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.

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.

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.

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.

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.

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.