Lasers in Surgery and Medicine 29:213±220 (2001)

Use of Osmotically Active Agents to Alter Optical Propertiesof Tissue: Effects on the Detected Fluorescence SignalMeasured Through Skin

Gracie Vargas, MS, Kin F. Chan, PhD, Sharon L. Thomsen, MD, and A.J. Welch, PhD

The Biomedical Engineering Program, The University of Texas at Austin, Austin, Texas 78712

Background and Objective: Hyper-osmotic chemicalagents were used to study the effects of transient tissuescattering on the remitted ¯uorescence emission intensityfrom a target placed under a tissue sample.Study Design/Materials and Methods: A ¯uorescent®lm was placed underneath in vitro and in vivo samples ofhamster skin, and the remitted ¯uorescent signal travel-ing to the tissue surface was monitored over time as thetissue was treated with an osmotically active agent.Results: The detected ¯uorescent signal increased as thescattering in tissue samples was substantially reduced.The increase was greater for dimethyl sulfoxide thanglucose or glycerol. It was not statistically different be-tween in vivo skin and in vitro skin.Conclusion: The study shows how chemical agents canbe used to improve the detected signal for a speci®c opticalapplication. It could be useful in a number of opticaltherapeutic and diagnostic applications that can bene®tfrom an increase in the penetration depth of light. LasersSurg. Med. 29:213±220, 2001. ß 2001 Wiley-Liss, Inc.

Key words: controlling optical properties; ¯uorescence;index matching; tissue optical clearing; glycerol; glucose;DMSO

INTRODUCTION

The ability to reversibly alter the optical properties ofnormally turbid tissues offers the potential for improvedlight-based diagnostic and therapeutic applications. Anincrease in light penetration depth will allow photonsto reach deep-lying target chromophores more effect-ively. This could be of bene®t to both laser therapeutictechniques and optical imaging applications. Throughapplication of hyper-osmotic chemical agents, scatteringin tissue can be temporarily modi®ed to improve light-mediated diagnostic and therapeutic techniques in speci®cinstances.

Previous studies have shown that osmotically activechemical agents are effective in changing the opticalproperties of in vitro and in vivo tissue [1±5]. Addition ofhyper-osmotic solutions of either glycerol, dimethyl sulf-oxide (DMSO), mannitol, or dextrose, among others causenormally turbid tissue such as skin, sclera, and aorta tobecome optically clear. Within minutes, the transmissionof light through the tissue begins to increase, and there is a

marked reduction in scattering [4]. Tissue shrinkageoccurs as a result of water loss; however, the change inlight transmission does not depend solely on the decreasein thickness. The primary change is a reduction in thescattering coef®cient [4]. We hypothesize that the agentsreduce scattering in tissue by index matching in two ways.The ®rst is by simply index matching the chemical agent(n� 1.47 for both anhydrous glycerol and pure DMSO, andn� 1.46 for 7 M glucose) to the main tissue constituentssuch as collagen (n� 1.5), adipose tissue (n� 1.48) andother high refractive index components. The second is bydehydration, which results in water loss from the inter-stitial space, both allowing components to be tightlypacked and increasing the concentration of glycosamino-proteins leading to increased refractive index. These couldlead to improved index matching as well.

Studies have shown that scattering and reabsorption inhighly turbid tissue can obscure the ¯uorescence spectra ofa ¯uorophore from within a tissue, both in magnitude ofthe ¯uorescence and in emission line shape, altering it inthe number of peaks [6±9]. Additionally, light in the UV tolower visible region of the spectrum has a short penetra-tion depth in tissue. While two-photon ¯uorescence allowsan increase in depth of penetration, this method is alsoassociated with a complex experimental setup.

The objective of this study is to assess the effects ofosmotically active chemical agents on ¯uorescence me-asurements made through skin in which the opticalscattering properties are transiently reduced. We hypothe-size that the decrease in scattering due to application ofthe optical clearing agents will lead to the detection ofmore emission photons. First, the decrease in scatteringwill allow more excitation photons to reach the ¯uorescenttarget. Second, a greater number of emission photons willreach the detector at the skin surface. Additionally, thedecrease in scattering will result in longer photon path-

Contract grant sponsor: National Science Foundation; Contractgrant number: BES29986296; Contract grant sponsor: the TexasHigher Education Coordinating Board; Contract grant number:BER-ATP-253; Contract grant sponsor: Albert and ClemmieCaster Foundation.

*Correspondence to: Gracie Vargas, Biomedical EngineeringProgram, ENS 610, The University of Texas at Austin, Austin,TX 78712.

Accepted 13 October 2000

ß 2001 Wiley-Liss, Inc.

lengths of the excitation and emission photons, leading to adecreased incidence of absorption. The technique could beinstrumental to the development of improved diagnosticprocedures using ¯uorescence spectroscopy, particularlybecause the change in optical properties is reversible.

MATERIALS AND METHODS

Animal Model

In vitro ¯uorescence experiments were performed onfreshly harvested dorsal skin samples from Golden Syrianhamsters. In vivo ¯uorescence measurements were per-formed on the hamster dorsal skin ¯ap window prepara-tion in an area void of blood vessels. The dorsal skin ¯apwindow preparation is described in Ref. [10]. Hamster skinwas chosen for its similarity to human skin in terms of itslayered structure and inhomogeneous makeup. The maindifference between hamster skin and human skin is thathamster skin contains an additional muscle layer under-neath the dermis not found in human skin, excludinghuman facial skin, which has an attached muscular layerbeneath the dermis/subcutaneus tissue. A single thicknessof skin was used in the window preparation, with the¯uorescent ®lm placed against the subdermal side as inthe in vitro studies. The probe with light excitation andcollection ®bers was placed against the epidermis in bothcases. A clearing agent was applied to the subdermal sideto reduce scattering in the skin. In vitro sample sizes were1 cm� 1 cm. The in vivo window preparation had a 1-cmdiameter circle of exposed skin.

Fluorophore Selection

A rhodamine ¯uorescent ®lm was chosen as a ¯uor-ophore for its inert properties and its relatively high

quantum yield. The ®lm had an absorption peak at 542 nmand an emission peak at 585 nm.

Intensity Measurements

This study investigated changes in magnitude of theremitted ¯uorescence centered at 585 nm arriving at thedetector through the skin from the ¯uorescent target. Theexperimental setup is shown in Fig. 1. A tunable pulsednitrogen dye laser (Laser Photonics, Orlando, FL) wasused as the excitation source at 542 nm. A band-pass ®lter(BPF) centered at 542 nm with a full-width-half-max(FWHM) bandwidth of 10 nm was used to further isolatethe laser light at 542 nm. Part of the excitation beam wassplit off with a microscope slide (BS) and used as areference signal. A dichroic beamsplitter (DBS) re¯ectedthe excitation light into the delivery end of the optical ®berbundle. Freshly excised hamster skin was placed directlyover the ¯uorescent target. The sample probe was placeddirectly against the epidermis, with a droplet of water inbetween as shown in Fig. 2.

The optical probe consisted of 25 ¯uorescence excitation®bers and 12 ¯uorescence emission collection ®bers arra-nged randomly within a central bundle (200-mm diameter,NA� 0.2). A ring of illumination and corresponding dif-fuse re¯ectance collection ®bers was arranged aroundthe central bundle. These were not used in this study. Thebundle is described in more detail in Ref. [11]. Fluores-cence emission collected by the optical ®ber was furtherisolated by passing the remitted light through a 565-nmlongpass (cutoff) ®lter. Detection of the total ¯uorescenceemission was achieved by using an avalanche photodiode(APD, Hamamatsu, Japan). The background signal takenwithout the skin or ¯uorescent ®lm in place was negligible.

Fig. 1. Experimental setup for measuring the emission signal

remitted from a ¯uorescent ®lm placed underneath in vivo

and in vitro skin samples. The ®lm was excited through the

skin sample with 542-nm light. The excitation light was

obtained from a pulsed nitrogen dye laser and passed through

band pass ®lter (BPF). Part of the light was re¯ected into a

reference detector using a microscope slide (BS). The rest of

the excitation light was then re¯ected by a dichroic beams-

plitter (DBS) and directed into the delivery end of a optical

®ber bundle and through the skin sample. Emission light from

the ¯uorescence ®lm was passed through a 565-nm cutoff

®lter (allowed wavelengths > 565 nm to be transmitted) and

collected with an avalanche photodiode (APD).

214 VARGAS ET AL.

Anhydrous glycerol (13 M), glucose (7 M), and anhydrousDMSO (14 M) referred to as `optical clearing agents', weretested to ensure they did not ¯uoresce at the wavelengthsof interest, nor did they quench the ¯uorescence. Thesample holder also did not ¯uoresce. These molar concen-trations of optical clearing agents were chosen on the basisof their ability to increase optical transmission throughtissue.

Measurement and Analysis

The ¯uorescent emission signal traveling through thenative skin from the ¯uorescence ®lm was measured threetimes for each sample (these were averaged), and ®vesamples were tested for each data set. A trial consisted ofmeasuring the ¯uorescence before and after application ofthe clearing agent at a given time interval (average of 5samples). The ``before'' measurement served as the control.Measurements of the ¯uorescence were made for times of0 seconds (native sample), 15, 30 seconds, 1, 5, 10, 15, and20 minutes after application of an optical clearing agent.The optical agents were applied to the subdermal side ofthe skin sample for both in vitro and in vivo cases. Thesame volume of agent (2 ml) was applied to all samples.The excess agent and displaced water were gently wipedwith gauze, and the sample was replaced over the ¯uore-scent ®lm, allowing the ¯uorescence magnitude to be mea-sured. The sample was then rehydrated in physiologicalphosphate-buffered saline solution. To eliminate errors dueto a ¯uctuating excitation source, the ratio of ¯uorescencedetected by the APD to the reference signal was taken forcomparison between trials (I¯uor/Iref). Comparison of the®ve sample values for each trial was performed by usingANOVA followed by Newman±Keuls multiple comparisonprocedure. ANOVA revealed if signi®cant differencesoccurred between data sets. If there were signi®cantdifferences, the Newman±Keuls multiple comparison pro-cedure [12] revealed which trials were signi®cantlydifferent. A signi®cance threshold of 0.05 was chosen.

The thickness of in vitro skin samples was measuredusing calipers (the sample was placed between two glass

slides, and the thickness of the slides was subtracted fromthe average of ®ve caliper measurements). For in vivosamples optical coherence tomography (OCT) was used todetermine changes in optical depth and compared to thoseof in vitro samples to determine changes in thickness. Thiswas compared with measurements taken with calipers.Control samples ranged from 0.9 to 2.02 mm in thickness,with an average of 1.39 mm and a standard deviationof 0.28 mm (sample size, ktot� 105). The ANOVA andNewman±Keuls multiple comparison procedures wereused to determine the signi®cance of changes in tissuethickness between time points for a given optical agent.Determination of sample lateral size changes was achievedby imaging several samples with a CCD attached to asurgical microscope. Each clearing agent was applied to asample for up to 40 minutes. The distance from edge to edgeof the sample was determined in two orthogonal directions.

Microscopic Effect of Optical Clearing Agentson Skin

Microscopic observation of the effects of the hyper-osmotic agents on frozen sections of rat skin wasaccomplished with increased contrast microscopy on atransmission light microscope (Zeiss Axiophot, Germany,Thornwood, NY). Rectangular sections were taken fromfreshly harvested rat skin, frozen, and cut on a cryostat(sections were 10-mm thick). The frozen sections were thenmounted on glass slides and immediately viewed under amicroscope with the condenser diaphragm aperturereduced to increase contrast. The test media included air,water, and the optical clearing agent, glycerol, DMSO, orglucose. Each frozen section was viewed and photographedin air and water for controls, and then the water wasdrained and replaced with one of the clearing agents.Spraque-Dawley rat skin was used for the microscopicobservation of glycerol. In the cases of DMSO and glucose,only fuzzy rat skin was available for imaging. The dif-ference between the two types of skin is that the fuzzy ratskin contains little hair and, therefore, few hair follicles.

Optical Clearing Agent Osmolality

The osmolalities of the three optical clearing solutionswere experimentally determined using an Osmette Aosmometer (Precision Systems, Inc., Natick, MA), whichuses the freezing-point method to determine solutionconcentration in units of osmole per kilogram of water.

RESULTS

Solutions of glycerol, glucose, or DMSO applied to skinresulted in an increase in detected ¯uorescence signals inall cases. The effect of 13 M glycerol, 14 M DMSO, and 7 Mglucose on the ¯uorescent signal collected in vitro is shownin the plot of Figure 3. According to the analysis usingANOVA followed by Newman±Keuls multiple compari-sons, the increase in ¯uorescent signal was signi®cant.Signi®cant differences in the means occurred between alltime periods when the skin was treated with DMSO,except between the short time periods of 15, 30 seconds,and 1 minute. In the case of glycerol, there were

Fig. 2. Close-up view of sample probe, skin, and ¯uorescent

®lm arrangement. The sample probe was placed against the

skin, with a water droplet used to maximize the detected

signal. The arrows represent the direction of light propagation

along the delivery and collection bundles, respectively.

CHEMICAL AGENTS TO ALTER TISSUE OPTICAL PROPERTIES 215

statistically signi®cant increases for 10, 15, and 20minutes. There were no statistically signi®cant differencesbetween 15, 30 seconds, 1, or 5 minutes. Finally, forglucose only 15 and 20 minutes applications resulted insigni®cant increases in ¯uorescence and were also differ-ent from each other. The shorter time periods had signalssigni®cantly smaller than these; however, their ¯uores-cence magnitudes were not statistically different fromeach other or from the native ¯uorescence.

The results for the in vivo cases are shown in Figure 4.For DMSO, the time periods that resulted in a signi®cantincrease in signal from the native case were 5, 10, 15, and20 minutes. There was no statistically signi®cant increasein the mean signal up to 1 minute. The same was true formeasurements made on in vivo skin treated with glycerol.In the case of glucose, only 15 and 20 minute applicationof the agent resulted in a signi®cant signal increase. The20-minute application was signi®cantly greater than the15-minute application.

DMSO resulted in the greatest increase in ¯uorescencesignal for both the in vitro and in vivo cases. The effects of

glycerol and glucose were analogous for in vivo and in vitromeasurements. There were no statistically signi®cantdifferences between in vivo and in vitro results.

Rehydration reversed the effect of the agentsÐthe¯uorescent signal decreased when physiological salinesolution was applied to the samples treated with glycerol,glucose, or DMSO. A histogram of the percent difference in®nal ¯uorescent signals after rehydration relative to con-trol values for all in vitro samples is shown in Figure 5.Each sample was rehydrated the same amount of time itwas in the optical clearing agent. The values on the x-axisare percent differences from the native case. For instance,

Fig. 3. Comparison of the percent increase in ¯uorescent

signal due to 13 M glycerol, 14 M DMSO, and 7 M glucose

in vitro.

Fig. 4. Comparison of the percent increase in ¯uorescent

signal due to 13 M glycerol, 14 M DMSO, and 7 M glucose

in vivo.

Fig. 5. Histograms showing the ®nal ¯uorescence signal

(expressed as a percentage change from the native case) of

samples after rehydration for the same amount of time the

tissue was treated with a chemical agent. Return in ®nal

¯uorescence after being treated with (a) glucose, and (b)

glycerol, and (c) DMSO.

216 VARGAS ET AL.

the bar between 0 and 10 represents the number ofsamples that, when rehydrated, returned to within � 10%of the original signal. As can be seen from this bar graph,values for the rehydrated samples are distributed aboutthe zero mark representing samples that returned to theoriginal signals after rehydration.

Since the thickness of the native control samples variedby at least 28%, the percent change in thickness wascalculated for each sample treated with the opticalclearing agents to determine shrinkage of the sample.The thickness of samples signi®cantly decreased with theaddition of glycerol. Newman±Keuls multiple comparisontest, using a signi®cance threshold of 0.05 revealed therewere signi®cant differences between thickness at thedifferent time points of glycerol application. Speci®cally,the change in thickness was most signi®cant for a 15- and20-minute application. A 5-minute application of glycerolresulted in an average 8% decrease in thickness. A 10-, 15-,and 20-minute application resulted in a thickness decreaseof 12, 15, and 22% decrease, respectively. For both DMSO

and glucose, ANOVA revealed that there was no signi®-cant difference in the means of the data sets for each timeperiod, because the P-value calculated from ANOVA washigher than the signi®cance threshold. This was true forthe in vivo and in vitro cases. There were no apparentlateral size changes in samples treated with glycerol orglucose. Those treated with DMSO resulted in an 18%average lateral shrinkage in width (a direction parallel tothe transverse section of the body) and a 7.8% increase inlength (in a direction parallel to the median planeseparating the right and left sides of the dorsal area).Measurements made on the osmometer revealed the 14 MDMSO solution had an osmolality of 15.2 Osm/kg. Thevalue for anhydrous glycerol was determined to be justunder one-half the value for DMSO (8.9 Osm/kg). The 7 Mglucose osmolality was found to be equal to 7.5 Osm/kg.

The histological effect of applying glycerol to skin can befound in frozen sections of rat skin viewed under highcontrast microscopy (Fig. 6, row 1). When in water, thereis much contrast between the skin constituents (Fig. 6b).

Fig. 6. Frozen sections of in vitro rat skin viewed under high

contrast microscopy. The images are categorized according to

agent used where Row 1 contains the three conditions for the

section 1 ®nally treated with glucose; Row 2, section 2 treated

with DMSO; and Row 3, section 3 treated with glycerol.

Column a, sections viewed in air; Column b, sections cover-

slipped with water; and Column c, section coverslipped with

optical clearing agent. The layers of the skin are denoted by

arrows: dermis ( ); adipose layer ( ); and muscle layer

( ).

CHEMICAL AGENTS TO ALTER TISSUE OPTICAL PROPERTIES 217

As soon as glycerol is applied, the collagen ®bers areless visible, and lipid and muscle layer begin to disappear(Fig. 6c). Similar effects are seen for glucose and DMSO(Fig. 6, rows 1 and 2), except there is less contrast insections treated with water (Fig. 6, column b, rows 2 and 3)due to the lack of a distinct adipose tissue layer which torein places and lack of hair follicles. The fuzzy rat skincontained signi®cantly fewer hair follicles than the skinused for glycerol observation. The follicles are the onlyobservable components that do not become less visiblewith the addition of the clearing agents.

DISCUSSION

Effect of Optical Clearing Agents onFluorescence Signal

Hyper-osmotic solutions of glycerol, glucose, and DMSOwere shown to signi®cantly increase the detected remitted¯uorescence from a subsurface target. The gradual in-crease in the signal detected over time is shown in Figures3 and 4. A 20-minute application of glycerol or glucose to invivo or in vitro skin caused the ¯uorescent signal toapproximately double, whereas DMSO increased it four-fold. Comparison of the agents applied to in vitro skin forthe same time periods reveals that the 14 M DMSOsolution was almost twice as effective as the glycerol orglucose solutions.

The agents used in this study were highly hyper-osmotic;however, the DMSO solution used had a much higherosmolality than the other two agents, accounting for themuch greater increases in measured ¯uorescence magni-tudes. The concentration of glycerol used is only slightlymore hyper-osmotic than the glucose solution and was onlyslightly more effective.

Mass Transport Considerations

The optical effect caused by the agents is a time-dependent process in that it occurs as a consequence ofthe transport of chemical agent and water in and out of thetissue, respectively. The mass transport that occurs withthe addition of highly hyper-osmotic agents is complex andinvolves various mechanisms. The higher osmotic poten-tial of the applied agents relative to tissue ¯uids causeswater to travel from areas of higher water potential and alower osmotic potential to a lower water potential andhigher osmotic potential. This means water leaves intra-cellular spaces if there is a hyper-osmotic agent in theextracellular space and leaves the bulk tissue when theagent surrounds it. Water diffusion will cease when anosmotic equilibrium is reached if the solutes outside areimpermeable. An additional factor to account for in thecase of the agents used in these studies is that they willdiffuse into the tissue although water is leaving and theyare membrane permeable. The transmembrane perme-ability times for glycerol, glucose, and DMSO are muchshorter compared with water (water is on the order of10ÿ 2 cm/minute, whereas glycerol and DMSO are on theorder of 10ÿ 5 cm/minute [13,14]) which accounts for aninitial decrease in cell volume as water leaves much faster

than the agent enters. Eventually, much of the intracel-lular water leaves the cell while the clearing agentcontinues to enter the cell, leading to a gradual increasein volume which stabilizes in time. The rate of water andagent transport out and into cells is a rate process limitedby the permeability of the water and the chemical agentsacross cell membranes and by the driving force (the im-balance in osmotic pressure caused by the hyper-osmoticagent) [15]. Permeability factors vary between the threeagents with DMSO permeating the fastest, and glucose theslowest [14,16]. Glucose is a hydrophilic molecule thatenters most cells by facilitated diffusion (moving down theconcentration gradient across the cell membrane with helpof a carrier protein). Since the number of glucose transportproteins is limited in the cell membrane, as the concentra-tion of glucose increases outside the cell its rate oftransport into the cell ®rst increases then reaches a peakand plateaus when all of the transport proteins are in use[17]. A plateau was not seen in the increase of ¯uorescenceduring the time glucose was applied to the skin, eitherindicating the role of cells in the overall changes were notdetectable or a 20-minute application was too short a time.Glycerol has been found to enter and exit cells byfacilitated diffusion in some cases such as in prokaryoticcells and erythrocytes; however, as a general rule it istransported by passive diffusion. DMSO penetrates mem-branes very rapidly, and even across the stratum corneumof skin [18] which the other two agents are not able to do.The mechanism is also by passive diffusion.

The mass transport processes become increasinglycomplex in bulk tissue. The transport processes at thecellular level in bulk tissue are different from those ofisolated cells [15]. For instance, water permeability isslower for cells in bulk tissue, speci®cally as they becomemore centrally located. Although membrane permeabilityfactors are important in the mass transport within bulktissue, diffusive processes become more important. In bulktissues, spatial concentration gradients occur, becausewater ef¯ux will occur at the surface ®rst and then deeperas the diffusion front moves [15].

The higher osmolality of DMSO used is thought to beaccountable for the larger increase in ¯uorescence signaldetected because the rate of water displacement is depen-dent on the imbalance in osmotic pressure present, whichwas greatest for DMSO. Additionally, the faster penetra-tion rate of DMSO over glycerol and glucose allows forsome replacement of interstitial water and creates a moreindex-matched environment between major tissue consti-tuents and the interstitial containing DMSO. The diffu-sion of DMSO into cells occurs quickly as well, although itis not clear how changes at the cellular level affect theoverall tissue-scattering properties of skin and othercollagenous tissues.

An issue that is important to the use of these opticalclearing agents in tissue is their toxicity levels, particu-larly at the concentrations used to cause a quick reductionin scattering. Much can be found in the cryopreservationliterature on this topic. The most signi®cant factor that willdetermine tissue viability is the osmotic stress induced on

218 VARGAS ET AL.

the tissue constituents, particularly cells. The concern intoxicity lies in osmotic effects rather than chemical toxicityeffects, particularly for glycerol and glucose [13,15,19]although instances of chemical toxicity have been sug-gested, such as in the study by Frim et al. [13], whichobserved the effects of glycerol on human granulocytes.High concentrations of the agents at the level of cells couldcause extreme volumetric deviations. One potential effectof glycerol is that it may modify the plasma membranelipid bilayer [18,20], but despite this, it is consideredchemically safe. DMSO has been found to have someharmful effects on tissue, which render it a toxic chemical;nevertheless, its lethal dose level is somewhat high,ranging from 2 to 12 g/kg BW for intravenous administra-tion and from 10±50 g when administered orally, sub-cutaneously, and intraperitoneally [18]. Various studieshave indicated DMSO can induce bradycardia, respiratoryproblems, and alterations in blood pressure. Morphologicalchanges occur in the lungs and liver. Administration ofDMSO to skin causes local irritation and may lead to localnecrosis. DMSO can potentially alter the chemical struc-ture and, hence the functional properties of cell mem-branes [20]. These reasons will affect the use of this agentin in vivo tissue; however, these agents help us elucidatethe mechanism of reduced scattering and mass transport.

Microscopic Observation

The contrast between the interstitium and tissuecomponents and the loss of contrast between the threemedia (air, water, and glycerol) are quite dramatic inFigure 6. The index-matching effect of glycerol can clearlybe observed in these slides. The glycerol seems to make theextracellular space better index matched to the collagen.Anhydrous glycerol, such as was used in these studies, hasa refractive index of 1.47 [21]. Collagen has a refractiveindex of 1.43 in the hydrated state and 1.53 in the dry state[22]. In the native state the extracellular ¯uid has arefractive index of 1.35 [23]; however, the presence ofglycerol or the loss of water due to glycerol will cause theextracellular ¯uid refractive index to become larger, betterindex matching collagen and other tissue constituents.Another area to note is the fatty layer consisting of adiposetissue, which becomes highly matching with the additionof glycerol. Adipose tissue has a refractive index of 1.46[24], very close to that of anhydrous glycerol. The presenceof skin appendages, such as the hair follicles seen inFigure 6 (row 3, column c), could impede imaging since theoptical clearing agents did not match them.

Mechanism of Reduced Scattering

More remains to be learned about the mechanism ofreduced scattering by these optical clearing agents. Thescattering in skin is due to the variations in refractiveindex found in the different tissue constituents. The highlyturbid nature of skin greatly limits the penetration depthof light. Although the optical clearing agents do closelyindex match major constituents of skin, such as collagenwhich makes up 77% of the dry weight of skin [25], theyalso act to pull water out of interstitial spaces and likely

out of intracellular spaces as discussed above. Thisdehydration contributes to some restructuring within theskin, and can also lead to a more closely index-matchedenvironment to occur. In addition, the ef¯ux of water fromthe extracellular spaces results in a highly concentratedextracellular solution [15] which will lead to an increasedrefractive index from that of the native tissue. Tissueshrinkage does occur when glycerol is applied to skin,leading to an increase in light transmission through theskin, however a previous study [4] has shown a reductionin the reduced scattering coef®cient, ms 0, is the main causeof altered optical properties. This previous study wasperformed by using a single integrating sphere in aspectrophotometer, which does not allow the determina-tion of the anisotropy factor, g, or scattering coef®cient, ms,to be determined (ms 0 � ms(1ÿ g)) simultaneously. There-fore, it is not known with certainty which of theseparameters is altered by the optical clearing agents.Double integrating sphere measurements are plannedfor the future to determine the degree of change in theanisotropy factor and the scattering coef®cient. However,if the anisotropy factor changes rather than the scatteringcoef®cient, its value will approach that of 1 (all forward-scattering, because the reduced scattering coef®cient isdecreased signi®cantly), and it will be dif®cult to differ-entiate scattered light from collimated transmittance. Theoverall contribution from cellular changes due to theagents on the change in optical properties has not beenfully studied, however studies in which the effect of theagents on the cell properties is underway.

One useful fact is that the decrease, then an increasein cell volume due to a hyperosmotic chemical agent isindicative of permeable substances. Sucrose is not perme-able, so cells only decrease in volume in its presence.Optical property measurements as a function of sucroseconcentration will be indicative of the role cells play on theoverall tissue scattering properties.

CONCLUSION

The technique used in this study allowed up to 200%increase in the ¯uorescent signal within 20 minutes ofapplication. The technique of reduced tissue scattering bythese optical clearing agents could be of bene®t to anumber of other optical diagnostic or therapeutic applica-tions. However, since the mechanism of tissue opticalclearing by these agents has not been fully established,more research will be conducted in this area. Additionalstudies on the effect of the optical clearing agents on¯uorescence emission spectra are of interest, because it isexpected that a reduction in tissue scattering will alter theremitted line shape.

ACKNOWLEDGMENTS

The authors would like to thank Christopher G.Rylander for his technical assistance. The authors alsothank Dr. Rebecca Richards-Kortum and Dr. Urs Utzingerfor use of the optical ®ber bundle used in these experi-ments. Ashley J. Welch is the Marion E. Forsman Cen-tennial Professor in Engineering. Funding for this

CHEMICAL AGENTS TO ALTER TISSUE OPTICAL PROPERTIES 219

research was provided by: National Science Foundation(9986296) Texas Higher Education Coordinating Board(003658-0185-1999).

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