is correctly connected. Nuts should be finger tight. No ions in the Masses per Reading window Coupling tubing not correctly connected. Check that coupling tubing is correctly connected. Nuts should be finger tight. No ions in the Masses per Reading window Blocked coupling tubing or blockage in the cone. Flush argon through the system by adjusting the makeup gas flow to 1.0 L/min in the Control Panel > HTI Setup > Chamber/Gases > Flushing Makeup gas flow. Flush for 1 min. Low chamber pressure. Leak in ablation chamber. Ensure that the ablation chamber lid is properly secured. Low chamber pressure. Low helium supply pressure. Ensure that the Check the helium supply pressure to ensure that it is 30 ±1 psi. High chamber pressure. The electrical connections in the pressure sensor may be disconnected. Contact Fluidigm Support. Poor transients. Tubing not correctly connected. Reinstall coupling tubing. Wide transient width. O-ring seals on coupling tubing connections not correctly made. Reinstall coupling tubing. In the HTI Status Panel check the chamber pressure and the helium flow. 106 Hyperion Imaging System:

107 Chapter 5: Maintenance Troubleshooting Symptom Possible Cause Recommended Solution Poor transients The injector/fitting may be pulled out slightly because the tubing is under tension. Check that injector and fitting are correctly installed. The coupling tubing should have a slight bend. Poor signal The injector/fitting may be pulled out a bit because the tubing is under tension. Check that injector and fitting are correctly installed. The tubing should have a slight bend. Alignment Errors Image cannot be brought into sharp focus. Microscope not aligned. In the HTI Camera window click Autofocus. Stage will not move or moves erratically. Stage not initialized correctly. Check the connectors on the instrument panel. Initial movement of the stage is large but otherwise it moves correctly. XY start position not set correctly. Click Camera > Action > Home XY Stage. If this does not resolve the issue you may need to power cycle the instrument. Shut down the software and then turn off the power on the laser to re-establish correct communication. Laser Not Firing Laser Not Firing Laser interlock tripped Turn the laser off and then on in the software. Laser Not Firing Laser alignment off. Contact a Fluidigm Service. Hyperion Imaging System: 107

108 Chapter 5: Maintenance Troubleshooting Symptom Possible Cause Recommended Solution No ablation on the slide. Laser alignment off. Contact a Fluidigm Service. Laser error message in HTI Acquisition mode. Signal Optimizing Errors Laser not communicating with software. Close the HTI Acquisition window. Reopen the window and turn on the laser. Click Laser Start. Begin the acquisition again. No ion signals (Looking for 1000 ions/shot. For example, at 20 Hz = counts/seconds on Masses Per Reading graph) Leak in chamber Check for gas leaks. Tighten fittings and nuts on the coupling tubing. Gases are set incorrectly Makeup gas should be approximately 0.82 L/min and helium should be approximately 0.15 L/min. These values will vary depending on the specific Hyperion Imaging System. Ablation chamber door may be open. Close the door. Flush the gases again. Ablation chamber cover not properly secured. Contact Fluidigm Support. Poor ablation. Too spaced out in either axis. Check for incorrect image analysis parameters. 108 Hyperion Imaging System:

109 Chapter 5: Maintenance Troubleshooting Symptom Possible Cause Recommended Solution Poor ablation. Too spaced out in either axis. Ensure that laser frequency is high enough. No ablation. Attenuator setting too low Increase the attenuator value. Lower than expected ion signals. XY position is not optimized. The XY position on Helios instrument has to be manually optimized. Lower than expected ion signals. Makeup gas setting not optimized. Optimize the makeup gas settings for better sensitivity. In the Data Acquisition Settings window select Makeup Gas in the drop-down and perform transient analysis. Low Lu175 dual counts or sensitivity Transient width not within acceptable range Transient width not within acceptable range Detector voltage setting not optimized. Current setting not optimized. AutoDV optimization performed with abnormally high Xe131 (or alternate isotope) levels. Helium flow is not optimum. Incorrect height of ablation stage. Use manual optimization of detector voltage in dry plasma mode. In the Data Acquisition Settings window select Detector Voltage and perform Transient analysis. Optimize the Gases/Current settings for better sensitivity. In the Data Acquisition Settings window select Current in the drop-down and perform transient analysis. Identify sources of leak and resolve. Check the torch for correct seal. Check coupling tube for damage. If the issue persists contact Fluidigm Support. Check that the helium gas line is connected correctly to the communication panel of the Hyperion Tissue Imager. Check that the gas cylinder is open and the gas line is connected correctly to the cylinder. Repeat the tuning procedure. Hyperion Imaging System: 109

110 Chapter 5: Maintenance Troubleshooting Symptom Possible Cause Recommended Solution Low mass peak means and signals. Sample not fully ablated. Select another region to ablate and test the efficiency. If the ablation is still not clean, power cycle the instrument. Chamber Pressure High chamber pressure reading in the Status Panel window (>2 psi), even with the sample door open. Damaged connector. Contact a Fluidigm Service. Other Poor transients Small fiber on one of the ends of, or inside of the coupling tubing, causing turbulent gas flow. Disconnect coupling tubing. Inspect with magnifying loop and remove any fiber(s) found. Poor transients Injector not inserted correctly. Check that the injector is firmly seated in the torch assembly, but also that the injector sealer cap may be tightly secured. Streaking in TIFF images Vibrations in the system. Check that the Hyperion Tissue Imager has been fully lowered to the ground. Check that the Helios instrument castors have been locked to minimize vibrations. Check that the correct Start Value has been used from the most recent tuning procedure. 110 Hyperion Imaging System:

111 Chapter 5: Maintenance Troubleshooting Symptom Possible Cause Recommended Solution Slow/irregular stage movement during ablation. Check that the slide has been secured to the stage with the clips. (The slide size must be compatible with the Hyperion Imaging System.) Power-cycle the instrument and try ablating a new area on the slide. Uneven spacing of marks on the glass slide. Vibrations in the system could cause this. Check that the Hyperion Tissue Imager has been fully lowered to the ground. Check that the Helios instrument castors have been locked to minimize the vibrations. If this continues to occur, Contact a Fluidigm Service. Flushing finishes prematurely. Software communications error. Click Chamber Flush on the Toolbar. The flush should take approximately 5 minutes. Irregular black pixels in TIFF image. Data cables in the data acquisition card disconnected or damaged. Tighten or replace the data cables from the Helios instrument to the computer tower. Verify that all data cables are correctly connected. High CVs (>50%) during tuning procedures. Multiple peaks in Plotviewer. High or low gain channel cables may be switched on the Helios preamplifier. The gain channel cables are located on the right side of the Helios instrument on the AC panel. The Low Out 1 and the High Out 2 cables may be switched. Hyperion Imaging System: 111

112 Chapter 5: Maintenance Troubleshooting Symptom Possible Cause Recommended Solution Laser produces a thin/wavy ablation pattern. Laser has not sufficiently warmed up. Allow 10 minutes for laser to warm up. Auto Focus not working. Iris aperture not centered. Check the centration of the ablation spot on the camera image. If the spot is not aligned contact your Fluidigm Service. Panorama images not appearing in HTI Acquisition window. No image to select ROIs. High number of panorama images reduces memory. Save the MCD file. Close the CyTOF software and restart. 112 Hyperion Imaging System:

113 Appendix A: Safety General Safety In addition to your site-specific safety requirements, Fluidigm recommends the following general safety guidelines in all laboratory and manufacturing areas: Laser instruments generate potentially hazardous UV radiation. Do not remove the top covers of the Hyperion Tissue Imager. Removal of the top cover and maintenance should not be performed by anyone other than a Fluidigm Field Service Engineer. Do not place fingers or hands in the ablation chamber when the sample door is opening or closing. The power supplies of the Hyperion Tissue Imager are capable of generating potentially lethal voltages. Removal of the side panel and maintenance on the electrical box should not be performed by anyone other than a Fluidigm Field Service Engineer. Know the locations of all safety equipment (fire extinguishers, spill kits, eyewashes/showers, first-aid kits, safety data sheets, etc.), emergency exit locations, and emergency/injury reporting procedures. Do not eat, drink, or smoke in lab areas. Maintain clean work areas. Wash hands before leaving the lab. Laser Safety The Hyperion Tissue Imager is a solid state pulsed laser and is classified as a Class 1 laser system, and complies with 21 CFR and except for deviations pursuant to Laser Notice No. 50, dated June 24, WARNING Do not modify this device. Unauthorized modifications may create a safety hazard. WARNING If this equipment is used in a manner not specified by Fluidigm Corporation, the protection provided by the equipment may be compromised. For more information regarding laser safety, consult the American National Standards Institute (ANSI) and the International Electrotechnical Commission s publication under ANSI Z136.1 and IEC , respectively. Hyperion Imaging System: 113

114 Appendix A: Safety General Safety Instrument Symbols The warnings provided in this manual must be observed during operation and maintenance of the Hyperion Tissue Imager. Table 1. Hazard symbols that may be observed in this manual as well on warning labels on the Hyperion Tissue Imager. Symbol Title Description Warning Indicates a hazardous situation that and result in death or serious injury if not avoided. Pinch hazard Laser radiation Finger cut hazard Tipping hazard Indicates where pinch hazards exist. Exercise caution when operating around these areas. The laser radiation of the Class laser system is eye-safe under all operating conditions. This product includes lasers of a higher class whose beams are confined within a suitable enclosure so that access to laser radiation is physically prevented. Broken glass may cause injury or cutting of fingers or hands. Caution when loading and unloading the sample slides. Movement or impact with the instrument may cause tipping. Trip hazard Trip hazards are lying about. Watch your step to avoid falling over objects. Compressed gas hazard Indicates the presence of any material contained under pressure, including compressed gas, dissolved gas, or gas liquefied by compression or refrigeration. A compressed gas cylinder can become a projectile when ruptured with the potential to cause significant damage. Conformity Mark Description Indicates conformity with TUV for safety certification in North America. 114 Hyperion Imaging System:

115 Appendix A: Safety General Safety Environmental Conditions Refer to the Environmental Requirements section of the Hyperion Tissue Imager Site Requirements Guide (PN ) for more detailed information on the recommended environmental conditions. Laboratory Ventilation The Hyperion Tissue Imager is an air cooled system. Any toxic combustion products, metal vapor, and ozone are vented through the Helios exhaust system. Therefore, an efficient exhaust system must be provided for your instrument. Detailed information on exhaust vents are described in the Helios Site Requirements Guide (PN ). Hyperion Imaging System: 115

116 Appendix A: Safety Electrical Safety Electrical Safety The Hyperion Tissue Imager is designed to protect the operator from potential electrical hazards. The following section describes recommended electrical safety guidelines. Table 2. Electrical hazard symbols on the Hyperion Tissue Imager. Symbol Title Description Electric shock hazard symbol This sign indicates high electricity levels and a threat of electric shock from machines and/or equipment in the vicinity. You may suffer severe injuries or death. Earth-ground symbol The earth-ground symbol represents any terminal intended for connection to an external conductor for protection against electrical shock or protective earth terminal. WARNING Lethal voltages are present at certain areas within the instrument. Installation and repair of the instrument should be performed only by a Fluidigm Field Service Engineer or those similarly authorized and trained by Fluidigm personnel. When the instrument is connected to line power, opening instrument covers are likely to expose live electrical parts. Water lines should be located away from electrical connections. Condensation and potential leaks may create an unsafe environment in the proximity of electrical connections. Capacitors inside the instrument may still be charged even if the instrument has been disconnected from all voltage sources. The instrument must be correctly connected to a suitable electrical supply (see the Hyperion Imaging System Site Requirements Guide for further details). The power supply must have a correctly installed protective conductor (earth-ground) and must be installed or checked by a qualified electrician before connecting the instrument. WARNING Any interruption of the protective conductor (earth-ground) inside or outside the instrument, or any disconnection of the protective conductor terminal, is likely to make the instrument dangerous. Do not operate the instrument with the covers or internal parts removed. Do not attempt to make internal adjustments or replacements. 116 Hyperion Imaging System:

117 Appendix A: Safety Chemical Safety Chemical Safety This section provides some general safety practices that you should observe when working with any chemicals. The responsible individuals must take the necessary precautions to ensure that the surrounding workplace is safe and that instrument operators are not exposed to hazardous levels of toxic substances. When working with any chemicals, refer to the applicable safety data sheets (SDSs) provided by the manufacturer or supplier. IMPORTANT Wear the appropriate personal protective equipment (PPE) at all times while handling chemicals. Use safety glasses (with side shields), goggles, or full-face shields, according to the types of chemicals you will be handling. When handling any chemical the following safe-handling guidelines should be strictly observed: Use, store, and dispose of chemicals in accordance with the manufacturer's recommendations and regulations applicable to the locality, state/ province, and/or country. When preparing chemical solutions, always work in a fume hood that is suitable for the chemicals you are using. Conduct sample preparation away from the instrument to minimize corrosion and contamination. Clean up spills immediately using the appropriate equipment and supplies, and follow the appropriate SDS guidelines. Store solvents in an approved cabinet (with the appropriate ventilation) away from the Hyperion Imaging System. Sample Handling and Preparation Sample preparation for formalin-fixed, paraffin-embedded tissue sections (FFPE) or frozen sections are no longer considered biologically active. It is recommended that you only use samples prepared in this way. WARNING BIOHAZARD. If you are putting biohazardous material on the instrument, use appropriate personal protective equipment and adhere to Biosafety in Microbiological and Biomedical Laboratories (BMBL) from the Centers for Disease Control and Prevention and to your lab s safety protocol to limit biohazard risks. If biohazardous materials are used, properly label the equipment as a biohazard. For more information, see the BMBL guidelines online at: cdc.gov/biosafety/publications/index.htm Hyperion Imaging System: 117

118 Appendix A: Safety Pressurized Gas Safety Pressurized Gas Safety Safe Handling of Gas Cylinders Argon gas used with the Hyperion Imaging System is normally stored in liquid argon tanks or pressurized containers. Carefully use, store, and handle compressed gases in cylinders. Gas cylinders can be hazardous if they are mishandled. Argon is neither explosive nor combustible. Helium gas is supplied in the non-liquefied or liquid form in a compressed gas cylinder for use with the Hyperion Imaging System. Contact the gas supplier for an SDS containing detailed information on the potential hazards associated with the gas. IMPORTANT If liquid argon or liquid helium is used, the gas cylinder must be fitted with an overpressure regulator, which will vent the cylinder as necessary to prevent it from becoming a safety hazard. WARNING Do not use electronic pressure regulator and auto switching valves, because they may affect the plasma stability and may also result in frequent loss of plasma. WARNING It is recommended to install an oxygen sensor in the room where the operator and gas storage are located. Safety Interlocks To prevent gas leakage from the system, there is a safety interlock on the sample introduction tubing connecting the Hyperion Tissue Imager to the Helios instrument. If the tubing is disconnected the laser power will be shut down. The sample loading door also has a safety interlock so that the laser will not operate if the sample loading door is not completely closed, thereby preventing gas leakage from the chamber. Other Safety Considerations WARNING. Tipping Hazard. The Hyperion Tissue Imager has a higher center of gravity, and therefore there is risk of tipping during moving of the instrument. 118 Hyperion Imaging System:

119 Appendix A: Safety Disposal of Products Disposal of Products Used tuning slides and sample slides should be handled and disposed of in accordance with federal, state, regional, and local laws for hazardous waste management and disposal. Do not dispose of this product in unsorted municipal waste. This equipment may contain hazardous substances that could affect health and the environment. Use appropriate takeback systems when disposing of materials and equipment. Learn more at fluidigm.com/compliance. Hyperion Imaging System: 119

120 Appendix B: Manual Tuning Disposal of Products Appendix B: Manual Tuning Manual Tuning (HTI Optimization) 1 From the Toolbar select Camera > Actions. In the Slide Move (µm) box enter a slide move value and scan the tuning slide using the up and down arrow keys to find a clean, unablated region for tuning 2 Create a panorama. Refer to section Create a Panorama. 3 Click the Panorama tab, drag and drop the translucent red box on a clean unablated region to select the ROI on the tuning slide for transient optimization. A minimum 1000 µm x 1000 µm region is recommended for tuning. 120 Hyperion Imaging System:

121 Appendix B: Manual Tuning Disposal of Products 4 Click the Log tab to see the dimensions of your panorama (labeled Ablation resolution). 5 In the Toolbar click Acq Settings. 6 In the Data Acquisition Settings window, right-click on the analytes table and choose Apply Template from the drop-down menu. Hyperion Imaging System: 121

122 Appendix B: Manual Tuning Disposal of Products 7 The Acquisition Templates window appears. Select Tuning Slide from the list. Click Select Template. (Check that Lu(175) is on the analyte list). IMPORTANT If switching from solution mode, it is recommended that you tune the XY parameters and check the transients. 8 The Base Template box updates with the template title (Tuning Slide). XY Parameter Optimization 1 In the Data Acquisition Settings window, under Parameter Name select XY -> Y from the drop-down. 2 Select Control Panel > Devices > XY1 Setup to obtain the X and Y parameters from the previous HTI sample run. 122 Hyperion Imaging System:

123 Appendix B: Manual Tuning Disposal of Products 3 In the Parameter box enter the following values: Parameter Start Value: Parameter End Value: Settling Time 5000: Step Value 5000: 4 Check the elements in the Element Table to ensure that the correct metals are selected. NOTE Alternatively, you may right-click on the Analytes table and select Element Table. Select the following five metals: Y(89), In(115), Ce(140), Eu(151), Eu(153), Lu(175). 5 In the Toolbar click Transient to open the Transients Per Reading window. Hyperion Imaging System: 123

124 Appendix B: Manual Tuning Disposal of Products 6 Select Lu(175) from the Analyte drop-down. This is the analyte selected by default. 7 Click Run. NOTE When the tuning is complete the transient peaks appear in the Transients Per Reading window. IMPORTANT You may need to repeat the tuning using a smaller Step Value and a Start Value and End Value range such as ± 10, Repeat the XY Parameter Optimization. In the Data Acquisition Settings window, under Parameter Name select XY -> X from the drop-down. Repeat Steps 1 7 above. Verify that the maximum value for Lu is >100,000. Makeup Gas Optimization 1 In the Data Acquisition Settings window, under Parameter Name select HTI -> Makeup Gas from the drop-down. 2 In the Parameter box enter the following values: Parameter Start Value: 0.5 Parameter End Value: 1.0 Parameter Step Value: Hyperion Imaging System:

125 Appendix B: Manual Tuning Disposal of Products 3 In the Toolbar click Transient to open the Transients Per Reading window. 4 Click Run. 5 Click Masses Per Reading to monitor the ion signals from the tuning slide. 6 Record the Makeup Gas value that corresponds to the highest Lu (175) response in the Masses Per Reading window. Subtract 0.1 from this value, 7 Select Control Panel > Analog Controls. In the Actual Current Value box, record this value. Current Optimization IMPORTANT Perform only if Lu(175) Maximum is <100, In the Data Acquisition Settings window, under Parameter Name select HTI -> Current from the drop-down. 2 In the Parameter box enter the following values: Parameter Start Value: 0 Parameter End Value: 10 Parameter Step Value: 0.5 Settling Time, ms: 20 Hyperion Imaging System: 125

126 Appendix B: Manual Tuning Disposal of Products 126 Hyperion Imaging System:

127 Appendix B: Manual Tuning Disposal of Products 3 Check the checkboxes for Dump to Masses File and for Dump to Transients File. Change the date and the parameters in the filename for each new tuning procedure. 4 In the Control toolbar click Transient to open the Transients Per Reading window. 5 Click Run. 6 Click Masses Per Reading to monitor the ion signals from the tuning slide. 7 When the tuning is complete the transient peaks appear in the Transients Per Reading window. Hyperion Imaging System: 127

128 Appendix B: Manual Tuning Disposal of Products 8 Record the value for the Current that corresponds to the highest Lu(175) response in the Masses Per Reading window. In Control Panel > Analog Controls, enter this value for Current. Click Set. 9 If the Lu(175) Maximum is <100,000 then repeat the XY Optimization. Helium Flow Optimization The Helium Flow Optimization allows users to set a range of values for helium flow, and for each helium flow value a corresponding transient curve is generated and displayed. Each curve represents the Dual Counts signal of 175 Lu at that particular helium flow. The optimal helium value is selected based on the curves generated. 1 In the Data Acquisition Settings window, under Parameter Name select HTI -> Helium Mass Flow from the drop-down. 2 In the Parameter box enter the following values: Parameter Start Value: 0.1 Parameter End Value: 0.25 Parameter Step Value: 0.01 Settling Time, ms: Ablation Energy, db: 0 NOTE The Ablation Power for transient analysis can vary between instruments and this will be determined during the installation of the instrument. Select 20 from the Ablation Frequency, Hz drop-down. Ablation Distance X, µm: 1. Ablation Distance Y, µm: Hyperion Imaging System:

129 Appendix B: Manual Tuning Disposal of Products IMPORTANT It is recommended that the Parameter Start Value and End Value range be from to provide a wide range to determine the optimal helium mass flow for the instrument. Once you have determined the optimal flow you can narrow the range during subsequent procedures. 3 Check the elements in the Element Table to ensure that the correct metals are selected. NOTE Alternatively, you may right click on the Analytes table and select Element Table. Select the five metals contained in the PMMA 5 Element Tuning Slide : Y(89), In(115), Ce(140), Eu(151), Eu(153), Lu(175). Hyperion Imaging System: 129

130 Appendix B: Manual Tuning Disposal of Products 4 In the Toolbar, click Transient to open the Transients Per Reading window. 5 Click Run. 6 When the tuning is complete the transient peaks appear in the Transients Per Reading window. Click the parameters (helium flow rates) in the legend to bold the trace on the graph. NOTE You may need to make adjustments if transients are not optimal. For example, check that the coupling tubing is correctly connected to the ablation chamber. Alternatively, you may need to adjust the alignment of the coupling tubing. Shut down the system before moving the Hyperion Tissue Imager. 130 Hyperion Imaging System:

131 Appendix B: Manual Tuning Disposal of Products Select the helium flow (Parameter) value with the optimal results. This corresponds to the maximum value obtained for Lu while also maintaining low values for Cross Talk 1 (CT1) and Cross Talk 2 (CT2). IMPORTANT The Cross Talk represents the amount of ions that are registered between two plumes. CT1 is the 5 ms window recorded if the laser is set to fire at a 200 Hz frequency, and CT2 is the next 5 ms window recorded if the laser is set to fire at a 100 Hz frequency. CT1 should be <0.15 and CT2 should be <0.05. Hyperion Imaging System: 131

132 Appendix B: Manual Tuning Disposal of Products Right-click on this value and click Select Value. A dialog box indicates Helium Mass Flow successfully set to the selected value. In this example the value is Click OK. IMPORTANT Copy and paste the entire Graph Summary table into Notepad file or a Microsoft Excel spreadsheet and save in a Transients folder as reference for subsequent tuning and calibration. IMPORTANT Record the optimal Helium Mass Flow selected and enter the Start Values into the Hyperion Software > Acquisition > Plume Start for single sample acquisition. NOTE If the values for Lu(175) in the Graph Summary are <100,000 it is recommended that you repeat the Transients and optimize the current by selecting Current from the Parameter drop-down list. NOTE If the first Helium Mass Flow value provides the maximum Lu(175) and cross talk values, perform the Transient analysis again and change the range of the helium flow to have that value in the middle of the range. 132 Hyperion Imaging System: