Snap-shot multspectral magng of vascular dynamcs n a mouse wndow chamber model Hansford C. Hendargo, 1,* Yuln Zhao, 1 Taylor Allenby, 2 and Gregory M. Palmer, 1 1 Department of Radaton Oncology, Duke Unversty Medcal Center, 203 Research Dr., Durham, NC 27710 2 Cornell Unversty, Ithaca, NY *Correspondng author: hansford.hendargo@duke.edu Receved Month X, XXXX; revsed Month X, XXXX; accepted Month X, XXXX; posted Month X, XXXX (Doc. ID XXXXX); publshed Month X, XXXX Understandng tumor vascular dynamcs through parameters such as blood flow and oxygenaton can yeld nsght nto tumor bology and therapeutc response. Hyperspectral mcroscopy enables optcal detecton of hemoglobn saturaton or blood velocty by ether acqurng multple mages that are spectrally dstnct or by rapd acquston at a sngle wavelength over tme. However, the seral acquston of spectral mages over tme prevents the ablty to montor rapd changes n vascular dynamcs and cannot montor concurrent changes n oxygenaton and flow rate. Here, we ntroduce snap shot-multspectral magng (SS-MSI) for use n magng the mcrovasculature n mouse dorsal wndow chambers. By spatally multplexng spectral nformaton nto a sngle mage capture, smultaneous acquston of dynamc hemoglobn saturaton and blood flow over tme s acheved down to the capllary level and provdes an mproved optcal tool for montorng rapd n vvo vascular dynamcs. 2015 Optcal Socety of Amerca OCIS Codes: (170.0170) Medcal optcs and botechnology, (110.0180) Mcroscopy, (110.4234) Multspectral and hyperspectral magng, (170.1460) Blood gas montorng. http://dx.do.org/10.1364/ol.99.099999 The study of tssue dynamcs s crtcal for understandng the processes leadng to tumor growth and development. The aberrant vasculature of tumors often leads to the development of a hypoxc mcroenvronment that may enable protectve and prolferatve effects [1]. Hypoxa acts as an upstream control for a number of molecular factors, such as hypoxa nducble factor-1 [2] and programmed death lgand-1 [3], that nduce tumor resstance to radaton and chemotherapy. However, the nteractons of hypoxa wth the mechanstc pathways governng tumor therapeutc response are complex and requre further study n order to develop more effectve clncal treatment of a varety of cancers. Optcal technques for observng and measurng tumor hemodynamcs have enabled the study of hypoxa n an n vvo settng wthout the need for nvasve probes. Photoacoustc tomography [4] and vsble-lght optcal coherence tomography [5] have been used to detect the oxygen saturaton levels n vvo by usng dfferences n the optcal absorpton propertes of oxygenated and deoxygenated hemoglobn. Hyperspectral magng has been developed wth spatal scannng setups [6] or a wavelength-tunable flter [7] and has been used to detect vascular oxygen levels n vvo. These methods suffer from slow acquston speeds and are affected by blood flow or respratory moton. Snap shot magng of hyperspectral data usng dsperson has been used to obtan vascular oxygen levels n the retna [8, 9], however, complex optcal hardware and processng was requred along wth a large number of wavelengths. Blood flow propertes may also provde addtonal nsght nto tumor growth and transport characterstcs of the vasculature. Laser Doppler magng measures blood perfuson over large areas, but does not resolve ndvdual vessels [10]. Optcal coherence tomography has demonstrated the ablty to mage three-dmensonal vascular structure and flow, but requres complex system desgn and long acquston tmes [11]. An automated post-processng method for measurng flow velocty from standard brghtfeld vdeo mcroscopy mages was prevously developed [12] but was unable to drectly correlate oxygenaton nformaton wth flow. Here, we demonstrate an mproved method termed snap shot-multspectral magng (SS-MSI) that only requres 4 smultaneously acqured wavelengths to enable computaton of hemoglobn saturaton maps from a sngle mage. Addtonally, concurrent acquston of blood flow velocty allows for mult-functonal hemodynamc data. Furthermore, the optcal setup uses smple desgn prncples that nterface wth standard mcroscopes allowng for potental wdespread use. Achevng snap shot acquston of hemoglobn saturaton requres smultaneous capture of suffcent spectral nformaton to determne the amount of optcal absorpton of blood n the vasculature. The domnant absorbers are oxy- and deoxyhemoglobn, and calculaton of ther relatve concentratons can be computed from multspectral mages as prevously detaled [13]. The absorpton, A, s computed as I cal ( ) A log, (1) Itssue ( ) n whch λ s the wavelength of lght, Ical s the transmtted ntensty of a calbraton mage of the source, and Itssue s the transmtted ntensty of the sample. A(λ) depends upon the concentraton of absorbers as λ b b μ λ ε λ A C, (2) o 1 eff where bo s a constant term accountng for overall changes caused by the source ntensty, b1 s a parameter
modulatng the effectve attenuaton coeffcent, μeff, as determned from dffuson theory, ε gves the attenuaton coeffcent of the th absorber, and C s the concentraton of the absorber modulated by the optcal path length. A least squares ft determnes the values of bo, b1, and C. Usng oxyhemoglobn (HbO2) and deoxyhemoglobn (HbR) as the prmary absorbers requres the selecton of at least 4 wavelengths over whch to perform the ft and thus determnes the mnmum number of wavelengths needed to form a multspectral dataset. Calculatng the rato CHbO2 to CHbO2 + CHbR yelds the fracton of oxyhemoglobn contaned n the blood. Addtonally, velocty maps of vascular flow can be computed wth SS-MSI by usng a prevously developed correlaton mappng algorthm usng mages at a sngle wavelength [12]. Brefly, movng scatterers n the blood cause ntensty fluctuatons n an mage over tme that are temporally correlated among neghborng pxels. Performng a pxel-by-pxel analyss and fndng the tme pont that maxmzes ths correlaton allows for computaton of the dstance traveled over tme for a partcular scatterer as well as ts drecton of moton. To further enhance ths processng method, mages were frst normalzed accordng to a spatal Fourer Transform lowpass flter of each mage to remove bulk ntensty varatons. A movng temporal average wth a 2 second wndow about each ndvdual frame was calculated and subtracted to enhance dfferences n the mages due to movng absorbers or scatterers. A Gabor flter was appled to the orgnal mages to create a mask of the vessel regons [14], whch was then appled to the processed mages to restrct analyss to vascular regons. These addtonal steps resulted n mproved flow vsualzaton n certan vessels and allowed SS-MSI to obtan smultaneous hemoglobn saturaton and velocty nformaton from the vasculature. Parallel detecton of dfferent wavelengths n SS-MSI was performed by optcally splttng and flterng the lght that was transmtted through a sample. A sequence of beamspltters mapped the feld of vew onto dfferent quadrants of the detector. Optcal bandpass flters were used to solate dfferent spectral components n each quadrant, thus allowng a sngle camera frame to contan a 4-wavelength multspectral dataset, as shown n Fg. 1. In post-processng, each camera frame was sub-dvded nto ts component wavelength mages, whch were then regstered to one another va an affne transformaton to ensure approprate spatal algnment of each mage. Performng the regstraton step on a sngle frame yelded the approprate transform parameters for all other frames acqured, ndependent of sample moton. A reference mage of the source dstrbuton was acqured by usng a neutral densty flter n place of the sample and acqurng an mage of the source output. Normalzng each ndvdual wavelength to ts correspondng reference mage compensated for varatons n the dstrbuton of the lght throughout the mage as well as dfferences n optcal loss between each channel. A non-negatve least squares ft of the multspectral mages acqured at each tme pont was then computed to determne the composton of oxyhemoglobn and deoxyhemoglobn present n the feld of vew. Acqurng mages over tme gave the ablty to detect dynamc changes n oxygenaton, the temporal resoluton of whch was lmted only by the exposure tme of the detector. SS-MSI was performed on a Zess Axo Observer nverted mcroscope. A halogen lamp provded the whte lght source for magng. A QV2 multchannel mager (Photometrcs) was attached va a sde documentaton port and was used to separate sample scattered lght nto 4 quadrants on a Hamamatsu Orca Flash4 CMOS camera by passng the lght through a sequence of beamspltters (30/70, 40/60, 50/50). Lght drected towards each camera quadrant was fltered at 540, 560, 580, and 610 nm, respectvely, each wth Δλ = 10 nm. The wavelengths were chosen at the local mnma and maxma of the absorpton spectra of hemoglobn (540, 560, and 580 nm). Addtonally, 610 nm was chosen as a wavelength wth a sgnfcant absorpton dfference between oxy/deoxyhemoglobn. Images acqured n the 580 nm channel were used for flow velocty calculatons. Valdaton of the ablty of SS-MSI to detect varyng levels of oxygenaton was carred out usng blood extracted from mce va cardac puncture. Blood samples were placed n heparnzed phosphate buffered salne (PBS, ph=7.2) wth 20 unts of heparn per ml of PBS. Blood samples were washed 3 tmes wth PBS and then placed nto a 48-well plate and ncubated at 37 C n varyng levels of po2 n a hypoxa chamber. The po2 level wthn the blood soluton was measured usng a fber probe oxygen sensor (OxyLte). Sample plates were sealed wth paraflm before beng removed from the hypoxa chamber and transferred to the mcroscope for magng. Fg. 1: Spectral data acquston and oxygen saturaton processng usng SS-MSI. Fg. 2: Measured so2 values for mouse blood smears versus po2 n soluton usng SS-MSI.
The samples were then returned to the hypoxa chamber for further ncubaton at dfferent oxygen levels. Images of the blood samples were acqured wth SS- MSI usng an exposure tme of 200 ms for all data ponts. The ndvdual wavelength mages were algned to the 580 nm mage as a reference. The least squares ft was computed wth the rato of oxyhemoglobn to the total hemoglobn content mapped onto each mage pxel. The relatve amount of oxyhemoglobn over the entre mage was averaged and plotted as a functon of the po2 level measured va the oxygen sensng probe n the hypoxa chamber as shown n Fg. 2. Errors were calculated as the standard devaton over each mage. The results were ft to the Hll equaton [15] and show a characterstc sgmod shape for the so2 level as measured va SS-MSI and are comparable wth publshed results for the mouse hemoglobn saturaton curve [16]. For n vvo demonstraton of SS-MSI, wndow chamber surgeres were performed on C57BL/6 mce and adhered to Insttutonal Anmal Care and Use Commttee gudelnes. Some anmals were njected wth ~100,000 Lews Lung carcnoma cells at the tme of surgery. Tumors were allowed to grow for 1 week before magng. Mce were anesthetzed durng magng sessons usng 1.5% soflurane mxed wth 21% O2 and balanced wth N2 admnstered va a nose cone. Body temperature was mantaned va a heatng pad set to 37 C. A mouse dorsal wndow chamber wth no tumor was maged usng both SS-MSI and hyperspectral magng usng a tunable flter as shown n Fg. 3. A ven and artery par wthn the wndow regon s shown. The oxygen saturaton n the arteres was hgher than that of the vens, although anesthesa effects may have caused overall lower than normal oxygen levels. Usng hyperspectral magng, mages were acqured sequentally for dfferent wavelengths usng an ntegraton tme of 100-300 ms per wavelength. Dfferences n ntegraton tmes compensated for sgnal loss caused by the spectral output of the source, tssue optcal propertes, and optcal losses due to the tunable flter. Due to the movement of blood n the vessels durng mage acquston, artfacts were created n calculatng the hemoglobn saturaton throughout the vasculature. In contrast, the SS-MSI mage, acqured n 500 ms, s free of these artfacts due to the smultaneous acquston of each wavelength, whch causes any moton from blood flow or respraton durng mage acquston to be equally averaged among each channel. Such mages can stll be successfully mapped together va the affne transform parameters orgnally obtaned. The so2 values between the hyperspectral and SS-MSI mages can be seen to have good correlaton throughout the mage, though slght dfferences are apparent n the man ven n the mddle of the mage. These dfferences may be caused by the moton artfacts n the hyperspectral dataset. Imagng a second mouse wth a tumor demonstrated the ablty of SS-MSI to detect changes n the vasculature over tme as shown n Fg. 4. Anesthesa was gven as prevously descrbed. Fg. 4(a) shows an mage of the wndow chamber regon under normoxc condtons. Images were contnuously acqured wth an exposure tme of 200 ms. Nearly 20 seconds after magng began, the O2 level was reduced to 12% to nduce hypoxa. Images were contnuously recorded for an addtonal 50 seconds. After magng, the mouse contnued to breathe hypoxc ar for an addtonal 3 mnutes. At that pont, a second set of mages was acqured as shown n Fg. 4(b), wth the O2 level adjusted back to normoxa after 17 seconds of magng. Fg. 3: Comparson of data acqured usng a tunable flter and usng SS-MSI. Whte arrows ndcate moton artfacts due to blood flow n the standard hyperspectral mage. SS- MSI mage s free of moton artfacts. Fg. 4: SS-MSI mages of oxygen saturaton n a mouse dorsal wndow chamber under (a) normoxc and (b) hypoxc condtons. (c) Plot of dynamc so2 changes over tme, averaged over the regons ndcated by the blue and red boxes n (a) and (b). Black lne ndcates tme at whch ar was changed from ether normoxa to hypoxa or vce versa.
the ven slows under hypoxa and shows a gradual ncrease as the ar returns to normoxc condtons. The reduced blood flow could occur as part of the overall response to a lack of oxygen as blood s dverted to more crtcal regons of the body. In concluson, we demonstrate a snap shot multspectral method that allows smultaneous acquston of vascular oxygen supply and blood flow mages n the mouse wndow chamber. SS-MSI only requres 4 wavelengths and uses relatvely smply hardware and software methods compared to other smlar technques. The ablty to mage dfferent parameters of n vvo vascular functon at the capllary level may provde a more comprehensve analyss of therapeutc effects on tumor development and angogeness. Ths work was supported by the North Carolna Botechnology Center grant No. 2013-MRG-1111 and the Natonal Insttute of Health grant No. NIH 5-R01- CA167250-02. Fg. 5: Maps of (a,b) speed and (c,d) drecton of blood flow n a mouse dorsal wndow chamber. Flow n the capllares was detected and appears as the thn, dagonal streaks throughout the mage. Whte arrow ndcates an artery wth a velocty too hgh to detect under normoxa but that slows under hypoxa. (e) Plot of average velocty from the regon ndcated by the whte box n (a) and (b) over tme as the O2 level s vared. Red lne ndcates tme when oxygen level was altered. Data was computed usng the same dataset n Fg. 4 at 580 nm. The oxygen saturaton level of a ven-artery par was tracked over tme and plotted n Fg. 4(c). It can be observed that notceable changes n vascular so2 occur nearly 10 seconds after envronmental O2 levels change. The so2 level n the artery also began to change before observable changes n the ven. The same dataset used n Fg. 4 was used to compute the flow velocty. Temporal correlaton mappng was performed on mages acqured at 580 nm. Fg. 5(a-d) shows the temporally averaged speed and drecton maps of the vascular flow over tme whle the mouse was breathng normoxc or hypoxc ar. Blood flow under hypoxa was observed to slow throughout the wndow chamber regon compared to normoxc flow. An artery at the bottom of Fg. 5(a) had a velocty too hgh to measure under normoxc condtons, but the flow slows under hypoxc condtons to allow velocty detecton n Fg. 5(b). Performng a short-tme Fourer transform along the temporal dmenson of the flow mages on sequences of 30 frames allowed computaton of the nstantaneous velocty over tme as shown n Fg. 5(e). The average velocty n
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