Exploring the future of regenerative medicine: Unveiling the potential of optical microscopy for structural and functional imaging of stem cells

Regenerative medicine, which utilizes stem cells for tissue and organ repair, holds immense promise in healthcare. A comprehensive understanding of stem cell characteristics is crucial to unlock their potential. This study explores the pivotal role of optical microscopy in advancing regenerative medicine as a potent tool for stem cell research. Advanced optical microscopy techniques enable an in‐depth examination of stem cell behavior, morphology, and functionality. The review encompasses current optical microscopy, elucidating its capabilities and constraints in stem cell imaging, while also shedding light on emerging technologies for improved stem cell visualization. Optical microscopy, complemented by techniques like fluorescence and multiphoton imaging, enhances our comprehension of stem cell dynamics. The introduction of label‐free imaging facilitates noninvasive, real‐time stem cell monitoring without external dyes or markers. By pushing the boundaries of optical microscopy, researchers reveal the intricate cellular mechanisms underpinning regenerative processes, thereby advancing more effective therapeutic strategies. The current study not only outlines the future of regenerative medicine but also underscores the pivotal role of optical microscopy in both structural and functional stem cell imaging.

optical microscopy techniques enable an in-depth examination of stem cell behavior, morphology, and functionality.The review encompasses current optical microscopy, elucidating its capabilities and constraints in stem cell imaging, while also shedding light on emerging technologies for improved stem cell visualization.Optical microscopy, complemented by techniques like fluorescence and multiphoton imaging, enhances our comprehension of stem cell dynamics.The introduction of label-free imaging facilitates noninvasive, real-time stem cell monitoring without external dyes or markers.By pushing the boundaries of optical microscopy, researchers reveal the intricate cellular mechanisms underpinning regenerative processes, thereby advancing more effective therapeutic strategies.The current study not only outlines the future of regenerative medicine but also underscores the pivotal role of optical microscopy in both structural and functional stem cell imaging.

| INTRODUCTION
Regenerative medicine stands at the forefront of modern healthcare, offering transformative strategies for treating a wide range of diseases, injuries, and age-related tissue damage.This rapidly evolving field is characterized by its potential to not only heal damaged tissues and organs but also to replace them, marking a significant shift in disease treatment paradigms [1,2].At its core, regenerative cell therapy revolves around the regeneration and reconstruction of tissues at the cellular level.Stem cells, with their remarkable ability to differentiate into various specialized cell types and sustain indefinite division, take center stage in regenerative cell therapy [1,2].These extraordinary cells are defined by their capacity to continuously self-renew and develop into specific cell lineages, making them invaluable assets in the pursuit of innovative treatments and therapies.Considering their transdifferentiation potential, stem cells can be divided into: (i) unipotent, (ii) multipotent, (iii) pluripotent, and (iv) totipotent [2].From regenerative applications point of view, stem cells can be divided to: (i) embryonic stem cells (ESCs), (ii) tissue specific progenitor stem cells, (iii) mesenchymal stem cells (MSCs), (iv) umbilical cord stem cells, (v) bone marrow stem cells, and (vi) induced pluripotent stem cells (iPSCs) [3] (see Figure 1).
The property of the self-renewing capacity of stem cells has given tremendous applications in advanced regenerative medicine [4,5].They are a specialized set of cells that can build out into a new organ or tissue.This ability of pluripotent or multipotent stem cells has currently been employed in the medical field to replace the damaged or diseased organs of patients or the affected individuals and helps to cure ailments [6].These stem cells have a predominant role in molecular biology and medicine.Owing to their widespread applications, stem cells have to be in constant observation once administered to the beneficiary people.
Development of safe and effective tissues or organs for replacement using stem cells requires controlled experiments.In these experiments, the methods for analyzing cells and tissues should enable real-time monitoring of biological samples with cellular or even subcellular spatial resolution without causing harm to them.To maintain continuous observation of administered cells or organs, invasive methods cannot be employed, as they may lead to tissue damage [7].Since we can get real-time information about the desired cells within no time, we can correct them immediately if there are any data gaps [8].As a result, noninvasive imaging technologies are vital.However, stem cells are required to be labeled to be imaged noninvasively in vivo [9].In conventional medical imaging methods, samples will be combined with special labels to enhance the contrast.The tissue sample can be labeled with a radionucleotide for SPECT and PET, and superparamagnetic iron oxide particles for MRI [10].However, these methods are extra-expensive and have a low spatial resolution.Optical imaging methods have evident benefits compared to MRI, PER, and SPECT.Optical imaging can be used without and with molecular labels.Up to now, a plethora of similar labels suitable for optical imaging have been created, including fluorescent proteins (FPs), such as green FPs (GFPs), red FPs, and others, which can be used in vivo [11,12].Also, bioluminescent proteins, and fluorescent nanoparticles can be used [13].
In the realm of optical imaging, the present review explores advanced techniques that offer unique advantages for studying stem cells within the context of regenerative medicine [6].Advanced optical imaging modalities such as stimulated-emission depletion microscopy (STED), structured illumination microscopy (SIM), spontaneous Raman Techniques, stochastic optical reconstruction microscopy (STORM), second harmonic generation (SHG) microscopy, and third harmonic generation (THG) microscopy provide key advantages (refer Table 1).STED microscopy allows for super-resolution imaging with unparalleled spatial detail, making it ideal for visualizing intricate cellular structures [38].SIM, on the other hand, offers high-resolution, fast imaging, enabling dynamic observation of cellular processes [39].Spontaneous Raman techniques provide label-free chemical information, which is valuable for understanding the molecular composition of stem cells.STORM and related techniques break the diffraction limit, offering nanoscale imaging of cellular structures [40].SHG and THG microscopy provide inherent contrast for specific biomolecules and structures.This review presents a comprehensive analysis of the applications and advantages of these advanced optical imaging modalities in the structural and functional imaging of stem cells in regenerative medicine, highlighting their role in advancing our understanding of these crucial components in the journey toward more effective regenerative therapies.

| Fluorescence microscopy
Fluorescence is based on the absorption of a photon with energy close to the energy of electron transition of a target molecule, followed by the emission of another photon with less energy than the absorbed photon energy.This difference in energy is known as Stokes shift.The fluorescent imaging technique provides measuring fluorescence signal with high temporal resolution (nanosecond range), spatial resolution, and high signal-to-noise ratio.Introduction of pinhole (see Figure 2A), a circular small aperture that is placed before the detector in order to avoid the out-of-focus signals or noise eventually improves the contrast and quality of the image.Even pinhole facilitates three-dimensional (3D) visualization of cellular and subcellular structures with better contrast [43].Noninvasive fluorescence imaging techniques provide the platform for comparing results with standard procedure results like immunohistochemistry and polymerase chain reaction.This technique has shown numerous benefits in the biomedical field like cell localization, tissue morphological study [44].
Fluorescence imaging of live cells using their labeling by FPs and dyes provides submicron resolution and serve as functional reporters.Highly sensitive camera to collect fluorescence signal were used to study low-level gene expression [45].Using transplantation of fluorescent-labeled pericytes and iPS cells allowed for confirmation of the angiogenesis in vivo [46].Stem cell therapy has been one of the best therapies for the repair of damaged organs [47].
Endomicroscopy imaging techniques have shown better efficacy and feasibility for tracking MSCs in RILI mice for restoring cells and healthy mice.Epithelial tissues of the lungs were the region of interest.The fiber optic • Enables visualization of intricate cellular structures.
• Provides nanoscale imaging capabilities.• Equipment can be complex and expensive.
• Limited penetration depth for deep tissue imaging.
• Suitable for dynamic observation of cellular processes.
• Less phototoxicity compared to some other techniques.• Resolution improvement over conventional microscopy is limited.
• Limited to relatively shallow imaging depths.
• May require specialized equipment.
• Ideal for detailed imaging of cellular structures.
• Provides molecular localization information.
• Requires specialized fluorophores and buffers.
• Slower imaging compared to conventional techniques.

FPS
• Offers high-resolution, 3D imaging of living specimens.
• Reduces photodamage and photobleaching compared to single-photon techniques.
• Requires specialized fluorophores for two-photon excitation.
• Complex setup and equipment.
• Limited to fluorescent samples.

FPS
• Label-free imaging, providing chemical information.
• Excellent for studying molecular composition of stem cells.
• Non-destructive and minimizes photodamage.• Lower sensitivity compared to fluorescence-based techniques.
• Longer acquisition times.
• Limited to specific molecular vibrations.
• Suitable for studying collagen and myosin.
• Limited to imaging noncentrosymmetric structures.
• Less versatility in contrast compared to fluorescence techniques.
• Offers complementary information to SHG.
• Noninvasive and label-free imaging.
• Limited to imaging noncentrosymmetric structures.
• Equipment may be costly.
• Can visualize tissue structures and functional information.
• Offers deep tissue penetration.
• Allows for functional and molecular imaging in regenerative medicine.• Typically requires specialized equipment.
• Limited to contrast from endogenous chromophores.[36,37] probe of endomicroscope was inserted through tracheotomy into the right lung.In both the mice, MSCs were detected which were injected via endotracheal (ET) and intravascular (IV) systems.ET group comprised more MSCs than IV group.In addition, a cell counting algorithm was developed to calculate the mean number of cells spotted by endomicroscopy video.The drawbacks of this technique comprise the non-distinguishable location of characters in the lung with accuracy [48].In vivo fluorescence imaging system consists of a camera in the confocal set-up that detects emission signals produced by a probe or fluorophore.Fluorescence imaging was performed with the help of tagging, labeling, or incorporating a GFP, reporter gene, or probe into the target cell.The use of the reporter gene can give information on cell viability and its ability to translate mRNA into protein for a longer period [6].Articular cartilages are one of the engineered tissues used for osteochondral defects as they possess the rheological property with respect to time.Fluorescence microscopy was used to assess articular cartilage characterization as well as extracellular matrix (ECM) evaluation during cartilage development.There was a significant difference observed in collagen type I and type II fluorescent spectrum with excitation and emission wavelengths at 450 nm and at 460-500 nm range, respectively.In addition, it possess chondroitin sulfate in ECM.There was minimal difference in articular cartilage and intervertebral disc, which was characterized by autofluorescence spectroscopy.In conclusion, it was stated that fluorescence, as well as autofluorescence microscopy, can be very beneficial for the assessment of engineered tissues and regenerative medicines [49].In another study, Fluorenylmethyloxycarbonyl fluorescence microscopy was used to assess the selfassembly of amyloid proteins in hydrogels.The amyloid hydrogel showed differentiation of MSCs into the neuronal lineage.The decreased fluorescence intensity and concomitant red shift depicted hydrogel gelation.The lesser the temperature, the more gelation was depicted by less fluorescence (as shown in Figure 3).It was also suggested that the fluorenyl group helped in gelation via π-π stacking [50].
Fluorescence microscopy is a rapid as well as a sensitive method for assessing molecular environments.It has the advantage of high sensitivity, specificity, and simplicity with lower cost compared to other techniques.It has a broad range of applications in regenerative medicine.Fluorescence microscopy depends on the components that produce fluorescence.The main disadvantage of fluorescence spectroscopy is that not all compounds exhibits fluorescence property.Hence, fluorescence spectroscopy can be only used for the compound that possesses fluorophore property.Fluorescence microscopy also does not provide deep-tissue imaging due to light scattering in heterogeneous tissues, which results in limiting its performance for in vivo imaging as its penetration depth ranges from 200 to 400 μm.To address these shortcomings, recent advances in fluorescent imaging techniques have led to the development of various tools like two-photon fluorescence microscopy (TPFM), fluorescent lifetime imaging microscopy (FLIM), fluorescent loss in photobleaching, and photoactivated localization microscopy (PALM).

| Two-photon fluorescence microscopy
Two-photon fluorescence microscopy (TPFM/TPM) is based on exiting fluorescence by absorbing two photons of infrared light simultaneously by molecule having electronic transitions in the UV-visible spectral range.This technique involves two photons of double the wavelength (half the energy) which is needed for excitation.Photobleaching and photocytotoxicity effects are reduced in TPFM due to using NIR photons by Ti:Saphire laser (as shown in Figure 2B).In addition, penetration into tissues is easier in TPFM compared to one-photon fluorescent microscopy due to less diffraction.TPFM is mainly used for live cell imaging, contributing to biomedical applications, diagnosis, co-localization of cell, and so on.TPFM helps researchers for delineation of morphology, viability, and differentiation of cells [51].
TPFM was used for imaging hMSC culture progression of differentiation factors by inducing hypoxia condition [52].TPF signal is contributed by NADPH, flavoprotein, and lipofuscin chromophores, which help to identify the quantity and oxygen concentration.TPF of NADPH and flavoprotein was used to assess redox ratio, metabolic activity, biochemical status, cell morphology, as well as oxidative stress.
In of collagen structure and its organization.It was found that collagen structure was disrupted in myocardial infarction affected patient's heart and the disruption increased with the fatal phase of the disease [54].In another study, TPM was used to assess gold nanocages labeled hMSCs in vitro as well as those migrated to glioblastoma region in nude mice in vivo for 4 weeks without altering the function of stem cells [55].A study with lipid droplet (LD) accumulating TP fluorescent probe named CBMC was designed for diagnosis of cancer cells.CBMC displayed weaker fluorescence due to lower polarity, vice versa in normal cells.In addition, fluorescence was higher in normal tissues compared to tumor.These results made it easier to locate the tumorigenic cells and establish the difference between normal and cancerous cells [56,57].Huang et al. used fluorescence dyes upon two-photon excitations to study the stem cell activity intracutaneously in mice (as shown in Figure 4).Some of the disadvantages of TPFM include excitation of dyes by broad excitation spectrum and, at the same time, absorption of light by the cells that can cause their damage.To overcome these shortcomings, techniques such as FLIM has been used.FLIM is a microscopic technique that offers imaging at superior resolution by exploiting the varying contrast present in the molecular environment of living samples.It has an advantage for sensing temperature changes, pH, viscosity, and ion concentration.It has high lifetime accuracy, simultaneous recording at different wavelength intervals and high photon efficiency.FLIM is often used to study cellular metabolism via autofluorescence in live animals and also used for fluorescence guided surgery [58].In a study, MSCs were grown with adipogenic differentiation factors and assessed via combination of TPFM and FLIM for metabolic co-factors like NADH, NADPH, and FAD.In addition, redox ratio of FAD:NADPH with bound or free NADH and bound NADPH was also evaluated by the same techniques.Meleshina et al. showed MSCs were assessed for differentiation in two directions, osteogenic and chondrogenic via evaluating similar optical redox ratio.Fluorescence increases with the ratio during differentiation due to increase in number of proteins and metabolic shift from oxidative phosphorylation to glycolytic pathway [59,60].The adipogenic stem cell differentiation and metabolic state was studied by combining TPFM with THG microscopy techniques (Figure 5) [61].enhanced spatial resolutions than the traditional optical microscopes and it is accomplished by spatially regulating the excitation radiation in a technique such as STED and by temporally regulating the emission fluorescent molecules as seen in PALM [18].In STED, the excitation spot is diffraction-limited; however, a concentric circular beam switches off the emission signal in the spot.As the excitation wavelength is filtered out, only a longer fluorescence wavelength is analyzed by the microscope.As the intensity increases, the focal spot emitting fluorescence becomes smaller [63].The image acquisition in STED is performed by using multiple scanning beams, wherein the spatial resolution can be adjusted by controlling the depletion laser radiation intensity.PALM, depicts the localization of the genetically encoded, single FPs based on temporal isolation combined with accurate positional details to reconstruct super-resolution images.Because PALM uses these FPs, it has a broad range of applicability and allows one to easily study a diverse range of proteins in cells and tissues.SRM relies on the capability of the technique to locate accurately the position of the molecules, whose collection provides a high spatial resolution.SRM predominantly used to image biological structures, this helps to refine the existing knowledge in understanding the underlying mechanistic principles [18].A microfluidics-based SR fluorescence microscopy was used to capture and characterize nanoscale selectin CD44 ligands, in hematopoietic stem/progenitor cells (HSPCs) homing.In this process, the transplanted HSPCs are moved to the bone marrow from the peripheral blood that is regulated by ligand-receptor interactions under external shear stress [64].

| Super-resolution microscopy
SIM employs a specialized optical setup designed to enhance spatial resolution in optical microscopy.The key components of a SIM setup include an illumination system, sample preparation, objective lens, and detector (as shown in Figure 2D).The illumination system generates a known structured pattern, often involving grid-like or sinusoidal interference fringes [65].This structured illumination pattern interacts with the sample, which is typically labeled with fluorescent markers.The objective lens, known for its high-quality optics, collects the emitted fluorescence from the sample and focuses it onto a sensitive camera or photodetector.In some variations of SIM, multiple images are acquired with different phases or orientations of the structured illumination pattern.These captured images are then subjected to advanced image reconstruction algorithms, which utilize the known pattern to extract high-frequency information from the acquired images [66].The final output is a super-resolved image that achieves significantly improved spatial resolution compared to traditional wide-field microscopy, providing researchers with the ability to visualize fine cellular structures and nanoscale features with exceptional clarity and detail.This technical setup forms the foundation of SIM, enabling its widespread application in various scientific disciplines.The SRM revealed the complex dynamics of ligand binding and rolling behavior.This microfluidicsbased, time-dependent SR microscopy experiment conducted at various time points revealed the nanoscale reorganization of the CD44 having a direct impact on the HSPCs rolling over E-selectins.It prompts a need for revision in the present knowledge about mechanisms of HSPCs homing, which, in turn, can improve the outcome of the blood disorders stem cell therapy.A study [67] has shown the utility and safety of silica-coated superparamagnetic iron particles (SiMAGs) via SR-based SIM.In this study, SIM has been used to determine their intracellular fate in human MSCs (hMSCs) in a pH-sensitive dynamicrange nanosensor.SIM results showed that SiMAGs were localized in the subcellular compartments in a proximity to the nucleus, in consonance with the lysosomal location.3D SIM images confirmed the co-localization of SiMAGs in the lysosomal intracellular space around the nucleus (as shown in Figure 6).SR microscopy, therefore, enables to study the subcellular location of SiMAGs in unprecedented detail.The implemented methodology allowed catching the degradation of SiMAGs particles used in hMSCs therapies, in turn enhancing the clinical benefits of particle-based therapeutics at a molecular level.
The STED microscopy has been employed for imaging two closely localized proteins from cardiomyocyte origin MSCs responsible for the process of mesenchymalto-endothelial transition as a response to cardiac ischemia [68].STED images revealed that the cardiomyocyte MSCs were positive for endothelial markers, indicating a transition.The images allowed one to distinguish surrounding cells that had a negative phenotype of these markers.As a result, SRM can be used to distinguish proteins and cell-specific markers that have the potential to enhance the therapeutic output of stem cell health and function.
STORM is a type of SR microscopy, in which the photo-switchable molecule in the activated state leads to emission of photons to allow precise localization before it enters the deactivated state.STORM was employed to study stem-cell-derived extracellular vesicles (EVs) using a photo-switchable lipid-dye [69].This method was referred as the d-STORM.EVs are responsible for intercell communication, including cancer cells.In contrast to previous studies, high-intensity STORM images showed that MSCs derived EVs reside in the size range of 20-250 nm, which in turn affected their expression.These results can be used in the further studies of the role of stem cell-derived EVs in blood cancers, which may serve as tumor biomarkers.A study by Stavenschi and Hoey [70] has explored the role of MSCs in a bone disorder, osteoporosis.They demonstrated that hydrostatic pressure is a mediator of osteogenic response in MSCs.The mechanotransduction process of osteogenesis was studied using STED microscopy.The intermediate filaments (IFs) that were examined showed breakdown and reorganization with translocation toward the perinuclear region.STED images in osteoporosis conditions were also captured, which showed that the IFs breakdown was disrupted in the condition.This study, therefore, prompts a new type of stem cell therapy to enhance the process of osteogenesis in such bone-loss diseases.One of the main limitations of SR microscopy is their inability to image structures that are distantly placed from the cover glass.The fluorescently labeled specimens in SIM, that are considerably distant do not show an illuminated stripe pattern, making it difficult to reconstruct the highresolution image.The requirement of an elevated focal plane, in order to obtain a clear image, is an additional disadvantage [71].

| Spontaneous Raman techniques
Raman spectroscopy uses Raman scattering, which occurs when the energy of emitted photon is distinct from the incident photon.Stokes and anti-Stokes shifts of emitted photon energy define two types of Raman scattering [72].A scattered photon with less energy and a lower frequency than the incident photon is produced by Stokes scattering.It takes place when energy from the incident photon is transferred to the molecule.In anti-Stokes scattering, the scattered photons have a higher energy compared to incident one.The Raman spectroscopy system consists of a monochromatic laser, a narrow-wavelength filter of this laser radiation in a scattered wave, a diffractive spectrophotometer for the scattered light spectral decomposition, and detectors like charge-coupled devices for obtaining the spectrum (as shown in Figure 7) [75].Several Raman techniques including surface enhanced Raman spectroscopy, coherent anti-Stokes Raman spectroscopy (CARS) and stimulated Raman scattering are present, each having their own uses and drawbacks.Raman microspectroscopy is the combination of Raman spectroscopy and microscopy to give an image with the morphological and molecular properties of the tissue or sample (as shown in Figure 8) [77][78][79][80].Raman spectroscopy is also combined with optic fibers for endoscopic studies [81].It is a noninvasive technique that only necessitates minimal sample processing and provides quantitative information about the biomolecules in tissues.Raman spectroscopy has the capability to perform real-time analysis of cells [82].Raman spectroscopy can be combined with optical fibers or any microscopy devices to increase the spatial resolution.
The human ESCs (hESCs) were differentiated from cardiomyocyte progeny by Raman spectroscopy [83,84].The Raman spectra of hESCs were found to be more similar to the human iPSCs than the progeny of hESCs after differentiation [81].The murine ESCs were studied during 16 days interval by Raman spectroscopy.The RNA and DNA peak intensity were reduced by 75 and 50%, respectively, during this period.Also, the ESCs have higher DNA and RNA peaks of Raman scattering [84].The differentiation of ESCs into hepatocytes was investigated by Raman spectroscopy [85].The ESCs Raman spectra differentiated hepatocytes after 17 days and the differentiating hepatocytes after 7 days of the experiment.The biochemical differences between these cells were established.Lipids and certain proteins were found to increase, but DNA, amino acids, and other proteins were found to decrease.The amount of LDs in hepatocytes also increased.The nuclear volume decreases during differentiation, resulting in decreased DNA content.Comparison of cell lines of primary stem cell and the identification of their biochemical profiles were done by Raman spectroscopy [86].It also allowed differentiating between the dead and live cells.The cell cycle stage could be identified by analyzing the DNA Raman scattering at 782, 788, and 1095 cm À1 .The lower intensity peaks corresponded to dead cells (see Figure 8) [76].
The MSC differentiation when forming bone minerals was observed with Raman spectroscopy [87].Peaks corresponding to phosphate (in calcium hydroxyapatite) formed in ECM, collagen, and carbonate were observed.The ratio of carbonate to phosphate and mineral to matrix obtained from the Raman spectra was used to identify the chemical nature of bones and boneforming cells.Significant difference was observed among the peaks of the nucleus and cytoplasm in ESCs and MSCs, due to their higher protein and lower RNA levels.Cells moved away from colonies, that is, lone cells lose their pluripotency, and the peaks of ESCs in colonies showed higher DNA and RNA peaks at 784 and 811 cm À1 , respectively [88,89].Hydroxyapatite production during the MSC osteogenic differentiation was studied with Raman spectroscopy [90].The calcium stain alizarin red S was employed to verify the state of MSCs during differentiation.The hydroxyapatite specific chemical shift changed 7-11 days post the beginning of differentiation in the Raman spectra indicating that this could be used as a marker for skeletal tissue formation [91].
The differentiation of skeletal stem cells was characterized by spectral markers.Octacalcium phosphate peaks were observed to decrease during differentiation and a β-tricalcium phosphate to appear after the differentiation [92].Adipose-derived stem cells (ADSCs) were also used for skeletal tissue regeneration and similar markers, as mentioned above, were used to characterize them [93].The differentiation ability of SSCs can be analyzed by studying newer single-cell-derived clones.The subtypes of the stromal bone marrow cells of humans were studied by Raman spectroscopy to assess their potential to differentiate [94].Dental pulp stem cells are used as a source of stem cells because they are capable of self-renewal, clonogenicity, and multipotent.However, they expand to give heterogeneous populations.Raman spectroscopy was used to differentiate DPSC subpopulations.The DPSCs were classified into populations, which have low proliferation or are unipotent (A1 and B1) and which have high proliferation or are multipotent (A3).The Raman spectra of these two populations were clearly distinguishable.The biochemical features of these cells were also highlighted in the Raman spectra.The spectral intensities of the A3 populations were higher than the A1 and B1.The DNA and protein content of the multipotent A3 populations were higher compared to unipotent population [95].The Raman spectra of several healthy cells (L132, MG63, SAOS2, and WS1) from various cell lines showed several typical protein peaks, including 623 and 1005 cm À1 for phenylalanine and 645 and 854 cm À1 for tyrosine.Despite the presence of DNA and RNA peaks, many important peaks of DNA and RNA were not found in the Raman spectra.Similarly, a number of key lipid peaks were absent.The latter could be due to the decreased sensitivity of this method, damage to cells present at the outer part of the pellet.Nonetheless, these peaks certainly indicate the biochemical properties of the cells and the difference among several cell lines.However, it could not be used to differentiate several different cell lines (Figure 9).Cell viability can also be studied by observing the Raman peaks, especially at 788 and 782 cm À1 .The former peak corresponding the O P O stretching, had a diminished energy and the latter is indicative of the ring stretching of the thyamine and cytosine residues [96].Raman spectroscopy was also used to differentiate immune cells such as T cells (CD 4+ and CD 8+ ) and NK cells.This technique was also used to investigate the activation of immune cells from an anergic state [97].However, this technique can cause photodamage and is not suitable for fixed or fluorescent samples [81].

| Coherent Raman techniques
CARS microscopy is a nonlinear optical technique, which employs a dual-laser system functioning at varied frequencies.The two laser beams were merged collinearly (as shown in Figure 7B), reach the multiphoton microscope, and the signals at the CARS wavelength are collected by a photomultiplier tube (PMT).CARS microscopy is around five times faster than conventional Raman spectroscopy.Since Raman spectroscopy excites a lower frequency vibration by a high-frequency laser, the molecular bonds fail to resonantly get excited and therefore, show a low signal level [93,98].Moreover, considering the low efficiency of Raman scattering (about 1 in 10 7 photons), CARS microscopy proves to be an important alternative, with its high sensitivity.
CARS microscopy was used to image human corneal fibroblasts, hMSCs, and collagen in a cell culture.The collagen formation is a biomarker for the differentiation of stem cells and ECM formation.The cells were identified by their lipidic structures.The membranes along with the lipid membranes present in them were visualized clearly at 2844 cm À1 , which depicted the CH 2 symmetric stretch Raman mode [99].Therefore, this technique can be used to study the cell morphology and collagen production in the 3D cultures of living cells (as shown in Figure 10).
ADSCs are used as a source of stem cells for regenerative medicine and as in vitro disease models.The presence of LDs is a feature of the differentiation of ADSCs to adipocytes.The CARS microscopy images were taken at regular intervals after induction of cell differentiation.Bright spots were observed in the images taken in the early days (2 days post-induction) depicting the presence of LDs at the nucleus periphery.The overall change in morphology combined with an increase in diameter of osteoblasts was also an indication of the differentiation.During the differentiation to osteoblasts, the ADSCs acquire an ECM made of collagen along with the visualization of hydroxyapatite.At an early stage after induction, the CARS images showed the cells elongation and the oxidization of flavoproteins leading to autofluorescence.At a later stage (7 days post-induction), collagen fibers were observed.CARS images also showed that the cells grow in several layers during this process [100].The high CH 2 and CH 3 stretching vibration signals of lipids were mainly detected with CARS [101].A coculture system of adipose-tumor epithelial cells was developed to study the mammary environment in vivo.CARS microscopy was used to determine the adipocyte viability.The analysis of MSC, SSC, and ADSC differentiation to adipocytes by CARS was done by studying the lipid content in these cells during the differentiation.The adipose tissue formed in this coculture system was viable, had morphological integrity and similar method of LD formation at the edges of epithelial cells like the in vivo environment [102].The MSCs differentiated to adipocytes were also observed by CARS microscopy to visualize the composition of vesicles and their respective lipid contents in 3D culture systems like Nichoids and flat substrates.The cell differentiation was greater in Nichoids than in flat surfaces.CARS was found to be more sensitive than the fluorescent microscopy for the quantification of lipid vesicles.On days 7 and 14 of experiment with CARS, the Nichoids were found to have 2 times and 3 times greater results, respectively.The flat surfaces had 8 times and 3 times greater results, respectively.The lipid vesicles were higher in number per cell on day 7 than day 14 for both Nichoids and flat surfaces.The sizes of LDs were found to be higher on day 14 than day 7 in Nichoids.Therefore, since Nichoids have a larger area filled with lipids, MSCs here have better adipogenesis, compared to flat substrates [103].Raman spectroscopy was used to study skeletal stem cells differentiating to adipocytes.The lipid vibrational frequency was determined by Raman spectroscopy, which was later imaged by CARS.The formation of lipids was studied by CARS microscopy in the differentiating skeletal stem cells as the lipid accumulates in the cells during adipogenesis.It was observed at a higher resolution in CARS compared to the Oil Red O-staining.CARS was also used to study the variation in the differentiation of skeletal stem cells upon using chemicals like bisphenol A diglycidyl ether and dexamethasone.The lipids droplets were observed at a high resolution even 24-72 h after induction of adipogenesis.The increased mass and volume of LDs were observed as differentiation progressed [104].CARS was used to visualize the differentiation of human neural-crest derived inferior turbinate stem cells (ITSCs) to osteogenic cells.The calcium and phosphate deposits including calcium apatite levels were analyzed.The colocalization of calcium apatite and collagen was observed too.Their 3D structures were also analyzed by CARS.The mineralized deposits were coated by a collagen matrix.The osteogenic differentiation was characterized by studying the PO 4 3À band at 959 cm À1 corresponding to calcium hydroxyapatite, which was appeared 14 days after induction of differentiation of ITSCs [105].Multiplex CARS was employed to get the Raman signal simultaneously from a region, where several spectrum peaks are present corresponding to the cell components.It allowed imaging the cartilage cells to understand the differentiation state.When proline was imaged with CARS, the peripheral areas of the cartilage cells were highlighted, indicating the formation of collagen in the ECM [106].However, CARS microscopy, at low powers, can cause photodamage due to high multi-photon absorption [93,98].

| Second harmonic generation (SHG) microscopy
SHG microscopy is second-order nonlinear optical technique, where two photons are coherently converted to comprise half the incident wavelength or twice the incident frequency.Unlike linear processes in conventional optical microscopy, SHG arises from the interaction of two photons to generate a single photon with double the frequency, resulting in the formation of the second harmonic signal [107].To efficiently collect generated SHG signal, a high-quality objective lens is typically used to focus the signal onto a photodetector, often a PMT or a photodiode.The SHG signal is collected in a nondescanned configuration, which enhances sensitivity and signal detection [108].The instrument setup may also include various filters (see Figure 10) and optics to precisely separate the SHG signal from the excitation light and other unwanted emissions.SHG microscopy is used extensively as a deep-tissue, noninvasive imaging technique.The advantage of this technique mainly lies in the ability to perform high contrast imaging without labeling (Figure 11).The tissue imaging with SHG is implemented without essential energy deposition and absorption, which reduces photobleaching and phototoxicity [111].
Pluripotent stem cells (PSCs)-derived cardiomyocytes (PSC-CMs) have the potential to be used in regenerative medicine.SHG microscopy was used to identify the stages of plated and suspension PSC-CMs.Based on their SHG signal, PSC-CMs differentiation was detected at 7.5 days due to SHG signal generation from myofilament network.The signal intensity is proportional to the maturity of the myofilament network.SHG signal was registered even from an individual myofilament.SHG can be used to differentiate hESCs and iPSCs.SHG allows identification of PSC-CMs faster than other techniques based on the absorption or Raman spectroscopies.SHG imaging can be used to obtain information about the contraction of PSC-CMs [112].When the epidermis and the dermal cells were taken out from the human and animal skin, acellular dermal matrices (ADMs) were obtained.They can be inserted into wounded tissues and used for burn injuries treatment and other skin reconstructions.It can be used as a scaffold, to which host cells can be introduced and functional tissues can be formed.MSCs can be seeded into the ADMs to help in wound healing applications.Collagen proteins constitute the majority of the acellular scaffolds.The collagen fiber density, orientation and shape can be determined by SHG microscopy (as shown in Figure 12).This technique has some additional advantages, including less invasiveness, high spatial resolution and contrast, 3D optical sectioning ability and absence of background interference [114].SHG also aids in visualizing cellular proteins like collagen I and myosin II used for studies skeletal and heart muscle tissues [115].Collagen I, a material used for scaffolding in tissue engineering, and its properties like fiber length, distribution, thickness and assembly, were studied extensively by SHG [116].However, this technique can only be used for a few molecules and certain biomaterials only.

| Third harmonic generation (THG) microscopy
THG microscopy involves tripling the frequency of the excitation wavelength, resulting in emitting of onephoton due to the combination of energy of three combined photons.The emitted photon will possess higher energy due to its wavelength being one third of the excitation wavelength.THG excitation chiefly arises at the interfaces such as cellular membranes, which forms the interface between lipid structures and aqueous interstitial fluids as well as between protein aggregates and water [117].THG microscopy utilizes a femtosecond laser as a light source to excite the sample.The high-quality objective lens collects the third harmonic signal generated by the sample, and optical filters separate it from the excitation laser light (as shown in Figure 11B).A photodetector, such as a PMT, captures the signal for image processing and analysis, allowing label-free, highresolution imaging of biological and material samples, particularly at interfaces and boundaries.THG microscopy has been widely used in the clinical field to assess dysfunction related to muscles and heart.Cardiovascular diseases are one of the major reasons for the increase in mortality rate in all over the world due to myocardium ischemic damage, which leads to atherosclerotic plaque following coronary artery occlusion.Cardiac stem cell therapy is widely used for remodeling ischemic myocardial tissue.In one of the cardiac stem cell therapy studies, THG was used to assess the multilamellar structure of densely folded cristae as wells as inner and outer mitochondrial membranes of single isolated cardiomyocyte.It helped in remodeling of cardiac tissue with a single cardiomyocyte as a reference entity.In addition, it will further be used to track the stem cell regeneration and development [118].In another study, reconstruction of posterior cornea via tissue engineering to treat diseased corneal endothelium was evaluated by harmonic imaging.The results were also compared with histopathological results to estimate the usefulness of harmonic imaging.THG allowed visualization of stromal and endothelial cells with dark nucleus, as stated in other studies.It also assessed the heterogeneity of refractive index of the cytoplasm owing to the presence of various organelles and compartments in the cytoplasm [119].This technique holds an advantage as it can be applied for living, dynamic, biological, and non-biological specimens [120].One of the major disadvantages of THG is its weak capability toward chemical specificity [121].With better development, THG can be then implemented in field research and clinical applications [117].

| Photoacoustic microscopy
In photoacoustic (PA) imaging, the use of near IR pulses of light and absence of background ultrasound (US) signal results in an enhanced depth resolution.The sample is radiated by a pulsed light, wherein heat expands and generates US waves instantly.The ability of this technique to image the human organs with more efficiency and maintain a high contrast and spatial resolution makes it rather different from other imaging techniques.The contrast in the images is provided due to the presence of endogenous proteins, engineered probes or nanomaterials like gold nanorods or NPs.Based on the foci type, PA imaging technique is classified into two types: acoustic foci and optical foci.The PA signals are produced at a point where the two foci overlap, providing maximum sensitivity.Using a transducer provides a higher central frequency, a broader range of bandwidth, higher spatial resolution below 100 μm.3D PA imaging has also been developed, due to which, it is easy to study the human cells labeled by a contrast agent used to examine gene expression, cell growth, and biological behavior in xenograft tumor mice (Figure 13).The combination with tomographic imaging techniques such as PA computed PA tomography and PA microscopy (PAM) surpasses the imaging limits of the fluorescence techniques, thus introducing a new dimension to study cell bases therapies [123].
Despite the significant research in the field of stem cell therapy, only a few have had a clinical implementation owing to the lack of imaging techniques to study the underlying mechanisms precisely.Several imaging techniques, such as magnetic resonance imaging (MRI), positron emission tomography, optical imaging, and US, have been used for obtaining images with high resolution, sensitivity, penetration depth, safety, and costefficiency.However, all of these techniques have a major drawback of obtaining images with poor contrast, low sensitivity and penetration depth, side effects of radiation and also cytotoxicity due to NPs.PA imaging on the other hand, addresses these challenges as it provides high contrast images by exploiting the different absorption levels in various tissue types.Also, it provides real-time imaging feedback during the therapy and monitor the challenges that obstruct the achievement of total therapeutic output [124].
Since the PA imaging technique has demonstrated a high potential to image biological specimens even at a greater tissue depth, it stands out to be one of the most convenient methods to be used in imaging/tracking cells.The ability of PAM to work in tandem to obtain images with better resolution makes this field of application very wide.Due to its ability to use an intrinsic contrast of specimens with enhanced spectral and lateral resolution, this imaging modality is rapidly emerging in the field of regenerative medicine.The capability of this technique to register images of only targeted cells allows it to be used for various purposes such as, stem cell tracking, longitudinal monitoring in vivo, evaluation of the vascularization in cells and most importantly to track and monitor the diseases in order to improve the therapeutic effectiveness of available treatment options.As a result of these growing applications, commercial PA systems will become more readily available for regenerative therapy research and development [125].
The similar study of Kubelick and Emelianov showed the usefulness of PA imaging for in vivo spinal cord regenerative therapy.Here, Prussian blue nanocubes (PBNCs) were used to label the stem cells before their delivery into the spinal cord [126].The combined US/PA images were captured by a Vevo LAZR with a 20-MHz linear array transducer, coupled to an optical fiber F I G U R E 1 2 Fibroblast-secreted collagen and scaffold fibers were visualized using second harmonic generation (SHG) emissions collected at depths of 10-40 μm (A).Yellow arrowheads visualize PCL fibers of the scaffold.Fibroblast-seeded constructs were fixed and visualized via SHG emissions (collagen and scaffold fibers in yellow) and DAPI staining (cell nucleus in blue) at depths of 10-40 μm (B).Fixed constructs were also visualized for fibroblast cytoskeleton (phalloidin-TRITC in red) proximity to secreted collagen (SHG in green) (C).SHG was obtained using 800 nm illumination, and all images were collected after 21 days of culture.White arrows indicate scaffold fiber orientation.(Figure reproduced from Reference [113] with permission from PLoS One).
bundle at a wavelength of 750 nm.The real-time US/PA images were obtained when the needle was injected into the spinal cord in vivo, showing accumulation of the PBNC-labeled cells.The 3D US/PA volumetric images were also obtained.This surgical guidance via PA imaging helps to track precisely the stem cells during the entire course of therapy.This technology is a great way to get feedback so that real-time adjustments can be made before the surgery is finished, improving outcomes.Therefore, the findings represent a significant advancement that will encourage the further development of imaging techniques in the area of spinal cord therapies.
Dhada et al. have employed PA imaging in order to determine the viability of the MSCs in mice via NP-based technique [127].Stem cells produce ROS upon death, which degrades the cell.A contrast agent made of inert gold nanorods coated with IR775c, an ROS-sensitive near IR dye, whose signal could assess the viability of the MSCs and also track their location were used.The MSCs were loaded with the NPs and transplanted into mice limb.A combined US and PA imaging revealed that a significant cell death occurred in 24 h and a 5% viability was observed after 10 days.Thus, nanoprobe-based PA imaging enables real-time, rapid tracking of stem cell viability, which improves our comprehension of stem cellbased therapies.It can improve the effectiveness of stem cell treatments currently being used in clinical settings.Donnelly et al. [128] have also exploited PA imaging for facilitating an image guided needle placement and monitoring of stem cells labeled with NPs during their delivery into the spinal cord.The US and PA combination helps in guiding needle for stem cell injection precisely with minimum risk of injury and maximum therapeutic output.In addition, the technique was also able to detect as few as 1000 cells and accurately localize them.Therefore, PA imaging becomes one of the best alternatives to already existed imaging modalities and allows real-time in vivo imaging.Therefore, this imaging platform could provide clinicians with more detailed, safe, and accurate information on creating compatible techniques for stem cell delivery.Similarly, another study [129] has explored the use of PA in vivo imaging for quantification of MSCs coated with silicagold NPs.Here, silica-coated gold nanorods have been playing the role of contrast agent for PA imaging.The silica coating facilitates the increased uptake of gold into cells with more than five folds, without toxicity, a decrease in rate of proliferation or pluripotency.Since PA offers low background, high-contrast, it allowed imaging of down to 100 000 cells even in an in vivo setting, with real-time monitoring, a spatial resolution of 340 μm and temporal resolution of 0.2 s, lower than any other traditional imaging techniques.These findings imply that, despite the use or presence of contrast agents, the therapeutic benefits of MSCs will not diminish.Thus, potency of the stem cells is not dependent on contrast agents, which in turn encourages the development of PA imaging in regenerative medicine.Qin et al. demonstrated the use of PA NPs (PANPs) incorporated with semiconducting polymers, as contrast agents for PA imaging of human ESC-derived cardiomyocytes (hESC-CMs) transplanted into living mouse hearts [130].Due to the ability of the semiconducting polymers to provide specific spectral features and strong PA signals, it was possible to detect the precise details of the cells and to distinguish the PANP labeled cells from the background tissues (as shown in Figure 14).PA imaging in combination with US has proved to be a great method to assist the delivery and engraftment of these hESC-CMs into the heart.In addition, PA images with an excellent spatial resolution of 100 μm, along for as few as 2000 labeled cells, were also registered.The PANP labeling is widely used to evaluate the transport of cells in various regenerative therapies because it had no negative impact on the cells.
In an another study, gold nanocages (AuNCs) were delivered in mouse ear tumors through glass capillaries in order to label human MSCs and examine their homing in the tumor [55].The PA amplitude had a linear relationship with the concentration of the AuNCs.Overtime, the previously labeled cells divided, due to which there was a decrease in the PA amplitude.A PA wavelength of 580 nm was recorded when the labeled hMSCs were injected into the mouse ear.A distinction between blood arteries and AuNCs labeled hMSCs was observed at 532 nm.Due to the blood vessel components' poorer absorption range, the tagged cells could only be seen at 638 nm.Thus, PAM was used to record signals from each of the components, which were easily separated out in the PA images.Therefore, this technique can be used to track labeled stem cells with high spectral and lateral resolution, further promoting their use in regenerative medicine.Bandwidth mismatch between the acoustic signal and the transducer on the surface of the sample, and background noise in the amplifier are some of the technological limitations that hinder the application of PAM in biological samples imaging.

| CONCLUSION
The field of regenerative medicine has made remarkable strides in harnessing the potential of stem cells, cell lines, scaffolds, and animal models for tissue repair and organ regeneration.However, amidst these advancements, it is vital to acknowledge the inherent challenges and opportunities that shape the future of regenerative medicine, particularly when considering the role of advanced optical microscopy techniques.The assessment of regenerative approaches necessitates a holistic evaluation of both their merits and limitations.In this context, techniques like confocal microscopy, with its remarkable ability to provide high-resolution, 3D images, STED, known for its super-resolution capabilities, SIM, which enhances resolution in live-cell imaging, STORM STORM, offering single-molecule precision, TPFM, which allows deep tissue imaging, and PAM, combining optical and acoustic imaging, characterized by their noninvasive, nontoxic, sensitive, and specific nature, have emerged as pivotal tools for structural and functional imaging of stem cells.Yet, a closer examination reveals challenges that require attention in the path ahead.
Foremost among these challenges is the need to enhance imaging depth, an issue that has persisted in optical microscopy.While these techniques offer superb resolution, their ability to visualize deep-seated tissues remains constrained.Researchers must focus on the development of innovative methods that enable deeper tissue visualization, thereby facilitating comprehensive studies of regenerative processes within organs and tissues situated deep within the body.Another challenge lies in the integration of multiple imaging modalities, a necessity for obtaining a complete understanding of regenerative processes.The seamless combination of optical microscopy with complementary techniques, such as MRI and computed tomography, holds the promise of yielding richer insights but demands intricate technical solutions.Real-time 3D imaging is another frontier, representing the future of regenerative medicine.The capacity to monitor dynamic regenerative events with high spatial and temporal resolution is transformative, yet the development of real-time 3D imaging capabilities remains an imposing challenge.
Translating the potential of regenerative medicine from research to clinical practice represents an ongoing endeavor.Bridging this gap necessitates the advancement of label-free optical techniques that facilitate noninvasive in vivo monitoring of stem cell therapies using PA imaging and spontaneous Raman techniques.This endeavor goes beyond the technical aspects to encompass regulatory, safety, and practical considerations.As the volume of imaging data continues to rise with the increasing sophistication of optical microscopy techniques, a further challenge arises in effective data management and analysis.Researchers are tasked with the development of robust data analysis methods and automated image interpretation to derive meaningful insights from the hidden details of data generated.Moreover, the inherent biological variability among individuals and biological systems poses a challenge.Regenerative processes are far from uniform, making the consideration of these variations vital.The future may involve the tailoring of regenerative therapies based on optical microscopy for individual patients, ushering in an era of personalized regenerative medicine.
However, as we look ahead, the future of optical imaging in regenerative medicine brims with potential.Researchers are poised to overcome these challenges and unlock the full potential of optical microscopy for the field.Enhancing imaging depth, seamless multimodal integration, real-time 3D imaging, and clinical translation represent transformative prospects.The development of advanced data analysis tools and the recognition of biological variability promise to enrich our understanding.Yet, perhaps the most promising prospect lies in interdisciplinary collaboration, uniting scientists, clinicians, and engineers across diverse fields to address the multifaceted challenges in regenerative medicine and optical microscopy.In conclusion, the convergence of regenerative medicine and advanced optical microscopy techniques presents a promising avenue for healthcare's future, one that hinges on researchers' collective efforts to address the challenges and embark on these future directions.This holds the potential to unlock the full capabilities of above discussed optical microscopy for the structural and functional imaging of stem cells, advancing regenerative medicine, deepening our comprehension of regenerative processes, and ushering in a new era of personalized and effective regenerative therapies.

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I G U R E 2 (A) Schematic representation of confocal fluorescence microscopy setup.(Figure reproduced from Reference [14] with kind permission from MDPI).(B) Schematic of the imaging setup of two-photon fluorescence microscopy.A mode-locked Ti:Sapphire laser at an output wavelength of 950 nm is used as the excitation source.(Figure reproduced from Reference [41] with kind permission from MDPI).(C) Schematic illustrations of the principle of stimulated-emission depletion (STED) microscopy.(D) Schematic illustrations of the principle of structured-illumination (SIM) microscopy.(Figures C and D reproduced from Reference [42] with permission from Frontiers).
another study, aggregation-induced emission of luminogen-loaded fluorescent nanoparticles (NPs) were synthesized for imaging of folate receptor overexpressed cancer cells via one-photon and two-photon fluorescence imaging.Internalization of NPs was observed in MCF-7 breast cancer cells via endocytosis and accumulation of folic acid-functionalized 2-(2,6-bis((E)-4-(phenyl(4 0 -(1,2,2-triphenylvinyl)-[1,1 0 -biphenyl]-4-yl)amino)styryl)-4H-pyran-4-ylidene) malononitrile loaded NPs were also seen in tumor tissues which were found to be key component of diagnosis.Fluorescence microscopy assisted in identifying diagnostic elements of cancer and the factors responsible for tumor progression [53].Cardiologists use TPM for monitoring cellular activities and whole heart valve tissues in myocardial infarction, as well as an ischemic heart for co-registered visualization of cardiomyocytes and collagen.A study with TPFM was used for observation F I G U R E 3 Immunostaining of neuronal markers of transplanted cell.The green, red, and blue channel represents the green fluorescent protein (GFP) signal from transplanted cells, the neuronal marker β-III-tubulin and nuclear stain DAPI, respectively.Cells transplanted into the SN stained positive for β-III-tubulin expression, indicating the neuronal lineage of differentiation.(Figure reproduced from Reference [50] with permission from Nature).
Super-resolution microscopy (SRM), also known as nanoscopy, is implemented by turning on and off different groups of fluorophores in a diffraction limited focal volume to collect signals with improved resolution[62].Due to this, the fluorescent molecules become bleached for a small duration, followed by photon emission.The recently developed super-resolution techniques are lightbased, which allow imaging of biological samples with F I G U R E 4 (A) Two-photon fluorescence images of interfollicular epidermis at different layers.(B) Order of keratinocyte activity in the respective layers of interfollicular epidermis.The Keratin 14 promoter drives the expression of green fluorescent protein (GFP) fused to histone H2B in a mouse strain.Red arrowheads that point to a stem cell division in the basal layer are spaced 20 min apart in time.(C) Telogen hair follicles in three dimensions.(D) A group of hair follicles and other cellular components of the skin are visible in a single optical plane taken from the dermis.Scale bars: 20 μm.(Figure reproduced from Reference [51] with permission from Wiley).

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I G U R E 5 Redox-based colorcoded images extracted from endogenous TPF images are superimposed on THG-based lipid content (magenta) images.The same scaffold is imaged after 2, 5, or 9 weeks in either adipogenic (top row) or propagation (bottom row) media.Scale Bar = 35 μm.Redox scale color bar is shown on the right.Endogenous silk fluorescence is shown in gray scale.(Figure reproduced from Reference [61] with permission from Elsevier).

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I G U R E 7 (A) Schematic diagram of detection platform based on Raman spectroscopy.Figure reproduced from Reference [73] with kind permission from MDPI.(B) Optical scheme of the CARS microscope.(Figure reproduced from Reference [74] with kind permission from OPTICA).

F I G U R E 8
Raman peaks showing the difference in DNA and protein content in A549 cells.(-▪-): measured values, (-•-): values corresponding to dead cells.(Figure reproduced from Reference [76] with permission).

F I G U R E 9
Raman scattering images of unstained, unlabeled living HeLa cells reconstructed using the distribution of Raman signals at (A) 753 cm À1 , (B) 1686 cm À1 , and (C) 2852 cm À1 , showing the distribution of cytochrome c, protein beta sheet, and lipid molecules, respectively.Image (D) was constructed by merging images (A) through (C) with color channels.The sample was irradiated with a light intensity of 3.3 Mw/μm 2 at the focal plane in 78 lines of exposure.The exposure time of each line was 5 s, and the images consist of 78 Â 281 pixels.(Figure reproduced from Reference [77] with kind permission from MDPI).

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I G U R E 1 0 Living human corneal fibroblast (A) and human mesenchymal stem cells (B) morphology in a 4-mm-thick fibrin hydrogel scaffolds (3D CARS imaging in red) and their collagen production (3D SHG imaging in white) at different days in culture (days 0, 7, 14, 21, 28).(Figure reproduced from Reference [99] with kind permission from Wiley).

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I G U R E 1 1 (A) Instrumentation setup of SHG microscopy (Figure reproduced from Reference [109] with kind permission from MDPI).(B) Schematic representation of the THG microscopy.Two paths are used for detection, one in reflection (for TPF signals) and the other in transmission mode (for THG signals).C, condenser lens; DM, dichroic mirror; F1; F2, filters; GM, galvanometric mirrors; L1; L2, telescope lenses; M, mirror; ND, neutral density filters; O, objective lens; PMT, photomultiplier tube.(Figure reproduced from Reference [110] with permission from OPTICA).

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I G U R E 1 3 (A) The basic setup of photoacoustic imaging (PAI) includes a pulse laser, ultrasound transducer, and data acquisition system.(Figure reproduced from Reference [122] with permission from MDPI).

F I G U R E 1 4
Imaging the engraftment of HeSC-CMs ex vivo.(A) A sectioned slide of the heart under bright field microscopy (Â2).(B) Photoacoustic imaging (PAI) detected the engrafted HeSC-CMs from a fixed heart before sectioning ex vivo.(C) Fluorescent microscopy directly identified the engrafted cells (brighter blue) from the host myocardium without any immunostaining.(D) Confocal images of the engrafted cells and the host myocardium with immunostaining to distinguish human cells from mouse cells.DAPI (blue) and cTnT (green) marked both host and engrafted cardiomyocytes, whereas human mitochondria antibody (purple) and photoacoustic nanoparticles (PANPs) fluorescence (red) distinguished the engrafted HeSC-CMs from the host cells.(Figure reproduced from Reference [130] with permission from Wiley publishers).
Comparison table showing the resolution, penetration depth, imaging speed, merits, and demerits of various optical imaging modalities used in stem cell research.
T A B L E 1