Multimodal Microscopy Research Articles

Motion-tolerant 3D volumetric multimodality microscopy imaging of human skin with subcellular resolution and extended field-of-view

The skin, the body’s largest organ, has a heterogeneous structure with various cell types and tissue layers. In vivo noninvasive 3D volumetric imaging with multi-contrast, high resolution, a large field-of-view (FOV), and no-motion artifacts is crucial for studying skin biology and diagnosing/evaluating diseases. Traditionally high-resolution in vivo skin microscopy methods capture images in the en-face (xy) plane parallel to the skin surface but are affected by involuntary motion, particularly during large-area volumetric data acquisition using xy-z mosaicking. In this work, we developed an xz-y imaging method that acquires images in the vertical (xz) plane and extends the FOV by moving the skin laterally along the y-direction. This approach is conceived based on our observation that involuntary skin movements are mostly along the vertical direction. Combined with a unique motion correction method, it enables 3D image reconstruction with subcellular resolution and an extended FOV close to a centimeter (8 mm). A multimodality microscopy system using this method provides simultaneous reflectance confocal, two-photon excited fluorescence, and second harmonic generation imaging, enabling multi-contrast capabilities. Using this system, we captured histology-like features of normal skin, vitiligo, and melanoma, demonstrating its potential for in vivo skin biology studies, clinical diagnosis, treatment planning and monitoring.

Five‐Modal Three‐Dimensional Optical Microscopy via Single 1550 nm Fiber Laser

Multimodal optical microscopy can be used to visualize multiple biological targets at the same location and at similar spatial resolutions, enabling more comprehensive studies of complex biological samples. However, current multimodal imaging systems typically use multiple or specially designed excitation lasers. Furthermore, previous studies have reported only three or four types of imaging modalities, limiting the number of imaging targets. This study develops an imaging system based on a single commercialized 1550 nm fiber laser and integrates five imaging modalities: second harmonic generation, third harmonic generation, two‐photon fluorescence, three‐photon fluorescence, and reflected confocal microscopy. Utilizing the characteristics of different imaging modalities, various biological components in biological tissues can be revealed simultaneously and cell movement in a complex biological environment can be traced noninvasively. The capacity of deep imaging has also been demonstrated in the mouse brain. The results show that the proposed multimodal microscopy system has great application potential in biomedical fields, such as tumor diagnosis and immunotherapy.

Tandem Raman microscopy: integration of spontaneous and coherent Raman scattering offering data fusion analysis to improve optical biosensing

We provide tandem Raman microscopy (TRAM), a cutting-edge multimodal microscope that integrates the methods of stimulated Raman scattering (SRS), coherent anti-Stokes Raman scattering (CARS), and spontaneous (resonance) Raman scattering [(R)RS]. The device facilitates sequential continuous wave (CW)-driven RS imaging to collect full spectra from every sample location and rapid pulsed-wave-driven SRS-CARS scanning at specific wavenumbers, offering a reliable and efficient analytical tool. The fingerprint spectral region can be included in the spectral imaging capabilities of CARS and SRS. Data collected from a sample area using several techniques can be integrated and analyzed, significantly increasing reliability and predictions. We analyzed the in vitro model of nonadherent leukocytes (LCs) to illustrate the capabilities of this unique system, emphasizing the benefits of measuring the same sample with three different Raman techniques without having to transfer it between microscopes. Data fusion allowed for the correct classification of two subtypes of LCs based on the partial least squares (PLS) discrimination, increasing the prediction accuracy from approximately 83% in the case of textural and morphological data (SRS) to 100% when combined with spectral data (SRS and RS). We also present RRS images of LC labeled with astaxanthin, and reference data from SRS and CARS microscopy. Additionally, polystyrene beads were investigated as a non-biological material. The advantages of each Raman technique are utilized when (R)RS, SRS, and CARS are combined into a single device. This paves the way for dependable chemical characterization in a wide range of scientific and industrial fields.

Self-Supervised and Zero-Shot Learning in Multi-Modal Raman Light Sheet Microscopy.

Advancements in Raman light sheet microscopy have provided a powerful, non-invasive, marker-free method for imaging complex 3D biological structures, such as cell cultures and spheroids. By combining 3D tomograms made by Rayleigh scattering, Raman scattering, and fluorescence detection, this modality captures complementary spatial and molecular data, critical for biomedical research, histology, and drug discovery. Despite its capabilities, Raman light sheet microscopy faces inherent limitations, including low signal intensity, high noise levels, and restricted spatial resolution, which impede the visualization of fine subcellular structures. Traditional enhancement techniques like Fourier transform filtering and spectral unmixing require extensive preprocessing and often introduce artifacts. More recently, deep learning techniques, which have shown great promise in enhancing image quality, face their own limitations. Specifically, conventional deep learning models require large quantities of high-quality, manually labeled training data for effective denoising and super-resolution tasks, which is challenging to obtain in multi-modal microscopy. In this study, we address these limitations by exploring advanced zero-shot and self-supervised learning approaches, such as ZS-DeconvNet, Noise2Noise, Noise2Void, Deep Image Prior (DIP), and Self2Self, which enhance image quality without the need for labeled and large datasets. This study offers a comparative evaluation of zero-shot and self-supervised learning methods, evaluating their effectiveness in denoising, resolution enhancement, and preserving biological structures in multi-modal Raman light sheet microscopic images. Our results demonstrate significant improvements in image clarity, offering a reliable solution for visualizing complex biological systems. These methods establish the way for future advancements in high-resolution imaging, with broad potential for enhancing biomedical research and discovery.

An integrated portable system for laser speckle contrast imaging and digital holographic microscopy

An integrated portable system for laser speckle contrast imaging and digital holographic microscopy

Imaging the cerebral vasculature at different scales: translational tools to investigate the neurovascular interfaces.

The improvements in imaging technology opened up the possibility to investigate the structure and function of cerebral vasculature and the neurovascular unit with unprecedented precision and gaining deep insights not only on the morphology of the vessels, but also regarding their function and regulation related to the cerebral activity. In this review we will dissect the different imaging capabilities regarding the cerebrovascular tree, the neurovascular unit and the hemodynamic response function and thus the vascular-neuronal coupling. We will discuss both clinical and preclinical setting, with a final discussion on the current scenery in cerebrovascular imaging where Magnetic Resonance Imaging and multimodal Microscopy emerge as the most potent and versatile tools respectively in the clinical and preclinical context.

Improving Chemical Composition Measurements from Microscale to Atomic Scale with Fused Multi-Modal Microscopy

Improving Chemical Composition Measurements from Microscale to Atomic Scale with Fused Multi-Modal Microscopy

Advanced Focused Ion Beam Methods and Multimodal Microscopy Techniques for the Analysis of Epoxy-Embedded and Native Bio-Samples

Advanced Focused Ion Beam Methods and Multimodal Microscopy Techniques for the Analysis of Epoxy-Embedded and Native Bio-Samples

Extracting and Utilizing Multimodal Microscopy Datasets of Images and Text with Foundation Models

Extracting and Utilizing Multimodal Microscopy Datasets of Images and Text with Foundation Models

Democratization of Multimodal Microscopy Imaging: Convergence Across Scales of Imaging Modalities from Single Instrument to Research Technology Centers. Advantages and Challenges

Democratization of Multimodal Microscopy Imaging: Convergence Across Scales of Imaging Modalities from Single Instrument to Research Technology Centers. Advantages and Challenges

Integrating multimodal Raman and photoluminescence microscopy with enhanced insights through multivariate analysis

This paper introduces a novel multimodal optical microscope, integrating Raman and laser-induced photoluminescence (PL) spectroscopy for the analysis of micro-samples relevant in Heritage Science. Micro-samples extracted from artworks, such as paintings, exhibit intricate material compositions characterized by high complexity and spatial heterogeneity, featuring multiple layers of paint that may be also affected by degradation phenomena. Employing a multimodal strategy becomes imperative for a comprehensive understanding of their material composition and condition. The effectiveness of the proposed setup derives from synergistically harnessing the distinct strengths of Raman and laser-induced PL spectroscopy. The capacity to identify various chemical species through the latter technique is enhanced by using multiple excitation wavelengths and two distinct excitation fluence regimes. The combination of the two complementary techniques allows the setup to effectively achieve comprehensive chemical mapping of sample through a raster scanning approach. To attain a competitive overall measurement time, we employ a short integration time for each measurement point. We further propose an analysis protocol rooted in a multivariate approach. Specifically, we employ non-negative matrix factorization as the spectral decomposition method. This enables the identification of spectral endmembers, effectively correlated with specific chemical compounds present in samples. To demonstrate its efficacy in Heritage Science, we present examples involving pigment powder dispersions and stratigraphic micro-samples from paintings. Through these examples, we show how the multimodal approach reinforces material identification and, more importantly, facilitates the extraction of complementary information. This is pivotal as the two optical techniques exhibit sensitivity to different materials. Looking ahead, our method holds potential applications in diverse research fields, including material science and biology.

An aldehyde-crosslinking mitochondrial probe for STED imaging in fixed cells

Fluorescence labeling of chemically fixed specimens, especially immunolabeling, plays a vital role in super-resolution imaging as it offers a convenient way to visualize cellular structures like mitochondria or the distribution of biomolecules with high detail. Despite the development of various distinct probes that enable super-resolved stimulated emission depletion (STED) imaging of mitochondria in live cells, most of these membrane-potential-dependent fluorophores cannot be retained well in mitochondria after chemical fixation. This lack of suitable mitochondrial probes has limited STED imaging of mitochondria to live cell samples. In this study, we introduce a mitochondria-specific probe, PK Mito Orange FX (PKMO FX), which features a fixation-driven cross-linking motif and accumulates in the mitochondrial inner membrane. It exhibits high fluorescence retention after chemical fixation and efficient depletion at 775 nm, enabling nanoscopic imaging both before and after aldehyde fixation. We demonstrate the compatibility of this probe with conventional immunolabeling and other strategies commonly used for fluorescence labeling of fixed samples. Moreover, we show that PKMO FX facilitates correlative super-resolution light and electron microscopy, enabling the correlation of multicolor fluorescence images and transmission EM images via the characteristic mitochondrial pattern. Our probe further expands the mitochondrial toolkit for multimodal microscopy at nanometer resolutions.

From Cell Populations to Molecular Complexes: Multiplexed Multimodal Microscopy to Explore p53-53BP1 Molecular Interaction.

Surpassing the diffraction barrier revolutionized modern fluorescence microscopy. However, intrinsic limitations in statistical sampling, the number of simultaneously analyzable channels, hardware requirements, and sample preparation procedures still represent an obstacle to its widespread diffusion in applicative biomedical research. Here, we present a novel pipeline based on automated multimodal microscopy and super-resolution techniques employing easily available materials and instruments and completed with open-source image-analysis software developed in our laboratory. The results show the potential impact of single-molecule localization microscopy (SMLM) on the study of biomolecules' interactions and the localization of macromolecular complexes. As a demonstrative application, we explored the basis of p53-53BP1 interactions, showing the formation of a putative macromolecular complex between the two proteins and the basal transcription machinery in situ, thus providing visual proof of the direct role of 53BP1 in sustaining p53 transactivation function. Moreover, high-content SMLM provided evidence of the presence of a 53BP1 complex on the cell cytoskeleton and in the mitochondrial space, thus suggesting the existence of novel alternative 53BP1 functions to support p53 activity.

A multi-modal microscope for integrated mapping of cellular forces and Brillouin scattering with high resolution

Mechanical forces and stiffness play key roles in the health and development of cells and tissue, but despite the physical connection between these quantities, they cannot be monitored in parallel in most cases. Here, we introduce a fully integrated microscope that combines a method for high-resolution cell force imaging (elastic resonator interference stress microscopy, ERISM) with non-contact mapping of the elastic properties of cells (via Brillouin microscopy). In order to integrate both techniques, we had to account for the strong back reflection on the surface of the microcavity used for ERISM measurements as well as the local destruction of the cavity under illumination for Brillouin microscopy measurements. Therefore, we developed an elastic optical microcavity with minimal absorption that can perform ERISM measurements without sustaining laser damage during Brillouin microscopy. Furthermore, an unequal-arm Michelson interferometer was designed to suppress the back reflection of the laser on the ERISM microcavity surface using division by amplitude interference to reduce the reflected light and enhance the Brillouin signal. We show the utility of our integrated microscope by simultaneously mapping cellular forces and Brillouin shifts in cultures of fibroblast cells.

MICROPHOTOGRAPHY- A REVIEW

Microphotographs are useful biology teaching aids. In laboratory activities that require microscope slide preparation, they are an effective tool for pre- and post-lab discussions. They are also useful mechanisms to enhance lectures that deal with the microscopic world.1Microphography is useful to generate high quality micrographs with high resolution with a larger field of view, that enable to view the crucial ultrastructure relevant for pathological diagnosis. Incorporation into correlation and multi-modal microscopy workflows will assist with solving problems not only in research but also in specific fields of pathology and medical diagnosis.2

Comparative morphology and function of Chloropidae (Diptera) tibial organ

The tibial organ of Chloropidae and Milichiidae flies is an obscure feature with taxonomic significance. This study provides the first in-depth investigation into the ultrastructure of the hind leg of 11 genera from all Chloropidae subfamilies using a multimodal microscopy approach. The modified dermal tissue associated with the tibial organ indicates glandular function, as evidenced by the presence of secretory vesicles containing non-proteinaceous elements, potentially indicating lipidic secretion. The overall similarity of the tibial organ between Chloropidae and Milichiidae indicates a shared homology. However, the evolutionary history of this structure is still contentious due to limitations in the phylogenetic relationships of both lineages. Moreover, our findings enable future comparative investigations of other Diptera leg organs that possess secretory ability, which could be homologous across schizophoran families, but not necessarily the organs themselves.

Automated Grain Boundary (GB) Segmentation and Microstructural Analysis in 347H Stainless Steel Using Deep Learning and Multimodal Microscopy

Automated Grain Boundary (GB) Segmentation and Microstructural Analysis in 347H Stainless Steel Using Deep Learning and Multimodal Microscopy

Elimination of HCV replication machinery early after antiviral treatment with DAA monitored by multimodal microscopy

Elimination of HCV replication machinery early after antiviral treatment with DAA monitored by multimodal microscopy

Multimodal Microscopy Characterisation of 2D Materials

Spatial characterisation of transition metal dichalcogenides (TMDCs) is crucial for understanding their optoelectronic properties and optimising film growth conditions. In this work, we present a multimodal microscopy platform combining conventional widefield, Raman, photoluminescence and second harmonic generation imaging for the characterisation of 2D materials.The platform was used to characterise CVD grown WSe2 crystals. Raman imaging identified the presence of both monolayer and multilayer WSe2 through a change in the intensity and position of the E12g / A1g phonon modes. Photoluminescence imaging confirmed the presence of monolayer WSe2 with emission at 780 nm and identified two distinct multilayer regions through changes in the photoluminescence wavelength and intensity. Lastly, second harmonic generation imaging under femtosecond laser excitation was used to obtain the relative growth orientation of the three identified domains in the crystal.Figure 1 highlights Raman imaging of WSe2. (a) Intensity of the E12g / A1g (250 cm-1) Raman band, (b) Peak position of the E12g / A1g (250 cm-1) Raman band, (c) least squares spectral matching revealing three distinct Raman spectral areas, (d) Averaged Raman spectra from areas A, B and C. Figure 1

Single source CARS-based multimodal microscopy system for biological tissue imaging [Invited

A coherent anti-Stokes Raman scattering (CARS)-based multimodality microscopy system was developed using a single Ti:sapphire femtosecond laser source for biological imaging. It provides three complementary and co-registered imaging modalities: CARS, MPM (multiphoton microscopy), and RCM (reflectance confocal microscopy). The imaging speed is about 1 frame-per-second (fps) with a digital resolution of 1024 × 1024 pixels. This microscopy system can provide clear 2-dimensional and 3-dimensional images of ex-vivo biological tissue samples. Its spectral selection initiates vibrational excitation in lipid cells (approximately 2850 cm-1) using two filters on the pump and Stokes beam paths. The excitation can be tuned over a wide spectral range with adjustable spectral filters. The imaging capability of this CARS-based multimodal microscopy system was demonstrated using porcine fat, murine skin, and murine liver tissue samples.