Search Results (48)

Three-dimensional (3D) cellular models, such as spheroids, serve as pivotal systems for understanding complex biological phenomena in histology, oncology, and tissue engineering. In response to the growing need for advanced imaging capabilities, we present a novel multi-modal Raman light sheet microscope designed to capture elastic (Rayleigh) and inelastic (Raman) scattering, along with fluorescence signals, in a single platform. By leveraging a shorter excitation wavelength (532 nm) to boost Raman scattering efficiency and incorporating robust fluorescence suppression, the system achieves label-free, high-resolution tomographic imaging without the drawbacks commonly associated with near-infrared modalities. An accompanying Deep Image Prior (DIP) seamlessly integrates with the microscope to provide unsupervised denoising and resolution enhancement, preserving critical molecular details and minimizing extraneous artifacts. Altogether, this synergy of optical and computational strategies underscores the potential for in-depth, 3D imaging of biomolecular and structural features in complex specimens and sets the stage for future advancements in biomedical research, diagnostics, and therapeutics. Full article

Emerging evidence from observational studies suggests that lifestyle modifications, particularly moderate-intensity exercise, may confer neuroprotective benefits against dementia, potentially by enhancing brain resistance through clearance mechanisms. Using light-sheet fluorescence microscopy (LSFM) with tissue clearing, we investigated the role of voluntary swimming in ameliorating β-amyloid pathology in a transgenic Alzheimer’s disease (AD) mouse model. Twenty 52-week-old hAPPsw mice were randomly divided into a 5-week voluntary swimming intervention group and a control group (each n = 10). Each session included a 10-min swim followed by a 10-min rest, escalating from one session per day in the first week to three sessions per day by the fifth week. The excised brains were prepared using tissue-clearing and volume immunostaining with thioflavin-S for β-amyloid. For LSFM imaging, the individual plaque area and volume, total plaque load, and morphological parameters were quantified via an Imaris-based three-dimensional (3D) volumetric surface model. Visual comparison revealed that the intervention group presented significantly lower β-amyloid accumulation. The total surface volume of β-amyloid accumulation in the intervention group was significantly lower than that of the control group (intervention, 122,180,948 μm³ [105,854,660–169,063,081]; control, 167,201,016 μm³ [139,367,765–193,535,450]; p = 0.043). There were no significant differences in the morphological parameters, such as ellipticity and sphericity. Our LSFM study demonstrated notable reductions in β-amyloid, as evidenced by a decrease in total surface volume, in 52-week-old transgenic mice after a 5-week structured swimming program, supporting the notion that even in advanced AD stages, leisure-time voluntary swimming serves as an efficacious intervention for augmenting resistance to pathology. Full article

Light microscopy has emerged as one of the fundamental methods to analyze biological systems; novel techniques of 3D microscopy and super-resolution microscopy (SRM) with an optical resolution down to the sub-nanometer range have recently been realized. However, most of these achievements have been made with fixed specimens, i.e., direct information about the dynamics of the biosystem studied was not possible. This stimulated the development of live cell microscopy imaging approaches, including Low Illumination Fluorescence Microscopy, Light Sheet (Fluorescence) Microscopy (LSFM), or Structured Illumination Microscopy (SIM). Here, we discuss perspectives, methods, and relevant light doses of advanced fluorescence microscopy imaging to keep the cells alive at low levels of phototoxicity. Full article

Trypanosoma cruzi, the etiological agent of Chagas disease, is a parasite known for its diverse genotypic variants, or Discrete Typing Units (DTUs), which have been associated with varying degrees of tissue involvement. However, aspects such as parasite attachment remain unclear. It has been suggested that the TcI genotype is associated with cardiac infection, the most common involved site in chronic human infection, while TcII is associated with digestive tract involvement. Traditional models for T. cruzi infection provide limited in vivo observation, making it challenging to observe the dynamics of parasite-host interactions. This study evaluates the cardiac attachment of trypomastigotes from TcI and TcII DTUs in zebrafish larvae. Labeled trypomastigotes were injected in the duct of Cuvier of zebrafish larvae and tracked by stereomicroscopy and light-sheet fluorescence microscopy (LSFM). Remarkably, it was possible to observe TcI parasites adhered to the atrium, atrioventricular valve, and circulatory system, while TcII trypomastigotes demonstrated adhesion to the atrium, atrioventricular valve, and yolk sac extension. When TcI and TcII were simultaneously injected, they both attached to the heart; however, more of the TcII trypomastigotes were observed attached to this organ. Although TcII DTU has previously been associated with digestive tissue infection, both parasite variants showed cardiac tissue attachment in this in vivo model. Full article

The detection and assessment of atherosclerosis and cardiovascular calcification can inform risk stratification and therapies to reduce cardiovascular morbidity and mortality. In this review, we provide an overview of current and emerging imaging techniques for assessing atherosclerosis and cardiovascular calcification in animal models. Traditional imaging modalities, such as computed tomography (CT) and magnetic resonance imaging (MRI), offer non-invasive approaches of visualizing atherosclerotic calcification in vivo; integration of these techniques with positron emission tomography (PET) imaging adds molecular imaging capabilities, such as detection of metabolically active microcalcifications with ¹⁸F-sodium fluoride. Photoacoustic imaging provides high contrast that enables in vivo evaluation of plaque composition, yet this method is limited by optical penetration depth. Light-sheet fluorescence microscopy provides high-resolution, three-dimensional imaging of cardiovascular structures and has been used for ex vivo assessment of atherosclerotic calcification, but its limited tissue penetration and requisite complex sample preparation preclude its use in vivo to evaluate cardiac tissue. Overall, with these evolving imaging tools, our understanding of cardiovascular calcification development in animal models is improving, and the combination of traditional imaging techniques with emerging molecular imaging modalities will enhance our ability to investigate therapeutic strategies for atherosclerotic calcification. Full article

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. Full article

This study presents an advanced integration of Multi-modal Raman Light Sheet Microscopy with zero-shot learning-based computational methods to significantly enhance the resolution and analysis of complex three-dimensional biological structures, such as 3D cell cultures and spheroids. The Multi-modal Raman Light Sheet Microscopy system incorporates Rayleigh scattering, Raman scattering, and fluorescence detection, enabling comprehensive, marker-free imaging of cellular architecture. These diverse modalities offer detailed spatial and molecular insights into cellular organization and interactions, critical for applications in biomedical research, drug discovery, and histological studies. To improve image quality without altering or introducing new biological information, we apply Zero-Shot Deconvolution Networks (ZS-DeconvNet), a deep-learning-based method that enhances resolution in an unsupervised manner. ZS-DeconvNet significantly refines image clarity and sharpness across multiple microscopy modalities without requiring large, labeled datasets, or introducing artifacts. By combining the strengths of multi-modal light sheet microscopy and ZS-DeconvNet, we achieve improved visualization of subcellular structures, offering clearer and more detailed representations of existing data. This approach holds significant potential for advancing high-resolution imaging in biomedical research and other related fields. Full article

Recent innovations in tissue clearing and light-sheet microscopy allow the rapid acquisition of intact micron-resolution images in fluorescently labeled samples. Automated, accurate, and high-throughput nuclear segmentation methods are in high demand to quantify the number of cells and evaluate cell-type specific marker co-labeling. Complete quantification of cellular level differences in genetically manipulated animal models will allow localization of organ structural differences well beyond what has previously been accomplished through slice histology or MRI. This paper proposes a nuclei identification tool for accurate nuclear segmentation from tissue-cleared microscopy images by regionally adaptive active learning. We gradually improved high-level nuclei-to-nuclei contextual heuristics to determine a non-linear mapping from local image appearance to the segmentation label at the center of each local neighborhood. In addition, we propose an adaptive fine-tuning (FT) strategy to tackle the complex segmentation task of separating nuclei in close proximity, allowing for the precise quantification of structures where nuclei are often densely packed. Compared to the current nuclei segmentation methods, we have achieved more accurate and robust nuclear segmentation results in various complex scenarios. Full article

Light Sheet Fluorescence Microscopy (LSFM) has emerged as a valuable tool for neurobiologists, enabling the rapid and high-quality volumetric imaging of mice brains. However, inherent artifacts and distortions introduced during the imaging process necessitate careful enhancement of LSFM images for optimal 3D reconstructions. This work aims to correct images slice by slice before reconstructing 3D volumes. Our approach involves a three-step process: firstly, the implementation of a deblurring algorithm using the work of K. Becker; secondly, an automatic contrast enhancement; and thirdly, the development of a convolutional denoising auto-encoder featuring skip connections to effectively address noise introduced by contrast enhancement, particularly excelling in handling mixed Poisson–Gaussian noise. Additionally, we tackle the challenge of axial distortion in LSFM by introducing an approach based on an auto-encoder trained on bead calibration images. The proposed pipeline demonstrates a complete solution, presenting promising results that surpass existing methods in denoising LSFM images. These advancements hold potential to significantly improve the interpretation of biological data. Full article

We present a combination of light-sheet excitation and two-dimensional fluorescence intensity ratio (FIR) measurements as a simple and promising technique for three-dimensional temperature mapping. The feasibility of this approach is demonstrated with samples fabricated with sodium yttrium fluoride nanoparticles co-doped with rare-earth ytterbium and erbium ions (NaYF₄:Yb³⁺/Er³⁺) incorporated into polydimethylsiloxane (PDMS) as a host material. In addition, we also evaluate the technique using lipid-coated NaYF₄:Yb³⁺/Er³⁺ nanoparticles immersed in agar. The composite materials show upconverted (UC) fluorescence bands when excited by a 980 nm near-infrared laser light-sheet. Using a single CMOS camera and a pair of interferometric optical filters to specifically image the two thermally-coupled bands (at 525 and 550 nm), the two-dimensional FIR and, hence, the temperature map can be readily obtained. The proposed method can take optically sectioned (confocal-like) images with good optical resolution over relatively large samples (up to the millimetric scale) for further 3D temperature reconstruction. Full article

Whole-brain imaging is important for understanding brain functions through deciphering tissue structures, neuronal circuits, and single-neuron tracing. Thus, many clearing methods have been developed to acquire whole-brain images or images of three-dimensional thick tissues. However, there are several limitations to imaging whole-brain volumes, including long image acquisition times, large volumes of data, and a long post-image process. Based on these limitations, many researchers are unsure about which light microscopy is most suitable for imaging thick tissues. Here, we compared fast-confocal microscopy with light-sheet fluorescence microscopy for whole-brain three-dimensional imaging, which can acquire images the fastest. To compare the two types of microscopies for large-volume imaging, we performed tissue clearing of a whole mouse brain, and changed the sample chamber and low- magnification objective lens and modified the sample holder of a light-sheet fluorescence microscope. We found out that light-sheet fluorescence microscopy using a 2.5× objective lens possesses several advantages, including saving time, large-volume image acquisitions, and high Z-resolution, over fast-confocal microscopy, which uses a 4× objective lens. Therefore, we suggest that light-sheet fluorescence microscopy is suitable for whole mouse brain imaging and for obtaining high-resolution three-dimensional images. Full article

Advances in imaging of microscopic structures are supported and complemented by adaptive visualization tools. These tools enable researchers to precisely capture and analyze complex three-dimensional structures of different kinds such as crystals, microchannels and electronic or biological material. In this contribution, we focus on 3D cell cultures. The new possibilities can play a particularly important role in biomedical research, especially here in the study of 3D cell cultures such as spheroids in the field of histology. By applying advanced imaging techniques, detailed information about the spatial arrangement and interactions between cells can be obtained. These insights help to gain a better understanding of cellular organization and function and have potential implications for the development of new therapies and drugs. In this context, this study presents a multi-modal light sheet microscope designed for the detection of elastic and inelastic light scattering, particularly Rayleigh scattering as well as the Stokes Raman effect and fluorescence for imaging purposes. By combining multiple modalities and stitching their individual results, three-dimensional objects are created combining complementary information for greater insight into spatial and molecular information. The individual components of the microscope are specifically selected to this end. Both Rayleigh and Stokes Raman scattering are inherent molecule properties and accordingly facilitate marker-free imaging. Consequently, altering influences on the sample by external factors are minimized. Furthermore, this article will give an outlook on possible future applications of the prototype microscope. Full article

The achievable resolution of a conventional imaging system is inevitably limited due to diffraction. Dealing with precise imaging in scattering media, such as in the case of biomedical imaging, is even more difficult owing to the weak signal-to-noise ratios. Recent developments in non-diffractive beams such as Bessel beams, Airy beams, vortex beams, and Mathieu beams have paved the way to tackle some of these challenges. This review specifically focuses on non-diffractive Bessel beams for ophthalmological applications. The theoretical foundation of the non-diffractive Bessel beam is discussed first followed by a review of various ophthalmological applications utilizing Bessel beams. The advantages and disadvantages of these techniques in comparison to those of existing state-of-the-art ophthalmological systems are discussed. The review concludes with an overview of the current developments and the future perspectives of non-diffractive beams in ophthalmology. Full article

Light sheet microscopy in live cells requires minimal excitation intensity and resolves three-dimensional (3D) information rapidly. Lattice light sheet microscopy (LLSM) works similarly but uses a lattice configuration of Bessel beams to generate a flatter, diffraction-limited z-axis sheet suitable for investigating subcellular compartments, with better tissue penetration. We developed a LLSM method for investigating cellular properties of tissue in situ. Neural structures provide an important target. Neurons are complex 3D structures, and signaling between cells and subcellular structures requires high resolution imaging. We developed an LLSM configuration based on the Janelia Research Campus design or in situ recording that allows simultaneous electrophysiological recording. We give examples of using LLSM to assess synaptic function in situ. In presynapses, evoked Ca²⁺ entry causes vesicle fusion and neurotransmitter release. We demonstrate the use of LLSM to measure stimulus-evoked localized presynaptic Ca²⁺ entry and track synaptic vesicle recycling. We also demonstrate the resolution of postsynaptic Ca²⁺ signaling in single synapses. A challenge in 3D imaging is the need to move the emission objective to maintain focus. We have developed an incoherent holographic lattice light-sheet (IHLLS) technique to replace the LLS tube lens with a dual diffractive lens to obtain 3D images of spatially incoherent light diffracted from an object as incoherent holograms. The 3D structure is reproduced within the scanned volume without moving the emission objective. This eliminates mechanical artifacts and improves temporal resolution. We focus on LLS and IHLLS applications and data obtained in neuroscience and emphasize increases in temporal and spatial resolution using these approaches. Full article

Two-photon excitation in light-sheet microscopy advances applications to live imaging of multicellular organisms. In a previous study, we developed a two-photon Bessel beam light-sheet microscope with a nearly 1-mm field of view and less than 4-μm axial resolution, using a low magnification (10×), middle numerical aperture (NA 0.5) detection objective. In this study, we aimed to construct a light-sheet microscope with higher resolution imaging while maintaining the large field of view, using low magnification (16×) with a high NA 0.8 objective. To address potential illumination and detection mismatch, we investigated the use of a depth of focus (DOF) extension method. Specifically, we used a stair-step device composed of five-layer annular zones that extended DOF two-fold, enough to cover the light-sheet thickness. Resolution measurements using fluorescent beads showed that the reduction in resolutions was small. We then applied this system to in vivo imaging of medaka fish and found that image quality degradation at the distal site of the beam injection could be compensated. This demonstrates that the extended DOF system combined with wide-field two-photon light-sheet microscopy offers a simple and easy setup for live imaging application of large multicellular organism specimens with sub-cellular resolution. Full article