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Confocal Raman microscopy provides composition and constitution of label-free samples at high spatial-resolution. In this study, the histology of atherosclerotic arteries was measured using a custom-built confocal Raman microscopy that was developed to improve imaging speed, diffraction efficiency, and spectral resolution. Additionally, the machine learning method was used to enhance the accuracy of classification. Results show that each layer of arteries and lipid-rich inflamed plaque can be successfully characterized. Further details can be found in the article by Jingchao Xing, Dong-Ryoung Lee, Jin Won Kim, and Hongki Yoo ( e202200243 ).

     

Fast denoising and lossless spectrum extraction in stimulated Raman scattering microscopy

Stimulated Raman scattering (SRS) microscopy is a nonlinear optical imaging method for visualizing chemical content based on molecular vibrational bonds. However, the imaging speed and sensitivity are currently limited by the noise of the light beam probing the Raman process. In this paper, we present a fast non-average denoising and high-precision Raman shift extraction method, based on a self-reinforcing signal-to-noise ratio (SNR) enhancement algorithm, for SRS spectroscopy and microscopy. We compare the results of this method with the filtering methods and the reported experimental methods to demonstrate its high efficiency and high precision in spectral denoising, Raman peak extraction and image quality improvement. We demonstrate a maximum SNR enhancement of 10.3 dB in fixed tissue imaging and 11.9 dB in vivo imaging. This method reduces the cost and complexity of the SRS system and allows for high-quality SRS imaging without use of special laser, complicated system design and Raman tags.     

Fast and sensitive delineation of brain tumor with clinically compatible moxifloxacin labeling and confocal microscopy

Delineation of brain tumor margins during surgery is critical to maximize tumor removal while preserving normal brain tissue to obtain optimal clinical outcomes. Although various imaging methods have been developed, they have limitations to be used in clinical practice. We developed a high‐speed cellular imaging method by using clinically compatible moxifloxacin and confocal microscopy for sensitive brain tumor detection and delineation. Moxifloxacin is a Food and Drug Administration (FDA) approved antibiotic and was used as a cell labeling agent through topical administration. Its strong fluorescence at short visible excitation wavelengths allowed video‐rate cellular imaging. Moxifloxacin‐based confocal microscopy (MBCM) was characterized in normal mouse brain specimens and visualized their cytoarchitecture clearly. Then, MBCM was applied to both brain tumor murine models and two malignant human brain tumors of glioblastoma and metastatic cancer. MBCM detected tumors in all the specimens by visualizing dense and irregular cell distributions, and tumor margins were easily delineated based on the cytoarchitecture. An image analysis method was developed for automated detection and delineation. MBCM demonstrated sensitive delineation of brain tumors through cytoarchitecture visualization and would have potentials for human applications, such as a surgery‐guiding method for tumor removal.      

Detection of oral squamous cell carcinoma with ex vivo fluorescence confocal microscopy: Sensitivity and specificity compared to histopathology

Real‐time microscopic imaging of freshly excised tissue enables a rapid bedside‐pathology. A possible application of interest is the detection of oral squamous cell carcinomas (OSCCs). The aim of this study was to analyze the sensitivity and specificity of ex vivo fluorescence confocal microscopy (FCM) for OSCCs and to compare confocal images visually and qualitatively with gold standard histopathology. Two hundred eighty ex vivo FCM images were prospectively collected and evaluated immediately after excision. Every confocal image was blindly assessed for the presence or absence of malignancy by two clinicians and one pathologist. The results were compared with conventional histopathology with hematoxylin and eosin staining. OSCCs were detected with a very high sensitivity of 0.991, specificity of 0.9527, positive predictive value of 0.9322 and negative predictive value of 0.9938. The results demonstrate the potential of ex vivo FCM in fresh tissue for rapid real‐time surgical pathology.     

Measurement of cartilage sub-component distributions through the surface by Raman spectroscopy-based multivariate analysis

Articular cartilage posesses unique material properties due to a complex depth-dependent composition of sub-components. Raman spectroscopy has proven valuable in quantifying this composition through cartilage cross-sections. However, cross-sectioning requires tissue destruction and is not practical in situ. In this work, Raman spectroscopy-based multivariate curve resolution (MCR) was employed in porcine cartilage samples (n = 12) to measure collagen, glycosaminoglycan, and water distributions through the surface for the first time; these were compared against cross-section standards. Through the surface Raman measurements proved reliable in predicting composition distribution up to a depth of approximately 0.5 mm. A fructose-based optical clearing agent (OCA) was also used in an attempt to further improve depth of resolution of this measurement method. However, it did not; mainly due to a high-spectral overlap with the Raman spectra of main cartilage sub-components. This measurement technique potentially could be used in situ, to better understand the etiology of joint diseases such as osteoarthritis (OA).     

Quadrature demodulation in line‐scan focal modulation microscopy for imaging three‐dimensional zebrafish neural structure

Visualizing biological processes in neuroscience requires in vivo functional imaging at single‐neuron resolution, high image acquisition speed and strong optical sectioning ability. However, due to light scattering of in tissue, very often conventional wide‐field fluorescence microscopes are unable to resolve cells in the presence of a strong out‐of‐focus background. Line‐scan focal modulation microscopy enables high temporal resolution and good optical sectioning ability at the same time. Here we demonstrate a quadrature demodulation method to extract the focal information with an extended frequency bandwidth and therefore higher spatial resolution. The performance of the demodulation scheme in line‐scan focal modulation microscope has been evaluated by performing imaging experiments with fluorescence beads and zebrafish neural structure. Reduced background, reduced artifacts and more detailed morphological information are evident in the obtained images.      

Vibrational two-photon microscopy for tissue imaging: Short-wave infrared surface-enhanced resonance hyper-Raman scattering

Multiphoton microscopy using short-wave infrared (SWIR) radiation offers nondestructive and high-resolution imaging through tissue. Two-photon fluorescence (TPF), for example, is commonly employed to increase the penetration depth and spatial resolution of SWIR imaging, but the broad spectral peaks limit its multiplexing capabilities. Hyper-Raman scattering, the vibrational analog of TPF, yields spectral features on the order of 20 cm^?1 and reporter-functionalized noble metal nanoparticles (NPs) provide a platform for both hyper-Raman signal enhancement and selective targeting in biological media. Herein we report the first tissue imaging study employing surface-enhanced resonance hyper-Raman scattering (SERHRS), the two-photon analog of surface-enhanced resonance Raman scattering. Specifically, we employ multicore gold-silica NPs (Au@SiO₂ NPs) functionalized with a near infrared-resonant cyanine dye, 3,3′-diethylthiatricarbocyanine iodide as a SERHRS reporter. SWIR SERHRS spectra are efficiently acquired from mouse spleen tissue. SWIR SERHRS combines two-photon imaging advantages with narrow vibrational peak widths, presenting future applications of multitargeted bioimaging.     

Cell and nucleus refractive‐index mapping by interferometric phase microscopy and rapid confocal fluorescence microscopy

We present a multimodal technique for measuring the integral refractive index and the thickness of biological cells and their organelles by integrating interferometric phase microscopy (IPM) and rapid confocal fluorescence microscopy. First, the actual thickness maps of the cellular compartments are reconstructed using the confocal fluorescent sections, and then the optical path difference (OPD) map of the same cell is reconstructed using IPM. Based on the co‐registered data, the integral refractive index maps of the cell and its organelles are calculated. This technique enables rapidly measuring refractive index of live, dynamic cells, where IPM provides quantitative imaging capabilities and confocal fluorescence microscopy provides molecular specificity of the cell organelles. We acquire human colorectal adenocarcinoma cells and show that the integral refractive index values are similar for the whole cell, the cytoplasm and the nucleus on the population level, but significantly different on the single cell level.     

Chemico‐mechanical imaging of Barrett's oesophagus

Barrett's oesophagus is a condition characterized by a change in the lining of the oesophagus that markedly increases the risk of adenocarcinoma. We demonstrate the first site‐matched application of Brillouin microscopy, Raman microscopy and FTIR micro‐spectroscopic imaging to ex‐vivo epithelial tissue – Barrett's oesophagus. The mechanical and chemical characters of the epithelium were assessed in histological sections from a patient subjected to endoscopic oesophageal biopsy. Previous studies have shown that both these properties change within the oesophageal wall, owing to the presence of distinct cellular and extracellular constituents which are putatively affected by oesophageal cancer. Brillouin microscopy enables maps of elasticity of the epithelium to be obtained, whilst Raman and FTIR imaging provide ’chemical images' without the need for labelling or staining. This site‐matched approach provides a valuable platform for investigating the structure, biomechanics and composition of complex heterogeneous systems. A combined Brillouin‐Raman device has potential for in‐vivo diagnosis of pathology.

First application of site‐matched micro Brillouin, Raman and FTIR spectroscopic imaging to epithelial tissue in Barrett's oesophagus     

Clearing,immunofluorescence, and confocal microscopy for the three-dimensional imaging of murine testes and study of testis biology

Optical clearing techniques provide unprecedented opportunities to study large tissue samples at histological resolution, eliminating the need for physical sectioning while preserving the three-dimensional structure of intact biological systems. There is significant potential for applying optical clearing to reproductive tissues. In testicular biology, for example, the study of spermatogenesis and the use of spermatogonial stem cells offer high-impact applications in fertility medicine and reproductive biotechnology. The objective of our study is to apply optical clearing, immunofluorescence, and confocal microscopy to testicular tissue in order to reconstruct its three-dimensional microstructure in intact samples. We used Triton-X/DMSO clearing in combination with refractive index matching to achieve optical transparency of fixed mouse testes. An antibody against smooth muscle actin was used to label peritubular myoid cells of seminiferous tubules while an antibody against ubiquitin C-terminal hydrolase was used to label Sertoli cells and spermatogonia in the seminiferous epithelium. Specimens were then imaged using confocal fluorescence microscopy. We were able to successfully clear testicular tissue and utilize immunofluorescent probes. Additionally, we successfully visualized the histological compartments of testicular tissue in three-dimensional reconstructions. Optical clearing combined with immunofluorescence and confocal imaging offers a powerful new method to analyze the cytoarchitecture of testicular tissue at histological resolution while maintaining the macro-scale perspective of the intact system. Considering the importance of the murine model, our developed method represents a significant contribution to the field of male reproductive biology, enabling the study of testicular function.     

Stereotaxic endoscopy for the ocular imaging of awake,freely moving animal models

Stereotaxic instruments are increasingly used in research animals for the study of disease, but typically require restraints and anesthetic procedures. A stereotaxic head mount that enables imaging of the anterior chamber of the eye in alert and freely mobile mice is presented in this study. The head mount is fitted based on computed tomography scans and manufactured using 3D printing. The system is placed noninvasively using temporal mount bars and a snout mount, without breaking the skin or risking suffocation, while an instrument channel stabilizes the ocular probes. With a flexible micro‐endoscopic probe and a confocal scanning laser microscopy system, <20 μm resolution is achieved in vivo with a field of view of nearly 1 mm. Discomfort is minimal, and further adaptations for minimally invasive neuroscience, optogenetics and auditory studies are possible.     

Detection and Phenotypic Characterization of Adult Neurogenesis

Studies of adult neurogenesis have greatly expanded in the last decade, largely as a result of improved tools for detecting and quantifying neurogenesis. In this review, we summarize and critically evaluate detection methods for neurogenesis in mammalian and human brain tissue. Besides thymidine analog labeling, cell-cycle markers are discussed, as well as cell stage and lineage commitment markers. Use of these histological tools is critically evaluated in terms of their strengths and limitations, as well as possible artifacts. Finally, we discuss the method of radiocarbon dating for determining cell and tissue turnover in humans.Detection of neurogenesis in vivo requires the ability to image at a cellular resolution, which currently precludes noninvasive imaging approaches, such as magnetic resonance imaging (MRI). In vivo microscopy, using deeply penetrating UV illumination with multiphoton microscopy, or by the recently available endoscopic confocal microscopy, may provide new opportunities for longitudinal studies of neurogenesis in the living animal with single-cell resolution. These newer microscopy approaches are particularly compelling when coupled with transgenic mice expressing phenotype-specific fluorescent reporter genes. Additionally, an advanced method using ¹⁴C carbon dating of postmortem DNA from specific cell populations of the brain revealed insights into adult human neurogenesis. Nevertheless, at present, the predominant approach for studying neurogenesis relies on traditional histological methods of fixation, production of tissue sections, staining, and microscopic analysis.This review discusses methodological considerations for detection of neurogenesis in the adult brain according to our current state of knowledge. This will include the use of exogenous or endogenous markers of cell cycle, as well as phenotype markers that contribute to resolving stages of neuronal lineage commitment. The accurate analysis of cell phenotype will be discussed, including suggestions for accurate detection and reliable quantification of cell numbers. Finally, we will present the newly developed ¹⁴C carbon dating of nuclear DNA for quantitative analysis of neurogenesis in human tissue.     

Multispectral near infrared absorption imaging for histology of skin cancer

Multispectral imaging combines the spectral resolution of spectroscopy with the spatial resolution of imaging and is therefore very useful for biomedical applications. Currently, histological diagnostics use mainly stainings with standard dyes (eg, hematoxylin + eosin) to identify tumors. This method is not applicable in vivo and provides low amounts of chemical information. Biomolecules absorb near infrared light (NIR, 800‐1700 nm) at different wavelengths, which could be used to fingerprint tissue. Here, we built a NIR multispectral absorption imaging setup to study skin tissue samples. NIR light (900‐1500 nm) was used for homogenous wide‐field transmission illumination and detected by a cooled InGaAs camera. In this setup, images I(x, y, λ) from dermatological samples (melanoma, nodular basal‐cell carcinoma, squamous‐cell carcinoma) were acquired to distinguish healthy from diseased tissue regions. In summary, we show the potential of multispectral NIR imaging for cancer diagnostics.      

Combined in vivo confocal Raman spectroscopy and confocal microscopy of human skin

In vivo confocal Raman spectroscopy is a noninvasive optical method to obtain detailed information about the molecular composition of the skin with high spatial resolution. In vivo confocal scanning laser microscopy is an imaging modality that provides optical sections of the skin without physically dissecting the tissue. A combination of both techniques in a single instrument is described. This combination allows the skin morphology to be visualized and (subsurface) structures in the skin to be targeted for Raman measurements. Novel results are presented that show detailed in vivo concentration profiles of water and of natural moisturizing factor for the stratum corneum that are directly related to the skin architecture by in vivo cross-sectional images of the skin. Targeting of skin structures is demonstrated by recording in vivo Raman spectra of sweat ducts and sebaceous glands in situ. In vivo measurements on dermal capillaries yielded high-quality Raman spectra of blood in a completely noninvasive manner. From the results of this exploratory study we conclude that the technique presented has great potential for fundamental skin research, pharmacology (percutaneous transport), clinical dermatology, and cosmetic research, as well as for noninvasive analysis of blood analytes, including glucose.     

Optical‐resolution photoacoustic microscopy with ultrafast dual‐wavelength excitation

Fast functional and molecular photoacoustic microscopy requires pulsed laser excitations at multiple wavelengths with enough pulse energy and short wavelength‐switching time. Recent development of stimulated Raman scattering in optical fiber offers a low‐cost laser source for multiwavelength photoacoustic imaging. In this approach, long fibers temporally separate different wavelengths via optical delay. The time delay between adjacent wavelengths may eventually limits the highest A‐line rate. In addition, a long‐time delay in fiber may limit the highest pulse energy, leading to poor image quality. In order to achieve high pulse energy and ultrafast dual‐wavelength excitation, we present optical‐resolution photoacoustic microscopy with ultrafast dual‐wavelength excitation and a signal separation method. The signal separation method is validated in numerical simulation and phantom experiments. We show that when two photoacoustic signals are partially overlapped with a 50‐ns delay, they can be recovered with 98% accuracy. We apply this ultrafast dual‐wavelength excitation technique to in vivo OR‐PAM. Results demonstrate that A‐lines at two wavelengths can be successfully separated, and sO₂ values can be reliably computed from the separated data. The ultrafast dual‐wavelength excitation enables fast functional photoacoustic microscopy with negligible misalignment among different wavelengths and high pulse energy, which is important for in vivo imaging of microvascular dynamics.     

Intracellular dynamics of topoisomerase I inhibitor,CPT-11, by slit-scanning confocal Raman microscopy

Most molecular imaging technologies require exogenous probes and may have some influence on the intracellular dynamics of target molecules. In contrast, Raman scattering light measurement can identify biomolecules in their innate state without application of staining methods. Our aim was to analyze intracellular dynamics of topoisomerase I inhibitor, CPT-11, by using slit-scanning confocal Raman microscopy, which can take Raman images with high temporal and spatial resolution. We could acquire images of the intracellular distribution of CPT-11 and its metabolite SN-38 within several minutes without use of any exogenous tags. Change of subcellular drug localization after treatment could be assessed by Raman imaging. We also showed intracellular conversion from CPT-11 to SN-38 using Raman spectra. The study shows the feasibility of using slit-scanning confocal Raman microscopy for the non-labeling evaluation of the intracellular dynamics of CPT-11 with high temporal and spatial resolution. We conclude that Raman spectromicroscopic imaging is useful for pharmacokinetic studies of anticancer drugs in living cells. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Optical coherence hyperspectral microscopy with a single supercontinuum light source

In the paper, we have developed an optical coherence hyperspectral microscopy with a single supercontinuum light source. The microscopy consists of optical coherence tomography (OCT) and hyperspectral imaging (HSI), which can visualize the structural and functional characteristics of biological tissues. The 500 to 700 nm band is selected for HSI and OCT imaging, where HSI enables imaging of oxygen saturation and hemoglobin (Hb) content, while OCT acquires structural characteristics to assess the morphology of biological tissues. The system performance of the optical coherence hyperspectral microscopy is verified by normal mice ears, and the practical applications of the microscopy is further performed in 4T1 and inflammation Balb/c mice ears in vivo. The experimental results demonstrate that the microscopy has potential to provide complementary information for clinical applications.     

Identification of cholesterol crystals in plaques of atherosclerotic mice using hyperspectral CARS imaging

The accumulation of lipids, including cholesterol, in the arterial wall plays a key role in the pathogenesis of atherosclerosis. Although several advances have been made in the detection and imaging of these lipid structures in plaque lesions, their morphology and composition have yet to be fully elucidated, particularly in different animal models of disease. To address this issue, we analyzed lipid morphology and composition in the atherosclerotic plaques of two animal models of disease, the low density lipoprotein receptor-deficient (LDLR(-/-)) mouse and the ApoE lipoprotein-deficient (ApoE(-/-)) mouse, utilizing hyperspectral coherent anti-Stokes Raman scattering (CARS) microscopy in combination with principal component analysis (PCA). Hyperspectral CARS imaging revealed lipid-rich macrophage cells and condensed needle-shaped and plate-shaped lipid crystal structures in both mice. Spectral analysis with PCA and comparison to spectra of pure cholesterol and cholesteryl ester derivatives further revealed these lipid structures to be pure cholesterol crystals, which were predominantly observed in the ApoE(-/-) mouse model. These results illustrate the ability of hyperspectral CARS imaging in combination with multivariate analysis to characterize atherosclerotic lipid morphology and composition with chemical specificity, and consequently, provide new insight into the formation of cholesterol crystal structures in atherosclerotic plaque lesions.     

Microstructural and mechanical insight into atherosclerotic plaques: an ex vivo DTI study to better assess plaque vulnerability

Non-invasive microstructural characterisation has the potential to determine the stability, or lack thereof, of atherosclerotic plaques and ultimately aid in better assessing plaques’ risk to rupture. If linked with mechanical characterisation using a clinically relevant imaging technique, mechanically sensitive rupture risk indicators could be possible. This study aims to provide this link–between a clinically relevant imaging technique and mechanical characterisation within human atherosclerotic plaques. Ex vivo diffusion tensor imaging, mechanical testing, and histological analysis were carried out on human carotid atherosclerotic plaques. DTI-derived tractography was found to yield significant mechanical insight into the mechanical properties of more stable and more vulnerable microstructures. Coupled with insights from digital image correlation and histology, specific failure characteristics of different microstructural arrangements furthered this finding. More circumferentially uniform microstructures failed at higher stresses and strains when compared to samples which had multiple microstructures, like those seen in a plaque cap. The novel findings in this study motivate diagnostic measures which use non-invasive characterisation of the underlying microstructure of plaques to determine their vulnerability to rupture.

Graphic abstract

     

Multicomposite super‐resolution microscopy: Enhanced Airyscan resolution with radial fluctuation and sample expansions

Either modulated illumination or temporal fluctuation analysis can assist super‐resolution techniques in overcoming the diffraction limit of conventional optical microscopy. As they are not contradictory to each other, an effective combination of spatial and temporal super‐resolution mechanisms would further improve the resolution of fluorescent images. Here, a super‐resolution imaging method called fluctuation‐enhanced Airyscan technology (FEAST) is proposed, which achieves ~40 nm lateral imaging resolution and is useful for a range of fluorescent proteins and organic dyes. It was demonstrated not only to obtain different subcellular super‐resolution images, but also to improve the accuracy of counting the average human epidermal growth factor receptor 2 (HER2) copy number for diagnosis in breast cancer. Furthermore, the combination of FEAST and sample expansion microscopy (Ex‐FEAST) improves the lateral resolution to ~26 nm.