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.

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.

Transformational change in the field of diffuse optics:From going bananas to going nuts

The concept of region of sensitivity is central to the field of diffuse optics and is closely related to the Jacobian matrix used to solve the inverse problem in imaging.It is well known that,in diffuse reflectance,the region of sensitivity associated with a given source-detector pair is shaped as a banana,and features maximal sensitivity to the portions of the sample that are closest to the source and the detector.We have recently introduced a dual-slope(DS)method based on a special arrangement of two sources and two detectors,which results in deeper and more localized regions of sensitivity,resembling the shapes of different kinds of nuts.Here,we report the regions of sensitivity associated with a variety of source-detector arrangements for DS measurements of intensity and phase with frequency-domain spectroscopy(modulation frequency:140 MHz)in a medium with absorption and reduced scattering coefficients of 0.1 and 12 cm^(-1),respectively.The main result is that the depth of maximum sensitivity,considering only cases that use source-detector separations of 25 and 35 mm,progressively increases as we consider single-distance intensity(2.0mm),DS intensity(4.6mm),single-distance phase(7.5mm),and DS phase(10.9 mm).These results indicate the importance of DS measurements,and even more so of phase measurements,when it is desirable to selectively probe deeper portions of a sample with diffuse optics.This is certainly the case in non-invasive optical studies of brain,muscle,and breast tissue,which are located underneath the superficial tissue at variable depths.

Reconstructing in vivo spatially offset Raman spectroscopy of human skin tissue using a GPU-accelerated Monte Carlo platform

As one type of spatially offset Raman spectroscopy(SORS), inverse SORS is particularly suited to in vivo biomedical measurements due to its ring-shaped illumination scheme. To explain inhomogeneous Raman scattering during in vivo inverse SORS measurements, the light–tissue interactions when excitation and regenerated Raman photons propagate in skin tissue were studied using Monte Carlo simulation. An eight-layered skin model was first built based on the latest transmission parameters. Then, an open-source platform, Monte Carlo e Xtreme(MCX), was adapted to study the distribution of 785 nm excitation photons inside the model with an inverse spatially shifted annular beam. The excitation photons were converted to emission photons by an inverse distribution method based on excitation flux with spatial offsets Δs of 1 mm, 2 mm, 3 mm and 5 mm. The intrinsic Raman spectra from separated skin layers were measured by continuous linear scanning to improve the simulation accuracy. The obtained results explain why the spectral detection depth gradually increases with increasing spatial offset, and address how the intrinsic Raman spectrum from deep skin layers is distorted by the reabsorption and scattering of the superficial tissue constituents. Meanwhile, it is demonstrated that the spectral contribution from subcutaneous fat will be improved when the offset increases to 5 mm, and the highest detection efficiency for dermal layer spectral detection could be achieved when Δs = 2 mm. Reasonably good matching between the calculated spectrum and the measured in vivo inverse SORS was achieved, thus demonstrating great utility of our modeling method and an approach to help understand the clinical measurements.

MCVM:MONTE CARLO MODELING OF PHOTON MIGRATION IN VOXELIZED MEDIA

The Monte Carlo code MCML(Monte Carlo modeling of light transport in multi-layered tissue)has been the gold standard for simulations of light transport in multi-layer tissue,but it is ineffective in the presence of three-dimensional(3D)heterogeneity.New techniques have been attempted to resolve this problem,such as MCLS,which is derived from MCML,and tMCimg,which draws upon image datasets.Nevertheless,these approaches are insufficient because of their low precision or simplistic modeling.We report on the development of a novel model for photon migration in voxelized media(MCVM)with 3D heterogeneity.Voxel crossing detection and refractive-index-unmatched boundaries were considered to improve the precision and eliminate dependence on refractive-index-matched tissue.Using a semi-infinite homogeneous medium,steady-state and time-resolved simulations of MCVM agreed well with MCML,with high precision(∼100%)for the total diffuse reflectance and total fractional absorption compared to those of tMCimg(<70%).Based on a refractive-index-matched heterogeneous skin model,the results of MCVM were found to coincide with those of MCLS.Finally,MCVM was applied to a two-layered sphere with multi-inclusions,which is an example of a 3D heterogeneous media with refractive-index-unmatched boundaries.MCVM provided a reliable model for simulation of photon migration in voxelized 3D heterogeneous media,and it was developed to be a flexible and simple software tool that delivers high-precision results.

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.

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.

Accurate analysis of limiting human dose of non-lethal laser weapons

In order to maximize the lethality and reversibility of the non-lethal laser weapons(NLLW) at the same time and thus provide a theoretical basis for the R&D of laser weapons in the future,this paper accurately analyzed the limiting biological dose of irreversible damage to human skin caused by the NLLW.Firstly,based on the burn theory in medicine and the actual tactical background,this paper redefines the evaluation criteria of the limiting laser dose of NLLW to the human body.Secondly,on the basis of anatomical knowledge,a 5-layer finite element model(FEM) of superficial skin is proposed,constructed and verified,which can accurately describe the limiting reversible damage.Based on the optimized Pennes bioheat transfer equation,the diffusion approximation theory,the modified Beer-Lambert law,the Arrhenius equation,and combined with dynamic thermophysical parameters,this paper highly restored the temperature distribution and accurately solved the necrotic tissue distribution inside the human skin irradiated by 1064 nm laser.Finally,it is concluded that the maximum human dose of the1064 nm NLLW is 8.93 J/cm^(2),8.29J/cm^(2),and 8.17 J/cm^(2) when the light spots are 5 mm,10 mm and15 mm,respectively,and the corresponding output power of the weapon is 46.74 W,173.72 W and384.77 W.Simultaneously,the temperature and damage distribution in the tissue at the time of ultimate damage are discussed from the axial and radial dimensions,respectively.The conclusions and analysis methods proposed in this paper are of great guiding significance for future research in military,medical and many other related fields.

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.

Time-reversed optical focusing through scattering mediaby dligital fullphase and amplitude recovery usinga single phase-only SLM

Focusing light though scattering media beyond the ballistic regime is a challenging task in biomedical optical imaging.This challenge can be overcome by wavefront shaping technique,in which a time reversed(TR)wavefront of scattered light is generated to suppress the scattering.In previous TR optical focusing experiments,a phase only spatial light modulator(SLM)has.been typically used to control the wavefront of incident light.Unfortunately,although the phase information is reconstructed by the phase-only SLM,the amplitude information is lost,resulting in decreased peak to-background ratio(PBR)of optical focusing in the TR wavefront recon-struction.A new method of TR optical focusing through scattering media is proposed here,which numerically reonstructs the full phase and amplitude of a simulated scattered light field by using a single phase only SLM.Simulation results and the proposed optical setup show that the time-reversal of a fully developed speckle field can be digially implemented with both phase and ampltude recovery,affording a way to improve the performance of light focusing through scattering media.

SPECTRAL IMAGING AND ANALYSIS TO YIELD TISSUE OPTICAL PROPERTIES

An introduction to the basics of spectral imaging as applied to biological tissues is presented.An example of a spectral image of a face is used to demonstrate the data and spectral analysis that specify the melanin content(M),blood content(B),tissue oxygen saturation(S),water content(W),fraction of scattering due to Rayleigh scattering(f)and due to Mie scattering(1−f),and the reduced scattering coefficient at 500-nm wavelength(µ
s 500 nm).The sensitivity of reflectance spectra to variation in the various parameters is illustrated.

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.

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.

Cherenkov radiation from electrons passing through human tissue

A method for investigating the optical properties of human tissues is suggested.The method is based on the measurement of Cherenkov radiation produced by relativistic electrons passing through the tissue.Monte-Carlo simulation of visible photon emission and propagation is carried out taking into account multiple electron and photon scattering processes.Sensitivity of the Cherenkov radiation to the optical characteristics of human tissues is demonstrated.

Phantom of Human Adipose Tissue and Studies of Light Propagation and Light Absorption for Parameterization and Evaluation of Noninvasive Optical Fat Measuring Devices

A method for noninvasive determination of fat and water content in the human body is examined. A spatially resolved spectroscopy method is used which can record low intensity near infrared spectra. This novel approach is compared to other methods for the determination of fat and water content. Monte Carlo simulations of light propagation in tissue are shown and the production and characterization of optical phantoms of adipose tissue are investigated.

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.

Measurement of optical penetration depth and refractive index of human tissue

Experimental techniques for measurement of optical penetration depth and refractive index of human tissue are presented, respectively. Optical penetration depth can be obtained from the measurement of the relative fluencc-depth distribution inside the target tissue. The depth of normal and carcinomatous human lung tissues irradiated with the wavelengths of 406.7, 632.8 and 674.4 nm in vitro are respectively determined. In addition, a novel simple method based on total internal reflection for measuring the refractive index of biotissue in vivo is developed, and the refractive indices of skin from people of different age, sex and skin color are measured. Their refractive indices are almost same and the average is 1.533.

New Approach for Noninvasive Determination of Tissue Optical Properties

A state-of-the-art radiative transfer model for the coupled air/tissue system is introduced. We then present a feasibility study of a new method for determining the tissue optical properties.