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This paper describes a methodology to differentiate morphea from lichen sclerosus based on examination with multiphoton microscopy (MPM) composed of two‐photon excited fluorescence (TPEF) and second harmonic generation (SHG). Subcellular‐resolution images were acquired by MPM from unstained lesion tissues then process spectral analysis to quantify the TPEF and SHG signals. Moreover, U‐Net was employed to segment elastic fiber in TPEF images to combine with collagen fiber in SHG images for precise fiber quantification. Predictions of segmentation showed excellent performance on several evaluation indicators. The mIoU, mPA, and F1 score reach 0.8516, 0.9281, and 0.941. The quantitative analysis demonstrated the increase of collagen fibers in morphea compared to that in lichen sclerosus cases. Meanwhile, the great diminution of elastic fiber in the dermis of lichen sclerosus was depicted based on MPM imaging. Thus, MPM was comparable to the histopathological examination and our experimental results accurately distinguish between morphea and lichen sclerosus. [ABSTRACT FROM AUTHOR]

Study Design.: Prospective cross-sectional study. Objective.: To determine if an accumulation of advanced glycation endproducts (AGEs) is associated with impaired paraspinal muscle composition. Background.: Impaired bone integrity and muscle function are described as osteosarcopenia. Osteosarcopenia is associated with falls, fragility fractures, and reduced quality of life. Bone integrity is influenced by bone quantity (bone mineral density) and quality (microarchitecture and collagen). The accumulation of AGEs stiffens collagen fibers and increases bone fragility. The relationship between paraspinal muscle composition and bone collagen properties has not been evaluated. Methods.: Intraoperative bone biopsies from the posterior superior iliac spine were obtained and evaluated with multiphoton microscopy for fluorescent AGE cross-link density (fAGEs). Preoperative magnetic resonance imaging measurements at level L4 included the musculus (m.) psoas and combined m. multifidus and m. erector spinae (posterior paraspinal musculature, PPM). Muscle segmentation on axial images (cross-sectional area, CSA) and calculation of a pixel intensity threshold method to differentiate muscle (functional cross-sectional area, fCSA) and intramuscular fat (FAT). Quantitative computed tomography was performed at the lumbar spine. Univariate and multivariable regression models were used to investigate associations between fAGEs and paraspinal musculature. Results: One hundred seven prospectively enrolled patients (50.5% female, age 60.7 y, BMI 28.9 kg/m2) were analyzed. In all, 41.1% and 15.0% of the patients demonstrated osteopenia and osteoporosis, respectively. Univariate linear regression analysis demonstrated a significant association between cortical fAGEs and CSA in the psoas (ρ=0.220, P =0.039) but not in the PPM. Trabecular fAGEs revealed no significant associations to PPM or psoas musculature. In the multivariable analysis, higher cortical fAGEs were associated with increased FAT (β=1.556; P =0.002) and CSA (β=1.305; P =0.005) in the PPM after adjusting for covariates. Conclusion.: This is the first investigation demonstrating that an accumulation of nonenzymatic collagen cross-linking product fAGEs in cortical bone is associated with increased intramuscular fat in the lumbar paraspinal musculature. [ABSTRACT FROM AUTHOR]

The tumor microenvironment (TME) is now recognized as an important participant of tumor progression. As the most abundant extracellular matrix component in TME, collagen plays an important role in tumor development. The imaging study of collagen morphological feature in TME is of great significance for understanding the state of tumor. Multiphoton microscopy (MPM), based on second harmonic generation (SHG) and two-photon excitation fluorescence (TPEF), can be used to monitor the morphological changes of biological tissues without labeling. In this study, we used MPM for large-scale imaging of early invasive breast cancer from the tumor center to normal tissues far from the tumor. We found that there were significant differences in collagen morphology between breast cancer tumor boundary, near tumor transition region and normal tissues far from the tumor. Furthermore, the morphological feature of eight collagen fibers was extracted to quantify the variation trend of collagen in three regions. These results may provide a new perspective for the optimal negative margin width of breast-conserving surgery and the understanding of tumor metastasis. [ABSTRACT FROM AUTHOR]

Two-photon microscopy (TPM) plays an important role in the study of the changes of the two important components of neurovascular units (NVU) – neurons and blood vessels after ischemic stroke (IS). IS refers to sudden neurological dysfunction caused by focal cerebral ischemia, which is one of the leading causes of death and disability worldwide. TPM is a new and rapidly developing high-resolution real-time imaging technique used in vivo that has attracted increasing attention from scientists in the neuroscience field. Neurons and blood vessels are important components of neurovascular units, and they undergo great changes after IS to respond to and compensate for ischemic injury. Here, we introduce the characteristics and pre-imaging preparations of TPM, and review the common methods and latest progress of TPM in the neuronal and vascular research for injury and recovery of IS in recent years. With the review, we clearly recognized that the most important advantage of TPM in the study of ischemic stroke is the ability to perform chronic longitudinal imaging of different tissues at a high resolution in vivo. Finally, we discuss the limitations of TPM and the technological advances in recent years. [ABSTRACT FROM AUTHOR]

Bacterial infections associated with implanted medical devices represents a healthcare crisis due to their persistence, antibiotic tolerance, and immune avoidance. Indwelling devices are rapidly coated with host plasma and extracellular matrix proteins which can then be exploited by bacterial pathogens for adherence and subsequent biofilm development. Our understanding of the host-pathogen interface that determines the fate of biofilm-mediated infections is limited to the experimental models employed by laboratories studying these organisms. Current in vivo models of biofilm-mediated infection, while certainly useful, are typically limited to end-point analyses of bacterial burden enumeration, immune cell profiling, and cytokine/chemokine analysis. Thus, with these models, the complex, real-time assessment of biofilm development and innate immune cell activity remains imperceptible. Here, we describe a novel murine biofilm infection model employing time-lapse intravital multiphoton microscopy which permits concurrent and real-time visualization of Staphylococcus aureus biofilm formation and immune cell activity. Using cell tracking, we found that S. aureus biofilms impede neutrophil chemotaxis, redirecting their migration patterns to prevent biofilm invasion. This approach is the first to directly examine device-associated biofilm development and host-pathogen interactions and will serve to both further our understanding of infection development and help reveal the effects of future antibiofilm treatment strategies.

Abstract Background While aging is the main risk factor for Alzheimer´s disease (AD), emerging evidence suggests that metabolic alterations such as type 2 diabetes (T2D) are also major contributors. Indeed, several studies have described a close relationship between AD and T2D with clinical evidence showing that both diseases coexist. A hallmark pathological event in AD is amyloid-β (Aβ) deposition in the brain as either amyloid plaques or around leptomeningeal and cortical arterioles, thus constituting cerebral amyloid angiopathy (CAA). CAA is observed in 85–95% of autopsy cases with AD and it contributes to AD pathology by limiting perivascular drainage of Aβ. Methods To further explore these alterations when AD and T2D coexist, we have used in vivo multiphoton microscopy to analyze over time the Aβ deposition in the form of plaques and CAA in a relevant model of AD (APPswe/PS1dE9) combined with T2D (db/db). We have simultaneously assessed the effects of high-fat diet-induced prediabetes in AD mice. Since both plaques and CAA are implicated in oxidative-stress mediated vascular damage in the brain, as well as in the activation of matrix metalloproteinases (MMP), we have also analyzed oxidative stress by Amplex Red oxidation, MMP activity by DQ™ Gelatin, and vascular functionality. Results We found that prediabetes accelerates amyloid plaque and CAA deposition, suggesting that initial metabolic alterations may directly affect AD pathology. T2D significantly affects vascular pathology and CAA deposition, which is increased in AD-T2D mice, suggesting that T2D favors vascular accumulation of Aβ. Moreover, T2D synergistically contributes to increase CAA mediated oxidative stress and MMP activation, affecting red blood cell velocity. Conclusions Our data support the cross-talk between metabolic disease and Aβ deposition that affects vascular integrity, ultimately contributing to AD pathology and related functional changes in the brain microvasculature.

Abstract Background Cytokinins are a class of phytohormone that play a crucial role in the development of plants. They are involved in the regulation of nearly every aspect of plant growth, from germination to senescence. The role of cytokinins in many developmental programs is complex and varies both spatially and temporally. Current techniques used to investigate the functions of cytokinins in plant development lack this spatial and temporal resolution required to observe cell-type specific effects. Results To this end, we present a method of activating a caged cytokinin in single cells. A caged benzyladenine was synthesized, along with caged adenine as a negative control. In vitro testing confirmed ultraviolet light-mediated uncaging, and subsequent root growth assays demonstrated that uncaging produced a cytokinin phenotype. This uncaging was confined to single cells using multiphoton confocal microscopy. Using an Arabidopsis thaliana cytokinin reporter line expressing TCSn::GFP, the resulting GFP expression was confined to the uncaging region, including in single cells. This study presents a novel cell-targeted method of cytokinin delivery, which has the potential to elucidate a broad range of processes in plant development. Conclusions We combined multiphoton confocal microscopy and a caged cytokinin treatment, allowing cell type-specific uncaging of a cytokinin in Arabidopsis roots.

Abstract Background The intestine is rich in food-derived and microbe-derived antigens. Regulatory T cells (Tregs) are an essential T-cell population that prevents systemic autoimmune diseases and inhibits inflammation by encountering antigens. Previously, it was reported that the functional loss of Tregs induces systemic inflammation, including inflammatory bowel disease and graft-versus-host disease in human and murine models. However, there is a dearth of information about how Tregs localize in different tissues and suppress effector cells. Main body The development of Tregs and their molecular mechanism in the digestive tract have been elucidated earlier using murine genetic models, infectious models, and human samples. Tregs suppress immune and other nonimmune cells through direct effect and cytokine production. The recent development of in vivo imaging technology allows us to visualize how Tregs localize and move in the settings of inflammation and homeostasis. This is important because, according to a recent report, Treg characterization and function are regulated by their location. Tregs located in the proximal intestine and its draining lymph nodes induce tolerance against food antigens, and those located in the distal intestine suppress the inflammation induced by microbial antigens. Taken together, various Tregs are induced in a location-specific manner in the gastrointestinal tract and influence the homeostasis of the gut. Conclusion In this review, we summarize how Tregs are induced in the digestive tract and the application of in vivo Treg imaging to elucidate immune homeostasis in the digestive tract.

The exocrine pancreas secretes fluid and digestive enzymes in response to parasympathetic release of acetylcholine (ACh) via the vagus nerve and the gut hormone cholecystokinin (CCK). Both secretion of fluid and exocytosis of secretory granules containing enzymes and zymogens are dependent on an increase in the cytosolic [Ca2+] in acinar cells. It is thought that the specific spatiotemporal characteristics of the Ca2+ signals are fundamental for appropriate secretion and that these properties are disrupted in disease states in the pancreas. While extensive research has been performed to characterize Ca2+ signalling in acinar cells, this has exclusively been achieved in ex vivo preparations of exocrine cells, where it is difficult to mimic physiological conditions. Here we have developed a method to optically observe pancreatic acinar Ca2+ signals in vivo using a genetically expressed Ca2+ indicator and imaged with multi‐photon microscopy in live animals. In vivo, acinar cells exhibited baseline activity in fasted animals, which was dependent on CCK1 receptors (CCK1Rs). Both stimulation of intrinsic nervous input and administration of systemic CCK induced oscillatory activity in a proportion of the cells, but the maximum frequencies were vastly different. Upon feeding, oscillatory activity was also observed, which was dependent on CCK1Rs. No evidence of a vago‐vagal reflex mediating the effects of CCK was observed. Our in vivo method revealed the spatial and temporal profile of physiologically evoked Ca2+ signals, which will provide new insights into future studies of the mechanisms underlying exocrine physiology and that are disrupted in pathological conditions. Key points: In the exocrine pancreas, the spatiotemporal properties of Ca2+ signals are fundamentally important for the appropriate stimulation of secretion by the neurotransmitter acetylcholine and gut hormone cholecystokinin.These characteristics were previously defined in ex vivo studies. Here we report the spatiotemporal characteristics of Ca2+ signals in vivo in response to physiological stimulation in a mouse engineered to express a Ca2+ indicator in acinar cells.Specific Ca2+ 'signatures' probably important for stimulating secretion are evoked in vivo in fasted animals, by feeding, neural stimulation and cholecystokinin administration.The Ca2+ signals are probably the result of the direct action of ACh and CCK on acinar cells and not indirectly through a vago‐vagal reflex. [ABSTRACT FROM AUTHOR]

Over the last half century, the autofluorescence of the metabolic cofactors NADH (reduced nicotinamide adenine dinucleotide) and FAD (flavin adenine dinucleotide) has been quantified in a variety of cell types and disease states. With the spread of nonlinear optical microscopy techniques in biomedical research, NADH and FAD imaging has offered an attractive solution to noninvasively monitor cell and tissue status and elucidate dynamic changes in cell or tissue metabolism. Various tools and methods to measure the temporal, spectral, and spatial properties of NADH and FAD autofluorescence have been developed. Specifically, an optical redox ratio of cofactor fluorescence intensities and NADH fluorescence lifetime parameters have been used in numerous applications, but significant work remains to mature this technology for understanding dynamic changes in metabolism. This article describes the current understanding of our optical sensitivity to different metabolic pathways and highlights current challenges in the field. Recent progress in addressing these challenges and acquiring more quantitative information in faster and more metabolically relevant formats is also discussed. [ABSTRACT FROM AUTHOR]

Background: Noninvasive skin examination evolved rapidly in recent years, with multiphoton microscopy (MPM) and reflectance confocal microscopy (RCM) being used to image in‐vivo skin at high resolution. The aim of this study is to compare the imaging clarity between the two techniques and measure the thickness of the epidermis in different body sites. We also measured the degree of skin aging with noninvasive tools. Methods: Fifty‐six volunteers were evaluated and measured at three different body sites, including the cheek, volar forearm, and back. We used RCM and MPM to evaluate the clarity of each skin layer, including stratum corneum, stratum granulosum, stratum spinosum, dermo‐epidermal junction, and dermis. We measured epidermal thickness (ET) at the three body sites in individuals of different ages and genders. We assessed skin aging by the second harmonic to autofluorescence aging index of dermis (SAAID), and multiple linear regression was used to analyze the factors affecting SAAID. Results: MPM had advantages in observation of stratum granulosum, collagen fiber, and elastic fiber (p < 0.001), but RCM provided better observation in dermo‐epidermal junction layer (p < 0.001). The epidermis was thicker in the cheek area than the volar forearm and back in both RCM and MPM detection, and the average ET measured by MPM was lower than RCM. ET varied among the three body sites with significant differences (p < 0.05). ET was significantly lower at almost all sites in individuals above 40y (p < 0.05). SAAID decreased with age, and more rapidly in women. Cheeks have lower SAAID scores than other body sites. Conclusion: MPM and RCM provide noninvasive methods for imaging skin and each method has its own advantages. Epidermal thickness and SAAID correlated with age, gender, and different body sites. MPM could also assess the degree of skin aging, which could guide the clinical treatment of patients with diffferent ages and genders in the above body sites. [ABSTRACT FROM AUTHOR]

Basal cell carcinoma (BCC) is the most common skin cancer, and its incidence is rising. Millions of benign biopsies are performed annually for BCC diagnosis, increasing morbidity, and healthcare costs. Non‐invasive in vivo technologies such as multiphoton microscopy (MPM) can aid in diagnosing BCC, reducing the need for biopsies. Furthermore, the second harmonic generation (SHG) signal generated from MPM can classify and prognosticate cancers based on extracellular matrix changes, especially collagen type I. We explored the potential of MPM to differentiate collagen changes associated with different BCC subtypes compared to normal skin structures and benign lesions. Quantitative analysis such as frequency band energy analysis in Fourier domain, CurveAlign and CT‐FIRE fibre analysis was performed on SHG images from 52 BCC and 12 benign lesions samples. Our results showed that collagen distribution is more aligned surrounding BCCs nests compared to the skin's normal structures (p < 0.001) and benign lesions (p < 0.001). Also, collagen was orientated more parallelly surrounding indolent BCC subtypes (superficial and nodular) versus those with more aggressive behaviour (infiltrative BCC) (p = 0.021). In conclusion, SHG signal from type I collagen can aid not only in the diagnosis of BCC but could be useful for prognosticating these tumors. Our initial results are limited to a small number of samples, requiring large‐scale studies to validate them. These findings represent the groundwork for future in vivo MPM for diagnosis and prognosis of BCC. [ABSTRACT FROM AUTHOR]

Alopecia areata (AA) is a disease affecting mostly young patients, being the second most prevalent cause of alopecia. For the assessment of AA patients, numerous non-invasive techniques are available. For diagnosing and treating AA, non-invasive hair follicle exploration is crucial. Trichoscopy, videodermoscopy, pull test, global scalp photography and scalp imaging techniques (reflectance confocal microscopy (RCM), multiphoton microscopy (MPM), and high-frequency ultrasonography (HF-USG) are non-invasive methods used to assess the hair loss. Short vellus hairs and yellow dots were the most commonly reported and sensitive trichoscopy signs of AA. Additionally, it was observed that AA activity was associated with black dots, micro-exclamation mark hairs and tapering hairs. A good prognosis of AA includes the transformation of vellus into terminal hairs. Reflectance confocal microscopy is a non-invasive imaging technique for in vivo evaluation of the skin and observation of cell morphology with a nearly histological resolution, which is helpful in diagnosis and management of AA. Additionally, MPM delivers information on hair follicles and scalp inflammation and can be utilized to assess hair growth while undergoing treatment. High-frequency ultrasonography allows distinguishing between hair cycle phases, which is undoubtedly important from the prognostic of AA. The current review aims to discuss the non-invasive methods of diagnosis of AA. [ABSTRACT FROM AUTHOR]

Neoadjuvant treatment is often considered in breast cancer patients with axillary lymph node involvement, but most of patients do not have a pathologic complete response to therapy. The detection of residual nodal disease has a significant impact on adjuvant therapy recommendations which may improve survival. Here, we investigate whether multiphoton microscopy (MPM) could identify the pathological changes of axillary lymphatic metastasis after neoadjuvant chemotherapy in breast cancer. And furthermore, we find that there are obvious differences in seven collagen morphological features between normal node and residual axillary disease by combining with a semi‐automatic image processing method, and also find that there are significant differences in four collagen features between the effective and no‐response treatment groups. These research results indicate that MPM may help estimate axillary treatment response in the neoadjuvant setting and thereby tailor more appropriate and personalized adjuvant treatments for breast cancer patients. [ABSTRACT FROM AUTHOR]

The tumor microenvironment (TME) is now recognized as an important participant of tumor progression. As the most abundant extracellular matrix component in TME, collagen plays an important role in tumor development. The imaging study of collagen morphological feature in TME is of great significance for understanding the state of tumor. Multiphoton microscopy (MPM), based on second harmonic generation (SHG) and two-photon excitation fluorescence (TPEF), can be used to monitor the morphological changes of biological tissues without labeling. In this study, we used MPM for large-scale imaging of early invasive breast cancer from the tumor center to normal tissues far from the tumor. We found that there were significant differences in collagen morphology between breast cancer tumor boundary, near tumor transition region and normal tissues far from the tumor. Furthermore, the morphological feature of eight collagen fibers was extracted to quantify the variation trend of collagen in three regions. These results may provide a new perspective for the optimal negative margin width of breast-conserving surgery and the understanding of tumor metastasis.

An analysis of the membrane organization and intracellular trafficking of lipids often relies on multiphoton (MP) and super-resolution microscopy of fluorescent lipid probes. A disadvantage of particularly intrinsically fluorescent lipid probes, such as the cholesterol and ergosterol analogue, dehydroergosterol (DHE), is their low MP absorption cross-section, resulting in a low signal-to-noise ratio (SNR) in live-cell imaging. Stimulated emission depletion (STED) microscopy of membrane probes like Nile Red enables one to resolve membrane features beyond the diffraction limit but exposes the sample to a lot of excitation light and suffers from a low SNR and photobleaching. Here, dynamic mode decomposition (DMD) and its variant, higher-order DMD (HoDMD), are applied to efficiently reconstruct and denoise the MP and STED microscopy data of lipid probes, allowing for an improved visualization of the membranes in cells. HoDMD also allows us to decompose and reconstruct two-photon polarimetry images of TopFluor-cholesterol in model and cellular membranes. Finally, DMD is shown to not only reconstruct and denoise 3D-STED image stacks of Nile Red-labeled cells but also to predict unseen image frames, thereby allowing for interpolation images along the optical axis. This important feature of DMD can be used to reduce the number of image acquisitions, thereby minimizing the light exposure of biological samples without compromising image quality. Thus, DMD as a computational tool enables gentler live-cell imaging of fluorescent probes in cellular membranes by MP and STED microscopy.

The intrinsic fluorescence of bacterial samples has a proven potential for label-free bacterial characterization, monitoring bacterial metabolic functions, and as a mechanism for tracking the transport of relevant components through vesicles. The reduced scattering and axial confinement of the excitation offered by multiphoton imaging can be used to overcome some of the limitations of single-photon excitation (e.g., scattering and out-of-plane photobleaching) to the imaging of bacterial communities. In this work, we demonstrate in vivo multi-photon microscopy imaging of Streptomyces bacterial communities, based on the excitation of blue endogenous fluorophores, using an ultrafast Yb-fiber laser amplifier. Its parameters, such as the pulse energy, duration, wavelength, and repetition rate, enable in vivo multicolor imaging with a single source through the simultaneous two- and three-photon excitation of different fluorophores. Three-photon excitation at 1040 nm allows fluorophores with blue and green emission spectra to be addressed (and their corresponding ultraviolet and blue single-photon excitation wavelengths, respectively), and two-photon excitation at the same wavelength allows fluorophores with yellow, orange, or red emission spectra to be addressed (and their corresponding green, yellow, and orange single-photon excitation wavelengths). We demonstrate that three-photon excitation allows imaging over a depth range of more than 6 effective attenuation lengths to take place, corresponding to an 800 micrometer depth of imaging, in samples with a high density of fluorescent structures.

ObjectivesNon-invasive visualization of hair follicles is important for proper diagnosis and management of alopecia; however, histological assessment remains the gold standard. Laser imaging technologies have made possible noninvasive in vivo evaluation of skin and hair follicle. The aim of this study was to evaluate the ability of multiphoton microscopy (MPM) to non-invasively identify morphological features that can distinguish scarring from non-scarring alopecia.MethodsMPM images were obtained from areas on the scalp affected by alopecia. Investigators blinded to the diagnosis analyzed hair follicle and shaft sizes. Patients were recruited and imaged at the UC Irvine Health Medical Center and the University of California, Irvine Beckman Laser Institute. Patients with androgenetic alopecia (AGA) and alopecia areata (AA), and scarring alopecia, in particular frontal fibrosing alopecia (FFA) were recruited and imaged from July 2016 to July 2017.ResultsWe imaged 5 normal scalp subjects and 12 patients affected by non-scarring (7 subjects) and scarring (5 subjects) alopecia. In normal and non-scarring alopecia patients, MPM identified presence of sebaceous glands associated with hair follicles. MPM images of scarring alopecia were characterized by the presence of inflammatory cells surrounding hair follicles. Measurements of hair follicle diameter sizes were found to be significantly smaller in scarring alopecia patients compared to normal (P

Abstract The development of optical organic nanoparticles (NPs) is desirable and widely studied. However, most organic dyes are water-insoluble such that the derivatization and modification of these dyes are difficult. Herein, we demonstrated a simple platform for the fabrication of organic NPs designed with emissive properties by loading ten different organic dyes (molar masses of 479.1–1081.7 g/mol) into water-soluble polymer nanosponges composed of poly(styrene-alt-maleic acid) (PSMA). The result showed a substantial improvement over the loading of commercial dyes (3.7–50% loading) while preventing their spontaneous aggregation in aqueous solutions. This packaging strategy includes our newly synthesized organic dyes (> 85% loading) designed for OPVs (242), DSSCs (YI-1, YI-3, YI-8), and OLEDs (ADF-1–3, and DTDPTID) applications. These low-cytotoxicity organic NPs exhibited tunable fluorescence from visible to near-infrared (NIR) emission for cellular imaging and biological tracking in vivo. Moreover, PSMA NPs loaded with designed NIR-dyes were fabricated, and photodynamic therapy with these dye-loaded PSMA NPs for the photolysis of cancer cells was achieved when coupled with 808 nm laser excitation. Indeed, our work demonstrates a facile approach for increasing the biocompatibility and stability of organic dyes by loading them into water-soluble polymer-based carriers, providing a new perspective of organic optoelectronic materials in biomedical theranostic applications.

Simple Summary: Efforts to identify lesions of head and neck cancers with high malignant potential are important to improve patient outcomes, as 5-year survival rates remain low due to late detection. Optical imaging approaches that provide direct cellular and structural atypia could be helpful in detection and pathology guidance. This study evaluates the method of multiphoton autofluorescence microscopy (MPAM) for its ability to reveal atypia associated with neoplasia in resected tumor samples without the need for exogenous dyes or tissue sectioning, showing a correlation with the corresponding histology. Depth-resolved label-free optical imaging by the method of multiphoton autofluorescence microscopy (MPAM) may offer new ways to examine cellular and extracellular atypia associated with epithelial squamous cell carcinoma (SCC). MPAM was evaluated for its ability to identify cellular and microstructural atypia in head and neck tissues from resected discarded tumor tissue. Three-dimensional image volumes were obtained from tissues from the floor of the mouth, tongue, and larynx, and were then processed for histology. MPAM micrographs were evaluated for qualitative metrics of cell atypia and quantitative measures associated with nuclear pleomorphism. Statistical analyses correlated MPAM endpoints with histological grade from each imaged site. Cellular overcrowding, discohesion, anisonucleosis, and multinucleated cells, as observed through MPAM, were found to be statistically associated with dysplasia and SCC grading, but not in histologically benign regions. A quantitative measure of the coefficient of variance in nuclear size in SCC and dysplasia was statistically elevated above histologically benign regions. MPAM also allowed for the identification of cellular heterogeneity across transitional areas and other features, such as inflammatory infiltrates. In the future, MPAM could be evaluated for the non-invasive detection of neoplasia, possibly as an adjunct to traditional conventional examination and biopsy. [ABSTRACT FROM AUTHOR]

Pre-eclampsia is a severe placenta-related complication of pregnancy with limited early diagnostic and therapeutic options. Aetiological knowledge is controversial, and there is no universal consensus on what constitutes the early and late phenotypes of pre-eclampsia. Phenotyping of native placental three-dimensional (3D) morphology offers a novel approach to improve our understanding of the structural placental abnormalities in pre-eclampsia. Healthy and pre-eclamptic placental tissues were imaged with multiphoton microscopy (MPM). Imaging based on inherent signal (collagen, and cytoplasm) and fluorescent staining (nuclei, and blood vessels) enabled the visualization of placental villous tissue with subcellular resolution. Images were analysed with a combination of open source (FIJI, VMTK, Stardist, MATLAB, DBSCAN), and commercially (MATLAB) available software. Trophoblast organization, 3D-villous tree structure, syncytial knots, fibrosis, and 3D-vascular networks were identified as quantifiable imaging targets. Preliminary data indicate increased syncytial knot density with characteristic elongated shape, higher occurrence of paddle-like villous sprouts, abnormal villous volume-to-surface ratio, and decreased vascular density in pre-eclampsia compared to control placentas. The preliminary data presented indicate the potential of quantifying 3D microscopic images for identifying different morphological features and phenotyping pre-eclampsia in placental villous tissue. [ABSTRACT FROM AUTHOR]

Corticosteroids, although highly effective for the treatment of both anterior and posterior ocular segment inflammation, still nowadays struggle for effective drug delivery due to their poor solubilization capabilities in water. This research work aims to develop nanostructured lipid carriers (NLC) intended for periocular administration of dexamethasone acetate to the posterior segment of the eye. Pre-formulation studies were initially performed to find solid and liquid lipid mixtures for dexamethasone acetate solubilization. Pseudoternary diagrams at 65 °C were constructed to select the best surfactant based on the macroscopic transparency and microscopic isotropy of the systems. The resulting NLC, obtained following an organic solvent-free methodology, was composed of triacetin, Imwitor® 491 (glycerol monostearate >90%) and tyloxapol with Z-average = 106.9 ± 1.2 nm, PDI = 0.104 ± 0.019 and zeta potential = −6.51 ± 0.575 mV. Ex vivo porcine sclera and choroid permeation studies revealed a considerable metabolism in the sclera of dexamethasone acetate into free dexamethasone, which demonstrated higher permeation capabilities across both tissues. In addition, the NLC behavior once applied onto the sclera was further studied by means of multiphoton microscopy by loading the NLC with the fluorescent probe Nile red. [ABSTRACT FROM AUTHOR]

Rose Bengal (RB) is a fluorescent dye with several potential biomedical applications, particularly in dermatology. Due to RB's poor physicochemical properties, several advanced delivery systems have been developed as a potential tool to promote its permeation across the skin. Nevertheless, no validated quantitative method to analyse RB within the skin is described in the literature. Considering RB exhibits a conjugated ring system, the current investigation proposes fluorescence-based techniques beneficial for qualitatively and quantitatively determining RB delivered to the skin. Notably, the development and validation of a fluorescence-coupled HPLC method to quantify RB within the skin matrix are herein described for the first time. The method was validated based on the ICH, FDA and EMA guidelines, and the validated parameters included specificity, linearity, LOD, LLOQ, accuracy and precision, and carry-over and dilution integrity. Finally, the method was applied to evaluate RB's ex vivo permeation and deposition profiles when loaded into dermatological formulations. Concerning qualitative determination, multiphoton microscopy was used to track the RB distribution within the skin strata, and fluorescence emission spectra were investigated to evaluate RB's behaviour when interacting with different environments. The analytical method proved specific, precise, accurate and sensitive to analyse RB in the skin. In addition, qualitative side-analytical techniques were revealed to play an essential role in evaluating the performance of RB's dermatological formulation. [ABSTRACT FROM AUTHOR]

To reduce the risk of post-hepatectomy liver failure in patients with hepatic pathologies, it is necessary to develop an approach to express the intraoperative assessment of the liver's regenerative potential. Traditional clinical methods do not enable the prediction of the function of the liver remnant. Modern label-free bioimaging, using multiphoton microscopy in combination with second harmonic generation (SHG) and fluorescence lifetime imaging microscopy (FLIM), can both expand the possibilities for diagnosing liver pathologies and for assessing the regenerative potential of the liver. Using multiphoton and SHG microscopy, we assessed the structural state of liver tissue at different stages of induced steatosis and fibrosis before and after 70% partial hepatectomy in rats. Using FLIM, we also performed a detailed analysis of the metabolic state of the hepatocytes. We were able to determine criteria that can reveal a lack of regenerative potential in violated liver, such as the presence of zones with reduced NAD(P)H autofluorescence signals. Furthermore, for a liver with pathology, there was an absence of the jump in the fluorescence lifetime contributions of the bound form of NADH and NADPH the 3rd day after hepatectomy that is characteristic of normal liver regeneration. Such results are associated with decreased intensity of oxidative phosphorylation and of biosynthetic processes in pathological liver, which is the reason for the impaired liver recovery. This modern approach offers an effective tool that can be successfully translated into the clinic for express, intraoperative assessment of the regenerative potential of the pathological liver of a patient. [ABSTRACT FROM AUTHOR]

Layer V neurons in the primary motor cortex (M1) are important for motor skill learning. Since pretreatment of either CNQX or APV in rat M1 layer V impaired rotor rod learning, we analysed training‐induced synaptic plasticity by whole‐cell patch‐clamp technique in acute brain slices. Rats trained for 1 day showed a decrease in small inhibitory postsynaptic current (mIPSC) frequency and an increase in the paired‐pulse ratio of evoked IPSCs, suggesting a transient decrease in presynaptic GABA release in the early phase. Rats trained for 2 days showed an increase in miniature excitatory postsynaptic current (mEPSC) amplitudes/frequency and elevated AMPA/NMDA ratios, suggesting a long‐term strengthening of AMPA receptor‐mediated excitatory synapses. Importantly, rotor rod performance in trained rats was correlated with the mean mEPSC amplitude and the frequency obtained from that animal. In current‐clamp analysis, 1‐day‐trained rats transiently decreased the current‐induced firing rate, while 2‐day‐trained rats returned to pre‐training levels, suggesting dynamic changes in intrinsic properties. Furthermore, western blot analysis of layer V detected decreased phosphorylation of Ser408–409 in GABAA receptor β3 subunits in 1‐day‐trained rats, and increased phosphorylation of Ser831 in AMPA receptor GluA1 subunits in 2‐day‐trained rats. Finally, live‐imaging analysis of Thy1‐YFP transgenic mice showed that the training rapidly recruited a substantial number of spines for long‐term plasticity in M1 layer V neurons. Taken together, these results indicate that motor training induces complex and diverse plasticity in M1 layer V pyramidal neurons. Key points: Here we examined motor training‐induced synaptic and intrinsic plasticity of layer V pyramidal neurons in the primary motor cortex.The training reduced presynaptic GABA release in the early phase, but strengthened AMPA receptor‐mediated excitatory synapses in the later phase: acquired motor performance after training correlated with the strength of excitatory synapses rather than inhibitory synapses.As to the intrinsic property, the training transiently decreased the firing rate in the early phase, but returned to pre‐training levels in the later phase.Western blot analysis detected decreased phosphorylation of Ser408‐409 in GABAA receptor β3 subunits in the acute phase, and increased phosphorylation of Ser831 in AMPA receptor GluA1 subunits in the later phase.Live‐imaging analysis of Thy1‐YFP transgenic mice showed rapid and long‐term spine plasticity in M1 layer V neurons, suggesting training‐induced increases in self‐entropy per spine. [ABSTRACT FROM AUTHOR]

Multi-photon microscopy (MPM) and coherent anti-Stokes Raman scattering (CARS) are two advanced nonlinear optical imaging techniques, which provide complementary information and have great potential in combination for noninvasive in vivo biomedical applications. This paper provides a detailed discussion of the basics, development and applications of these technologies for in vivo skin research, covering the following topics: The principle and advantage of MPM and CARS, instrumentation development for in vivo applications, MPM and CARS of normal skin, application of MPM and CARS in skin cancer and disease diagnosis; application of MPM in skin disease intervention, i.e., imaging guided two-photon photothermolysis. [ABSTRACT FROM AUTHOR]

Multiphoton microscopy (MPM) has emerged as a vital tool in neuroscience, enabling deeper imaging with a broader field of view, as well as faster and sub-cellular resolution. Recent innovations in ultrafast fiber laser technology have revolutionized MPM applications in living brains, offering advantages like cost-effectiveness and user-friendliness. In this review, we explore the progress in ultrafast fiber laser technology, focusing on its integration into MPM for neuroscience research. We also examine the utility of femtosecond fiber lasers in fluorescence and label-free two- and three-photon microscopy applications within the field. Furthermore, we delve into future possibilities, including next-generation fiber laser designs, novel laser characteristics, and their potential for achieving high spatial and temporal resolution imaging. We also discuss the integration of fiber lasers with implanted microscopes, opening doors for clinical and fundamental neuroscience investigations.

Rheumatoid arthritis is a chronic form of arthritis that causes bone destruction in joints such as the knees and fingers. Over the past two decades, the clinical outcomes of rheumatoid arthritis have improved substantially with the development of biological agents and Janus kinase inhibitors. Osteoclasts are myeloid lineage cells with a unique bone-destroying ability that can lead to joint destruction. On the other hand, osteoclasts play an important role in skeletal homeostasis by supporting bone remodeling together with osteoblasts in the bone marrow under steady-state conditions. However, the same osteoclasts are considered to participate in physiological bone remodeling and joint destruction. We found that pathological osteoclasts have different differentiation pathways and regulatory transcription factors compared to physiological osteoclasts. We also identified arthritis-associated osteoclastogenic macrophages (AtoMs), which are common progenitors of pathological osteoclasts in mice and humans that develop specifically in inflamed synovial tissue. This review presents details of the newly identified AtoMs and the original intravital imaging systems that can visualize synovial tissue and pathological osteoclasts at the pannus–bone interface.

Hilar cholangiocarcinoma (HCC) is a common malignant tumor of the biliary system. The structural characteristics of the bile duct tissue can reflect the changes in its function. The visualization of these specific features is of special significance for understanding the degree of invasion of HCC and tumor borders. Radical R0 resection is the only cure for HCC. Currently, the commonly used medical imaging diagnostic methods can only provide a rough tumor range and require a histopathological analysis. Multiphoton microscopy (MPM) not only has an ultra-high spatial resolution but is also extremely sensitive to collagen fibers with noncentrosymmetric structures. In this study, MPM is applied to image the boundaries of HCC tumors. First, the experimental results show that MPM can clearly reveal the existence of residual cancer cells at the surgical margin. Second, the density of the collagen fibers and the dispersion of the 2D direction angle of the collagen fibers of normal and cancerous tissues are further calculated quantitatively. The collagen fiber signals before and after cancer cell invasion into the different tissues are found to be different. Finally, through a logistic regression prediction curve, combined with the collagen fiber density and 2D direction angle as indicators, it is further judged whether the resection margin is negative, which is closely related to the prognosis of HCC. The experimental results indicate that MPM imaging can serve as a new tool for diagnosing whether the HCC tumor boundary reaches R0 resection.

Multi-photon microscopy (MPM) and coherent anti-Stokes Raman scattering (CARS) are two advanced nonlinear optical imaging techniques, which provide complementary information and have great potential in combination for noninvasive in vivo biomedical applications. This paper provides a detailed discussion of the basics, development and applications of these technologies for in vivo skin research, covering the following topics: The principle and advantage of MPM and CARS, instrumentation development for in vivo applications, MPM and CARS of normal skin, application of MPM and CARS in skin cancer and disease diagnosis; application of MPM in skin disease intervention, i.e., imaging guided two-photon photothermolysis.

Optical spectroscopy and microscopy techniques are widely used for basic studies of living systems. However, their application in clinical practice has two fundamental limitations. First, the depth of probing biological tissues with light is small and varies from tenths to several of millimeters. Secondly, it is difficult to use exogenous labels, which increase the sensitivity and specificity of pathological tissue detection, in vivo measurements on patients. This raises the question of the place of biomedical photonics among other physical diagnostic methods used in clinical practice. This article presents an review of optical methods and relatively new certified commercially available medical devices that use photonics to solve intraoperative diagnostic problems, i.e., the discrimination between pathological and healthy tissue sites in vivo and ex vivo by an endogenous optical response. This work discusses a wide range of medical fields in which researchers and engineers have been able to achieve high rates of sensitivity and specificity in solving the problem of classifying such tissues. The advantages and disadvantages of optical imaging and diagnostic methods, which determine their place in clinical practice, are discussed by the example of intraoperative diagnostics. [ABSTRACT FROM AUTHOR]

Multiscale neurophysiology reveals that simple motor actions are associated with changes in neuronal firing in virtually every brain region studied. Accordingly, the assessment of focal pathology such as stroke or progressive neurodegenerative diseases must also extend widely across brain areas. To derive mechanistic information through imaging, multiple resolution scales and multimodal factors must be included, such as the structure and function of specific neurons and glial cells and the dynamics of specific neurotransmitters. Emerging multiscale methods in preclinical animal studies that span micro- to macroscale examinations fill this gap, allowing a circuit-based understanding of pathophysiological mechanisms. Combined with high-performance computation and open-source data repositories, these emerging multiscale and large field-of-view techniques include live functional ultrasound, multi- and single-photon wide-scale light microscopy, video-based miniscopes, and tissue-penetrating fiber photometry, as well as variants of post-mortem expansion microscopy. We present these technologies and outline use cases and data pipelines to uncover new knowledge within animal models of stroke, Alzheimer's disease, and movement disorders. Preclinical multiscale imaging and electrophysiological technology enable exploration of the distributed functional connectivity that underlies symptomology and explain the pathophysiology of neurological disease. These emerging technologies and example use cases are presented in the context of stroke and neurological disorders. [ABSTRACT FROM AUTHOR]

Multiphoton microscopy (MPM) imaging relies on the nonlinear interaction between ultrashort optical pulses and the samples to achieve image contrast. Featuring larger penetration depth, less phototoxicity, 3-dimensional sectioning capability, no need for labeling, MPM become a powerful medical imaging technique that can identify structural characteristics of tissues at the cellular and subcellular levels. In this review paper, we introduce the working principle of MPM imaging, present the current results of MPM imaging applied to the study of gastric tumors, and discuss the future prospects of this interdisciplinary research field. [ABSTRACT FROM AUTHOR]

Background: Cytokinins are a class of phytohormone that play a crucial role in the development of plants. They are involved in the regulation of nearly every aspect of plant growth, from germination to senescence. The role of cytokinins in many developmental programs is complex and varies both spatially and temporally. Current techniques used to investigate the functions of cytokinins in plant development lack this spatial and temporal resolution required to observe cell-type specific effects. Results: To this end, we present a method of activating a caged cytokinin in single cells. A caged benzyladenine was synthesized, along with caged adenine as a negative control. In vitro testing confirmed ultraviolet light-mediated uncaging, and subsequent root growth assays demonstrated that uncaging produced a cytokinin phenotype. This uncaging was confined to single cells using multiphoton confocal microscopy. Using an Arabidopsis thaliana cytokinin reporter line expressing TCSn::GFP, the resulting GFP expression was confined to the uncaging region, including in single cells. This study presents a novel cell-targeted method of cytokinin delivery, which has the potential to elucidate a broad range of processes in plant development. Conclusions: We combined multiphoton confocal microscopy and a caged cytokinin treatment, allowing cell type-specific uncaging of a cytokinin in Arabidopsis roots. [ABSTRACT FROM AUTHOR]

Background: While aging is the main risk factor for Alzheimer´s disease (AD), emerging evidence suggests that metabolic alterations such as type 2 diabetes (T2D) are also major contributors. Indeed, several studies have described a close relationship between AD and T2D with clinical evidence showing that both diseases coexist. A hallmark pathological event in AD is amyloid-β (Aβ) deposition in the brain as either amyloid plaques or around leptomeningeal and cortical arterioles, thus constituting cerebral amyloid angiopathy (CAA). CAA is observed in 85–95% of autopsy cases with AD and it contributes to AD pathology by limiting perivascular drainage of Aβ. Methods: To further explore these alterations when AD and T2D coexist, we have used in vivo multiphoton microscopy to analyze over time the Aβ deposition in the form of plaques and CAA in a relevant model of AD (APPswe/PS1dE9) combined with T2D (db/db). We have simultaneously assessed the effects of high-fat diet-induced prediabetes in AD mice. Since both plaques and CAA are implicated in oxidative-stress mediated vascular damage in the brain, as well as in the activation of matrix metalloproteinases (MMP), we have also analyzed oxidative stress by Amplex Red oxidation, MMP activity by DQ™ Gelatin, and vascular functionality. Results: We found that prediabetes accelerates amyloid plaque and CAA deposition, suggesting that initial metabolic alterations may directly affect AD pathology. T2D significantly affects vascular pathology and CAA deposition, which is increased in AD-T2D mice, suggesting that T2D favors vascular accumulation of Aβ. Moreover, T2D synergistically contributes to increase CAA mediated oxidative stress and MMP activation, affecting red blood cell velocity. Conclusions: Our data support the cross-talk between metabolic disease and Aβ deposition that affects vascular integrity, ultimately contributing to AD pathology and related functional changes in the brain microvasculature. [ABSTRACT FROM AUTHOR]

Hilar cholangiocarcinoma (HCC) is a common malignant tumor of the biliary system. The structural characteristics of the bile duct tissue can reflect the changes in its function. The visualization of these specific features is of special significance for understanding the degree of invasion of HCC and tumor borders. Radical R0 resection is the only cure for HCC. Currently, the commonly used medical imaging diagnostic methods can only provide a rough tumor range and require a histopathological analysis. Multiphoton microscopy (MPM) not only has an ultra-high spatial resolution but is also extremely sensitive to collagen fibers with noncentrosymmetric structures. In this study, MPM is applied to image the boundaries of HCC tumors. First, the experimental results show that MPM can clearly reveal the existence of residual cancer cells at the surgical margin. Second, the density of the collagen fibers and the dispersion of the 2D direction angle of the collagen fibers of normal and cancerous tissues are further calculated quantitatively. The collagen fiber signals before and after cancer cell invasion into the different tissues are found to be different. Finally, through a logistic regression prediction curve, combined with the collagen fiber density and 2D direction angle as indicators, it is further judged whether the resection margin is negative, which is closely related to the prognosis of HCC. The experimental results indicate that MPM imaging can serve as a new tool for diagnosing whether the HCC tumor boundary reaches R0 resection. [ABSTRACT FROM AUTHOR]

Fibrous meningiomas, a common type of brain tumor, present surgical challenges due to their variable hardness, which is crucial for complete resection and patient prognosis. This study explores the use of label-free multiphoton microscopy (MPM) for the objective assessment of the texture of fibrous meningiomas. Fresh tumor samples from 20 patients were analyzed using both multichannel and lambda mode MPM, with quantitative image analysis algorithms determining collagen content and multi-peak spectral fitting providing additional optical collagen metrics. The study compared medium and hard fibrous meningiomas, utilizing receiver operating characteristic analysis to evaluate predictive performance. Microstructural features were clearly visualized, enabling accurate diagnosis. Collagen-related parameters significantly differentiated between moderate and hard tumors (p < 0.05). High predictive values were observed for collagen content, collagen-to-NADH-free ratio, and collagen-to-FAD ratio (AUC = 0.748-0.839). A multivariate logistic model combining these biomarkers significantly improved diagnostic accuracy (AUC = 0.907). The findings suggest that MPM, with its ability to visualize and quantify microstructures such as collagen and cells without the need for staining, holds strong potential for rapid, objective, and accurate assessment of tumor texture during neurosurgery. The integration of MPM with multiphoton endoscopy paves the way for potential in vivo applications in the future., (© 2024 Wiley‐VCH GmbH.)

Phyllodes tumors (PTs) are rare breast stroma neoplasms, and their accurate identification at various stages is essential for personalized patient treatment. In this study, multiphoton microscopy (MPM) with two-photon excited fluorescence (TPEF) and second harmonic generation (SHG) imaging was used for label-free detection and differentiation of PTs and normal breast tissue. An automated image processing strategy was developed to quantify changes in collagen fiber morphology within the stroma and boundary of PTs, establishing optical diagnostic characteristics of PTs using MPM. The results demonstrated that MPM could be used for the detection of different stages of PTs, and the morphological alterations in collagen fibers could serve as critical indicators of PT malignancy, offering new insights for the diagnosis and grading of benign, borderline, and malignant PTs. It lays the groundwork for the future application of compact MPM for the rapid detection and diagnosis of PTs., (© 2024 Wiley‐VCH GmbH.)

Lymph-node status is important in decision-making during early gastric cancer (EGC) treatment. Currently, endoscopic submucosal dissection is the mainstream treatment for EGC. However, it is challenging for even experienced endoscopists to accurately diagnose and treat EGC. Multiphoton microscopy can extract the morphological features of collagen fibers from tissues. The characteristics of collagen fibers can be used to assess the lymph-node metastasis status in patients with EGC. First, we compared the accuracy of four deep learning models (VGG16, ResNet34, MobileNetV2, and PVTv2) in training preprocessed images and test datasets. Next, we integrated the features of the best-performing model, which was PVTv2, with manual and clinical features to develop a novel model called AutoLNMNet. The prediction accuracy of AutoLNMNet for the no metastasis (Ly0) and metastasis in lymph nodes (Ly1) stages reached 0.92, which was 0.3% higher than that of PVTv2. The receiver operating characteristics of AutoLNMNet in quantifying Ly0 and Ly1 stages were 0.97 and 0.97, respectively. Therefore, AutoLNMNet is highly reliable and accurate in detecting lymph-node metastasis, providing an important tool for the early diagnosis and treatment of EGC., (© 2024 Wiley Periodicals LLC.)

High-resolution visualization of the deep brain is still a challenging and very significant issue. Multiphoton microscopy (MPM) holds great promise for high-spatiotemporal deep-tissue imaging under NIR-III and NIR-IV excitation. However, thus far, their applications have been seriously restricted by the scarcity of efficient organic probes. Herein, we designed and synthesized two donor-acceptor-donor-type conjugated small molecules ( TNT and TNS ) for in vivo mouse deep-brain imaging with three- and four-photon microscopy under 1700 and 2200 nm excitation. With a selenium (Se) substitution, we synthesized two conjugated small molecules to promote their emission into the deep near-infrared region with high quantum yields of 55% and 20% in THF solvent, respectively, and their water-dispersive nanoparticles have relatively large absorption cross-sections in the 1700 and 2200 nm windows, respectively, with good biosafety. With these superiorities, these organic NPs achieve high-resolution deep-brain imaging via three-photon and four-photon microscopy with excitation at 1700 and 2200 nm windows, and 1620 μm deep in the brain vasculature can be visualized in vivo . This study demonstrates the efficiency of NIR-emissive conjugated small molecules for high-performance MPM imaging in the NIR-III and NIR-IV window and provides a route for the future design of organic MPM probes.

The pre-clinical validation of cell therapies requires monitoring the biodistribution of transplanted cells in tissues of host organisms. Real-time detection of these cells in the circulatory system and identification of their aggregation state is a crucial piece of information, but necessitates deep penetration and fast imaging with high selectivity, subcellular resolution, and high throughput. In this study, multiphoton-based in-flow detection of human stem cells in whole, unfiltered blood is demonstrated in a microfluidic channel. The approach relies on a multiphoton microscope with diffractive scanning in the direction perpendicular to the flow via a rapidly wavelength-swept laser. Stem cells are labeled with metal oxide harmonic nanoparticles. Thanks to their strong and quasi-instantaneous second harmonic generation (SHG), an imaging rate in excess of 10 000 frames per second is achieved with pixel dwell times of 1 ns, a duration shorter than typical fluorescence lifetimes yet compatible with SHG. Through automated cell identification and segmentation, morphological features of each individual detected event are extracted and cell aggregates are distinguished from isolated cells. This combination of high-speed multiphoton microscopy and high-sensitivity SHG nanoparticle labeling in turbid media promises the detection of rare cells in the bloodstream for assessing novel cell-based therapies., (© 2024 The Author(s). Small published by Wiley‐VCH GmbH.)