Lightsheet Microscopy

Dyer, Laura A.; Parker, Andrew; Paphiti, Keanu; Sanderson, Jeremy

doi:10.1002/cpz1.448

Cited by 9 publications

(5 citation statements)

References 295 publications

(300 reference statements)

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“…Light‐sheet microscopy, known as state‐of‐the‐art fluorescence microscopy, is a technique in which a sheet of excitation light is emitted from the side to obtain a cross‐sectional image of the light [ 21 , 22 ], which is essential for the spatial visualization of cleared bulk tumors [ 19 , 23 ]. If this tissue clearing and 3D fluorescence imaging technology can be applied to the pathological diagnosis of human cancers, it may lead to the visualization of the diversity of cancer cells and the distribution of various immune cells in cancer tissue [ 24 ], and to cancer diagnosis to realize personalized medicine suitable for each patient.…”

Section: Discussionmentioning

confidence: 99%

Volumetric imaging of the tumor microvasculature reflects outcomes and genomic states of clear cell renal cell carcinoma

Kaneko,

Masuda,

Takamatsu

et al. 2024

The Journal of Pathology CR

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Tumor structure is heterogeneous and complex, and it is difficult to obtain complete characteristics by two‐dimensional analysis. The aim of this study was to visualize and characterize volumetric vascular information of clear cell renal cell carcinoma (ccRCC) tumors using whole tissue phenotyping and three‐dimensional light‐sheet microscopy. Here, we used the diagnosing immunolabeled paraffin‐embedded cleared organs pipeline for tissue clearing, immunolabeling, and three‐dimensional imaging. The spatial distributions of CD34, which targets blood vessels, and LYVE‐1, which targets lymphatic vessels, were examined by calculating three‐dimensional density, vessel length, vessel radius, and density curves, such as skewness, kurtosis, and variance of the expression. We then examined those associations with ccRCC outcomes and genetic alteration state. Formalin‐fixed paraffin‐embedded tumor samples from 46 ccRCC patients were included in the study. Receiver operating characteristic curve analyses revealed the associations between blood vessel and lymphatic vessel distributions and pathological factors such as a high nuclear grade, large tumor size, and the presence of venous invasion. Furthermore, three‐dimensional imaging parameters stratified ccRCC patients regarding survival outcomes. An analysis of genomic alterations based on volumetric vascular information parameters revealed that PI3K‐mTOR pathway mutations related to the blood vessel radius were significantly different. Collectively, we have shown that the spatial elucidation of volumetric vasculature information could be prognostic and may serve as a new biomarker for genomic alterations. High‐end tissue clearing techniques and volumetric immunohistochemistry enable three‐dimensional analysis of tumors, leading to a better understanding of the microvascular structure in the tumor space.

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Section: Discussionmentioning

confidence: 99%

Volumetric imaging of the tumor microvasculature reflects outcomes and genomic states of clear cell renal cell carcinoma

Kaneko,

Masuda,

Takamatsu

et al. 2024

The Journal of Pathology CR

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“…These are key to understanding the improvements of this architecture with respect to the state of the art. Recent manuscripts show how SPIM can acquire high-resolution images with low-phototoxicity [ 40 , 41 ]. In particular, the inclusion of the two Galvo motors allows for imaging with a static sample stage.…”

Section: Discussionmentioning

confidence: 99%

Single Plane Illumination Microscopy for Microfluidic Device Imaging

et al. 2022

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Three-dimensional imaging of live processes at a cellular level is a challenging task. It requires high-speed acquisition capabilities, low phototoxicity, and low mechanical disturbances. Three-dimensional imaging in microfluidic devices poses additional challenges as a deep penetration of the light source is required, along with a stationary setting, so the flows are not perturbed. Different types of fluorescence microscopy techniques have been used to address these limitations; particularly, confocal microscopy and light sheet fluorescence microscopy (LSFM). This manuscript proposes a novel architecture of a type of LSFM, single-plane illumination microscopy (SPIM). This custom-made microscope includes two mirror galvanometers to scan the sample vertically and reduce shadowing artifacts while avoiding unnecessary movement. In addition, two electro-tunable lenses fine-tune the focus position and reduce the scattering caused by the microfluidic devices. The microscope has been fully set up and characterized, achieving a resolution of 1.50 μm in the x-y plane and 7.93 μm in the z-direction. The proposed architecture has risen to the challenges posed when imaging microfluidic devices and live processes, as it can successfully acquire 3D volumetric images together with time-lapse recordings, and it is thus a suitable microscopic technique for live tracking miniaturized tissue and disease models.

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“…Just as with single‐photon confocal microscopy and selective plane illumination (lightsheet) microscopy (Dyer, Parker, Paphiti, & Sanderson, 2022), it can be advantageous to optically clear tissue prior to acquiring images. Benzyl alcohol (Clendenon, Young, Ferkowicz, Phillips, & Dunn, 2011), benzyl alcohol with benzyl benzoate (BABB; see Becker, Jährling, Saghafi, Weiler, & Dodt, 2012), methyl salicylate (MacDonald & Rubel, 2008), and mineral oil with glycerol (Wilson, Degan, Warren, & Fischer, 2012) have been used to clear thick tissues to reduce scattering.…”

Section: Fundamental Principlesmentioning

confidence: 99%

Multi‐Photon Microscopy

Sanderson

2023

Current Protocols

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In this series of papers on light microscopy imaging, we have covered the fundamentals of microscopy, super-resolution microscopy, and lightsheet microscopy. This last review covers multi-photon microscopy with a brief reference to intravital imaging and Brainbow labeling. Multi-photon microscopy is often referred to as two-photon microscopy. Indeed, using two-photon microscopy is by far the most common way of imaging thick tissues; however, it is theoretically possible to use a higher number of photons, and three-photon microscopy is possible. Therefore, this review is titled "multi-photon microscopy." Another term for describing multi-photon microscopy is "non-linear" microscopy because fluorescence intensity at the focal spot depends upon the average squared intensity rather than the squared average intensity; hence, non-linear optics (NLO) is an alternative name for multi-photon microscopy. It is this non-linear relationship (or third exponential power in the case of three-photon excitation) that determines the axial optical sectioning capability of multi-photon imaging. In this paper, the necessity for two-photon or multi-photon imaging is explained, and the method of optical sectioning by multi-photon microscopy is described. Advice is also given on what fluorescent markers to use and other practical aspects of imaging thick tissues. The technique of Brainbow imaging is discussed. The review concludes with a description of intravital imaging of the mouse.

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Lightsheet Microscopy

Cited by 9 publications

References 295 publications

Volumetric imaging of the tumor microvasculature reflects outcomes and genomic states of clear cell renal cell carcinoma

Volumetric imaging of the tumor microvasculature reflects outcomes and genomic states of clear cell renal cell carcinoma

Single Plane Illumination Microscopy for Microfluidic Device Imaging

Multi‐Photon Microscopy

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