Tissue optical clearing enhances efficacy of vascular targeted photodynamic therapy of mouse dorsal skin

Vascular-targeted photodynamic therapy(V-PDT)is an effective treatment for port wine stains(PWS).However,repeated treatment is usually needed to achieve optimal treatment outcomes,possibly due to the limited treatment light penetration depth in the PWS lesion.The optical clearing technique can increase light penetration in depth by reducing light scattering.This study aimed to investigate the V-PDT in combination with an optical clearing agent(OCA)for the therapeutic enhancement of V-PDT in the rodent skinfold window chamber model.Vascular responses were closely monitored with laser speckle contrast imaging(LSCI),optical coherence tomography angiography,and stereo microscope before,during,and after the treatment.We further quantitatively demonstrated the effects of V-PDT in combination with OCA on the blood flow and blood vessel size of skin microvasculature.The combination of OCA and V-PDT resulted in significant vascular damage,including vasoconstriction and the reduction of blood flow.Our results indicate the promising potential of OCA for enhancing V-PDT for treating vascular-related diseases,including PWS.

MACS-W:A modified optical clearing agent for imaging 3D cell cultures

Three-dimensional(3D)cell cultures have contributed to a variety of biological research fields by filling the gap between monolayers and animal models.The modern optical sectioning microscopic methods make it possible to probe the complexity of 3D cell cultures but are limited by the inherent opaqueness.While tissue optical clearing methods have emerged as powerful tools for investigating whole-mount tissues in 3D,they often have limitations,such as being too harsh for fragile 3D cell cultures,requiring complex handling protocols,or inducing tissue deformation with shrinkage or expansion.To address this issue,we proposed a modified optical clearing method for 3D cell cultures,called MACS-W,which is simple,highly efficient,and morphology-preserving.In our evaluation of MACS-W,we found that it exhibits excellent clearing capability in just 10 min,with minimal deformation,and helps drug evaluation on tumor spheroids.In summary,MACS-W is a fast,minimally-deformative and fluorescence compatible clearing method that has the potential to be widely used in the studies of 3D cell cultures.

QUANTITATIVE CONTROL OF OPTICAL CLEARING EFFECTS STUDIED WITH TISSUE-LIKE PHANTOM

Tissue optical clearing by use of optical clearing agents(OCAs)has been proven to have potential to reduce the highly scattering effect of biological tissues in optical techniques.However,the difference in tissue samples could lead to unreliable results,making it difficult to quantitatively control the dose of OCAs during the course of tissue optical clearing.In this work,in order to study the effects of optical clearing,we customized tissue-like phantoms with optical properties of some biological tissue.Diffuse reflectance and total transmittance of tissue-like phantoms with different OCAs(DMSO or glycerol)and porcine skin tissues were measured.Then optical property parameters were calculated by inverse adding-doubling(IAD)algorithm.Results showed that OCAs could lead to a reduction in scattering of tissue-like phantoms as it did to porcine skin tissue in vitro.Furthermore,a series of relational expressions could be fit to quantitatively describe the relationship between the doses of OCAs and the reduction of scattering effects.Therefore,proper tissue-like phantom could facilitate optical clearing to be used in quantitative control of tissue optical properties,and further promote the application potential of optical clearing to light-based noninvasive diagnostic and therapeutic techniques.

ULTRASOUND ENHANCED SKIN OPTICAL CLEARING:MICROSTRUCTURAL CHANGES

Our previous studies demonstrated the ultrasound-induced skin optical clearing enhancement with topical application of optical clearing agents on in vitro porcine skin and in vivo human skin.The objective of this study was to investigate the possible mechanisms of the enhanced skin optical clearing by ultrasound medications.Optical clearing effects of ex vivo guinea pig abdomen skin topically applied with 60% glycerol or the combination of 60% glycerol and ultrasound were studied by optical coherence tomography(OCT).Microstructure of skin surface was examined by scanning electron microscopy(SEM).Ultrasound with a frequency of 1MHz and a power of 0.75W over a 3-cm probe was simultaneously applied with glycerol solution for 15min.The combination of 60% glycerol and ultrasound results in a 19% increase in OCT 1/e light penetration depth after 30min,which is much better than 60% glycerol alone.SEM images demonstrated that changes in skin microstructure due to the tight order of the lipid bilayers in the stratum corneum disrupted and the separation of keratinocytes by the application of ultrasound contribute to the ultrasound-enhanced intact skin optical clearing effects.

Improving sampling depth of laser speckle imaging by topical optical clearing:A theoretical and in vivo study

The effect of optical cleaning method combined with laser speckle imaging(LSI)was discussed to improve the detection depth of LSI due to high scattering characteristics of skin,which limit its clinical application.A double-layer skin tissue model embedded with a single blood vessel was established,and the Monte Carlo method was used to simulate photon propagation under the action of light-permeating agent.808 nm semiconductor and 632.8 nm He–Ne lasers were selected to study the e®ect of optical clearing agents(OCAs)on photon deposition in tissues.Results show that the photon energy deposition density in the epidermis increases with the amount of tissue°uid replaced by OCA.Compared with glucose solution,polyethylene glycol 400(PEG 400)and glycerol can considerably increase the average penetration depth of photons in the skin tissue,thereby raising the sampling depth of the LSI.After the action of glycerol,PEG 400,and glucose,the average photon penetration depth is increased by 51.78%,51.06%,and 21.51%for 808nm,68.93%,67.94%,and 26.67%for 632.8 nm lasers,respectively.In vivo experiment by dorsal skin chamber proves that glycerol can cause a substantial decrease in blood°ow rate,whereas PEG 400 can signicantly improve the capability of light penetration without a®ecting blood velocity,which exhibits considerable potential in the monitoring of blood°ow in skin tissues.

IMPROVE OPTICAL CLEARING OF SKIN IN VITRO WITH PROPYLENE GLYCOL AS A PENETRATION ENHANCER

In order to enhance the optical clearing effect of topically applied optical clearing agents(OCAs),we evaluated the effect of propylene glycol(PG)as a chemical penetration enhancer(PE)on optical clearing of skin in vitro by observation and measurement of optical-transmittance and diffuse-reflectance spectra.Three OCAs,i.e.,glycerol,D-sorbitol and PEG400,and two other penetration enhancers,Azone and Thiazone,were used in this study.The results indicated that the decrease of reduced scattering coefficient caused by OCA/PG was larger than that by pure OCA,and the change by OCA/water was the least after the same treatment time.There were significant differences for the reduced scattering coefficient at 630 nm after 120 min application of agents between OCA and OCA/PG.The efficacy of optical clearing caused by OCA/PG depended on the OCA itself.When PEG400 was mixed with three different PEs,we found the optical clearing were different.The penetration enhancing ability of PG was much better compared to Azone,and suboptimal to Thiazone.Also,this study provides evidence for the use of PG as a PE in order to improve skin optical clearing.

Ex-vivo confocal Raman microspectroscopy of porcine skin with 633/785-NM laser excitation and optical clearing with glycerol/water/DMSO solution

Confocal Raman microspectroscopy(CRM)with 633-and 785-nm excitation wavelengths combined with optical clearing(OC)technique was used for ex-vivo study of porcine skin in the Raman fingerprint region.The optical clearing has been performed on the skin samples by applying a mixture of glycerol and distilled water and a mixture of glycerol,distilled water and chemical penetration enhancer dimethyl sulfoxide(DMSO)during 30 min and 60 min of treat-ment.It was shown that the combined use of the optical clearing technique and CRM at 633 nm allowed one to preserve the high probing depth,signal-to-noise ratio and spectral resolution simultaneously.Comparing the effect of different optical clearing agents on porcine skin showed that an optical clearing agent containing chemical penetration enhancer provides higher optical clearing efficiency.Also,an increase in treatment time allows to improve the optical clearing efficiency of both optical clearing agents.As a result of optical clearing,the detection of the amide-Ⅲ spectral region indicating well-distinguishable structural differences between the type-Ⅰ and type-Ⅳ collagens has been improved.

ASSESSMENT OF OPTICAL CLEARING INDUCED IMPROVEMENT OF LASER SPECKLE CONTRAST IMAGING

Laser Speckle Contrast Imaging(LSCI)plays an important role in studying blood flow,but suffers from limited penetration depth of light in turbid tissue.The strong scattering of tissue obviously reduces the image contrast which decreases the sensitivity to flow velocity.Some image processing or optical clearing methods have been proposed to lessen the deficiency,but quantitative assessment of improvement is seldom given.In this study,LSCI was applied to monitor the blood flow through a capillary embedded within various tissue phantoms at depths of 0.25,0.45,0.65,0.85 and 1.05 mm,and the flow velocity in capillary was controllable from 0 to 4mm/s.Here,glycerol,a common optical clearing agent,was mixed with Intralipid at different volume ratio to make the reduced scattering coefficient of tissue phantom decrease from 13.00 to 0.50 cm−1.The quantitative analysis demonstrates that the optical clearing method can obviously enhance the image contrast,imaging depth,and sensitivity to blood flow velocity.Comparing the Laser Speckle Contrast Analysis methods and the optical clearing method,we find that for typical turbid tissue,the sensitivity to velocity estimated by the Laser Speckle Temporal Contrast Analysis(LSTCA)is twice of that by the Laser Speckle Spatial Contrast Analysis(LSSCA);while the sensitivity to velocity estimated by using the two analysis methods has a 10-fold increase,respectively,if addition of glycerol makes the reduced scattering coefficient of tissue phantom decrease by 30%.Combining the LSTCA and the optical clearing method,the sensitivity to flow velocity will be further enhanced.

PARAMETRIC STUDY OF TISSUE OPTICAL CLEARING BY LOCALIZED MECHANICAL COMPRESSION USING COMBINED FINITE ELEMENT AND MONTE CARLO SIMULATION

Tissue Optical Clearing Devices(TOCDs)have been shown to increase light transmission through mechanically compressed regions of naturally turbid biological tissues.We hypothesize that zones of high compressive strain induced by TOCD pins produce localized water displacement and reversible changes in tissue optical properties.In this paper,we demonstrate a novel combined mechanical finite element model and optical Monte Carlo model which simulates TOCD pin compression of an ex vivo porcine skin sample and modified spatial photon fluence distributions within the tissue.Results of this simulation qualitatively suggest that light transmission through the skin can be significantly affected by changes in compressed tissue geometry as well as concurrent changes in tissue optical properties.The development of a comprehensive multi-domain model of TOCD application to tissues such as skin could ultimately be used as a framework for optimizing future design of TOCDs.

Combining CUBIC Optical Clearing and Thy1-YFP-16 Mice to Observe Morphological Axon Changes During Wallerian Degeneration

Objective:Wallerian degeneration is a pathological process closely related to peripheral nerve regeneration following injury,and includes the disintegration and phagocytosis of peripheral nervous system cells.Traditionally,morphological changes are observed by performing immunofluorescence staining after sectioning,which results in the loss of some histological information.The purpose of this study was to explore a new,nondestmetive,and systematic method for observing axonal histological changes during Wallerian degeneration.Methods:Thirty male Thy1-YFP-16 mice(SPF grade,6 weeks old,20±5 g)were randomly selected and divided into clear,unobstructed brain imaging cocktails and computational analysis(CUBIC)optical clearing(n=15)and traditional method groups(n=15).Five mice in each group were sacrificed at 1st,3rd,and 5th day following a crush operation.The histological axon changes were observed by CUBIC light optical clearing treatment,direct tissue section imaging,and HE staining.Results:The results revealed that,compared with traditional imaging methods,there was no physical damage to the samples,which allowed for three-dimensional and deep-seated tissue imaging through CUBIC.Local image information could be nicely obtained by direct fluorescence imaging and HE staining,but it was difficult to obtain image information of the entire sample.At the same time,the image information obtained by fluorescence imaging and HE staining was partially lost.Conclusion:The combining of CUBIC and Thy1-YFP transgenic mice allowed for a clear and comprehensive observation of histological changes of axons in Wallerian degeneration.

Quantitative evaluation of enhanced laser tattoo removal by skin optical clearing

Lasers have been widely used for tattoo removal,but the limited light penetration depth calused by high skin scattering property restricts the therapeutic outcome of deep tattoo.Skin optical clearing method,by introducing optical clearing agent(OCA)into skin,has shown some im-provement in the effect of laser tattoo removal.In this study,the enhanced laser tattoo removal has been quantitatively assessed.OCA was applied to the skin of tattoo animal model and Q switched Nd:YAG laser(1064 nm)irradiation was used to remove the tattoo.The skin evaluation instrument(Mexameter probe,MPA580)was applied to measure the content of tattoo pigment before and after laser treatment,and then the clearance rate of pigment was calculated.Further,Monte Carlo(MC)method was utilized to simulate the efecet of skin optical clearing on light transmission in tattoo skin model.By comparing the pigment change of tattoo areas respectively treated with OCA plus laser and single laser,it was found that pigment clearance of the former tattoo area was increased by 1.5-fold.Further,the MC simulation verifed that the reduced light scattering in skin could increase the effective dose of lumninous fux reaching to the deep tattoo regions.It can be concluded from both experiment and theoretical simulations that skin optical clearing technique could improve the outcome of laser tattoo re moval,which should be beneficial for clinical laser tattoo removal and other laser pigment elimination.

Tissue optical clearing imaging for structural changes of neuromuscular junctions after mice ischemic stroke[Invited]

Ischemic stroke causes long-term disability and results in motor impairments.Such impairments are associated with structural changes in the neuromuscular junction(NMJ),including detailed morphology and three-dimensional(3D)distribution.However,previous studies only explored morphological changes of individual NMJs after stroke,which limits the understanding of their role in post-stroke motor impairment.Here,we examine 3D distributions and detailed morphology of NMJs in entire mouse muscles after unilateral and bilateral strokes induced by photothrombosis.The results show that 3D distributions and numbers of NMJs do not change after stroke,and severe unilateral stroke causes similar levels of NMJ fragmentation and area enlargement to bilateral stroke.This research provides structural data,deepening the understanding of neuromuscular pathophysiology after stroke.

Skull optical clearing window for in vivo imaging of the mouse cortex at synaptic resolution

Imaging cells and microvasculature in the living brain is crucial to understanding an array of neurobiological phenomena.Here,we introduce a skull optical clearing window for imaging cortical structures at synaptic resolution.Combined with two-photon microscopy,this technique allowed us to repeatedly image neurons,microglia and microvasculature of mice.We applied it to study the plasticity of dendritic spines in critical periods and to visualize dendrites and microglia after laser ablation.Given its easy handling and safety,this method holds great promise for application in neuroscience research.

ALTERATIONS IN AUTOFLUORESCENCE SIGNAL FROM RAT SKIN EX VIVO UNDER OPTICAL IMMERSION CLEARING

For the first time,the changes in autofluorescence spectra of ex vivo rat skin have been experimentally investigated using the combination of fluorescence spectroscopy and optical immersion clearing.The glucose,glycerol and propylene glycol solutions were used as clearing agents.The optical clearing was performed from the dermal side of skin imitating the in vivo injection of clearing agent under the dermal layers.In this contribution,the common properties of autofluorescence variation during optical immersion clearing were determined.The tendency of autofluorescence signal to decrease with reduction of scattering in tissue was noticed and discussed in detail.However,the differences in the shape of spectral curves under application of different clearing agents showed that optical clearing affects the autofluorescence properties of tissue differently depending on the type of clearing liquid.The results obtained are useful for the understanding of tissue optical clearing mechanisms and for improving techniques such as fluorescence spectroscopy.

Glycerol effects on optical,weight and geometrical properties of skin tissue

Complex study of glycerol effects on the skin tissue was performed.The change in optical,weight and geometrical parameters of the rat skin under the action of the glycerol solutions was studied ex vivo.Possible mechanisms of the skin optical clearing under the action of glycerol solutions of different concentrations were discussed.The results can be helpful for refinement of models developed to evaluate the effective diffusion coefficients of glycerol in tissues.

A"messenger zone hypothesis"based on the visual three-dimensional spatial distribution of motoneurons innervating deep limb muscles

Coordinated contraction of skeletal muscles relies on selective connections between the muscles and multiple classes of the spinal motoneuro ns.Howeve r,current research on the spatial location of the spinal motoneurons innervating differe nt muscles is limited.In this study,we investigated the spatial distribution and relative position of different motoneurons that control the deep muscles of the mouse hindlimbs,which were innervated by the obturator nerve,femoral nerve,inferior gluteal nerve,deep pe roneal nerve,and tibial nerve.Locations were visualized by combining a multiplex retrograde tracking technique compatible with three-dimensional imaging of solvent-cleared o rgans(3DISCO)and 3-D imaging technology based on lightsheet fluorescence microscopy(LSFM).Additionally,we propose the hypothesis that"messenger zones"exist as interlaced areas between the motoneuron pools that dominate the synergistic or antagonist muscle groups.We hypothesize that these interlaced neurons may participate in muscle coordination as messenger neurons.Analysis revealed the precise mutual positional relationships among the many motoneurons that innervate different deep muscles of the mouse.Not only do these findings update and supplement our knowledge regarding the overall spatial layout of spinal motoneurons that control mouse limb muscles,but they also provide insights into the mechanisms through which muscle activity is coordinated and the architecture of motor circuits.

OPTICAL MEA SUREMENTS OF RAT MUSCLE SAMPLES UND ER TREATMENT WITH ETHYLENE GLYCOL AND GLUCOSE

With the objective to study the variation of optical properties of rat muscle during optical clearing,we have performed a set of optical measurements from that kind of tissue.The.measurements performed were total tr ansmittance,ollimated transmit tance,specular reflec-tance and total reflectance.This set of measurements is suficient to determine diffuse reflectance and absorbance of the sample,also necessary to est imate the optical properties.All the per formed measurements and calculated quantities will be used later in inverse Monte Carlo(IMC)simu-lations to determine the evolution of the optical properties of muscle during treatments with ethylene glycol and glucose.The results obt ained with the measurements already provide some information about the optical c learing treatments applied to the muscle and translate the mechanisms of turning the tissue more transparent and sequence of regimes of optical clearing.

Effects of delayed repair of peripheral nerve injury on the spatial distribution of motor endplates in target muscle

Motor endplates(MEPs) are important sites of information exchange between motor neurons and skeletal muscle, and are distributed in an organized pattern of lamellae in the muscle. Delayed repair of peripheral nerve injury typically results in unsatisfactory functional recovery because of MEP degeneration. In this study, the mouse tibial nerve was transected and repaired with a biodegradable chitin conduit, immediately following or 1 or 3 months after the injury. Fluorescent α-bungarotoxin was injected to label MEPs. Tissue optical clearing combined with light-sheet microscopy revealed that MEPs were distributed in an organized pattern of lamellae in skeletal muscle after delayed repair for 1 and 3 months. However, the total number of MEPs, the number of MEPs per lamellar cluster, and the maturation of single MEPs in gastrocnemius muscle gradually decreased with increasing denervation time. These findings suggest that delayed repair can restore the spatial distribution of MEPs, but it has an adverse effect on the homogeneity of MEPs in the lamellar clusters and the total number of MEPs in the target muscle. The study procedures were approved by the Animal Ethics Committee of the Peking University People's Hospital(approval No. 2019 PHC015) on April 8, 2019.

Varying of up-conversion nanoparticles luminescence from the muscle tissue depth during the compression

The current work is focused on the study of optical clearing of skeletal muscles under local compression.The experiments were performed on in vitro bovine skeletal muscle.The time dependence of optical clearing was studied by monitoring the luminescence intensity of NaYF_(4)∶Er,Yb upconverting particles located under tissue layers.This study shows the possibility to use upconverting nanoparticles(UCNPs)both for studying the dynamics of the optical clearing of biological tissue under compression and to detect moments of cell wall damage under excessive pressure.The advantage of using UCNPs is the presence of several bands in their luminescence spectra,located both at close wavelengths and far apart.

Quantification of glucose and glycerol diffusion in myocardium

The results on determination of glucose and glycerol difusion cofficients in myocardium tissuse are presented.The method is based on the measurement and analysis of temporal dependence of tissue optical collimated transmittance umder action of a hyperosmotic agent.This temporal tissue response is related to the rate of the agent and water diffusion in a tissue.The diffusion coefficients for tssue fAuid fluxes at glucose and glycerol application to the myocardium at 20℃ have been estimated as(4.75±3.40)×10^(-7) and(7.71±4.63)×10^(-7) cm^(2)/s,respectively.