Light sheet fluorescence microscopy (LSFM): past, present and future

The Analyst ◽  
2014 ◽  
Vol 139 (19) ◽  
pp. 4758-4768 ◽  
Author(s):  
John Lim ◽  
Hwee Kuan Lee ◽  
Weimiao Yu ◽  
Sohail Ahmed
Keyword(s):  
Fluorescence Microscopy ◽  
Imaging Modality ◽  
Light Sheet ◽  
Light Sheet Fluorescence Microscopy ◽  
Over Time

Light sheet fluorescence microscopy (LSFM) has emerged as an important imaging modality to follow biology in live 3D samples over time with reduced phototoxicity and photobleaching.

Use of high-refractive index hydrogels and tissue clearing for large biological sample imaging (Preprint)

2021 ◽  
Author(s):  
Alexander Richardson ◽  
Sandra Fok ◽  
Victoria Lee ◽  
Kerry-Anne Rye ◽  
Nick Di Girolamo ◽  
...  
Keyword(s):  
Refractive Index ◽  
Fluorescence Microscopy ◽  
Water Content ◽  
Light Sheet ◽  
High Water ◽  
High Refractive Index ◽  
High Water Content ◽  
Tissue Clearing ◽  
Light Sheet Fluorescence Microscopy ◽  
Over Time

UNSTRUCTURED Recent advances in tissue clearing and light sheet fluorescence microscopy have improved insights into and understanding of tissue morphology and disease pathology by imaging large samples without the requirement of histological sectioning. However, sample handling and conservation of sample integrity during lengthy staining and acquisition protocols remains a challenge. This study overcomes these challenges with acrylamide hydrogels synthesised to match the refractive index of solutions typically utilised in aqueous tissue clearing protocols. These hydrogels have a high-water content (82.0+/-3.7% by weight). The gels are stable over time and FITC-IgG readily permeated into, and effluxed out of them. Whilst the gels deformed and/or swelled over time in some commonly used solutions, this was overcome by using a previously described custom refractive index matched solution. To validate their use, CUBIC cleared mouse tissues and whole embryos were embedded in hydrogels, stained using fluorescent small molecule dyes, labels and antibodies and successfully imaged using light sheet fluorescence microscopy. In conclusion, the high-water content, high refractive index hydrogels described in this study have a broad applicability to research that delves into pathophysiological processes by stabilising and protecting large and fragile samples.

scholarly journals Use of high-refractive index hydrogels and tissue clearing for large biological sample imaging

2020 ◽  
Author(s):  
Alexander Richardson ◽  
Sandra Fok ◽  
Victoria Lee ◽  
Kerry-Anne Rye ◽  
Nick Di Girolamo ◽  
...  
Keyword(s):  
Refractive Index ◽  
Fluorescence Microscopy ◽  
Water Content ◽  
Light Sheet ◽  
High Water ◽  
High Refractive Index ◽  
High Water Content ◽  
Tissue Clearing ◽  
Light Sheet Fluorescence Microscopy ◽  
Over Time

ABSTRACTRecent advances in tissue clearing and light sheet fluorescence microscopy have improved insights into and understanding of tissue morphology and disease pathology by imaging large samples without the requirement of histological sectioning. However, sample handling and conservation of sample integrity during lengthy staining and acquisition protocols remains a challenge. This study overcomes these challenges with acrylamide hydrogels synthesised to match the refractive index of solutions typically utilised in aqueous tissue clearing protocols. These hydrogels have a high-water content (82.0±3.7% by weight). The gels are stable over time and FITC-IgG readily permeated into, and effluxed out of them. Whilst the gels deformed and/or swelled over time in some commonly used solutions, this was overcome by using a previously described custom RIMS solution. To validate their use, CUBIC cleared mouse tissues and whole embryos were embedded in hydrogels, stained using fluorescent small molecule dyes, labels and antibodies and successfully imaged using light sheet fluorescence microscopy. In conclusion, the high-water content, high refractive index hydrogels described in this study have a broad applicability to research that delves into pathophysiological processes by stabilisating and protecting large and fragile samples.

scholarly journals Use of High-Refractive Index Hydrogels and Tissue Clearing for Large Biological Sample Imaging

Gels ◽  
2022 ◽  
Vol 8 (1) ◽  
pp. 32
Author(s):  
Alexander Richardson ◽  
Sandra Fok ◽  
Victoria Lee ◽  
Kerry-Anne Rye ◽  
Nick Di Girolamo ◽  
...  
Keyword(s):  
Refractive Index ◽  
Fluorescence Microscopy ◽  
Water Content ◽  
Light Sheet ◽  
High Water ◽  
High Refractive Index ◽  
High Water Content ◽  
Tissue Clearing ◽  
Light Sheet Fluorescence Microscopy ◽  
Over Time

Recent advances in tissue clearing and light sheet fluorescence microscopy have improved insights into and understanding of tissue morphology and disease pathology by imaging large samples without the requirement of histological sectioning. However, sample handling and conservation of sample integrity during lengthy staining and acquisition protocols remains a challenge. This study overcomes these challenges with acrylamide hydrogels synthesised to match the refractive index of solutions typically utilised in aqueous tissue clearing protocols. These hydrogels have a high-water content (82.0 ± 3.7% by weight). The gels are stable over time and FITC-IgG readily permeated into and effluxed out of them. Whilst the gels deformed and/or swelled over time in some commonly used solutions, this was overcome by using a previously described custom refractive index matched solution. To validate their use, CUBIC cleared mouse tissues and whole embryos were embedded in hydrogels, stained using fluorescent small molecule dyes, labels and antibodies and successfully imaged using light sheet fluorescence microscopy. In conclusion, the high water content, high refractive index hydrogels described in this study have broad applicability to research that delves into pathophysiological processes by stabilising and protecting large and fragile samples.

scholarly journals Three-photon light-sheet fluorescence microscopy

2018 ◽  
Author(s):  
Adriá Escobet-Montalbán ◽  
Federico M. Gasparoli ◽  
Jonathan Nylk ◽  
Pengfei Liu ◽  
Zhengyi Yang ◽  
...  
Keyword(s):  
Fluorescence Microscopy ◽  
Imaging Modality ◽  
Light Sheet ◽  
Wide Field ◽  
Excitation Light ◽  
Volumetric Imaging ◽  
Cellular Spheroids ◽  
Light Sheet Microscopy ◽  
Photon Excitation ◽  
Light Sheet Fluorescence Microscopy

We present the first demonstration of three-photon excitation light-sheet fluorescence microscopy. Light-sheet fluorescence microscopy in single- and two-photon modes has emerged as a powerful wide-field, low photo-damage technique for fast volumetric imaging of biological samples. We extend this imaging modality to the three-photon regime enhancing its penetration depth. Our present study uses a standard conventional femtosecond pulsed laser at 1000 nm wavelength for the imaging of 450 µm diameter cellular spheroids. In addition, we show, experimentally and through numerical simulations, the potential advantages in three-photon light-sheet microscopy of using propagation-invariant Bessel beams in preference to Gaussian beams.

scholarly journals Cytotoxic effects and tolerability of gemcitabine and axitinib in a xenograft model for c-myc amplified medulloblastoma

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Stefanie Schwinn ◽  
Zeinab Mokhtari ◽  
Sina Thusek ◽  
Theresa Schneider ◽  
Anna-Leena Sirén ◽  
...  
Keyword(s):  
Fluorescence Microscopy ◽  
Tumor Growth ◽  
Intensive Therapy ◽  
Xenograft Model ◽  
Light Sheet ◽  
Tumor Control ◽  
Orthotopic Model ◽  
Group 3 ◽  
Light Sheet Fluorescence Microscopy

AbstractMedulloblastoma is the most common high-grade brain tumor in childhood. Medulloblastomas with c-myc amplification, classified as group 3, are the most aggressive among the four disease subtypes resulting in a 5-year overall survival of just above 50%. Despite current intensive therapy regimens, patients suffering from group 3 medulloblastoma urgently require new therapeutic options. Using a recently established c-myc amplified human medulloblastoma cell line, we performed an in-vitro-drug screen with single and combinatorial drugs that are either already clinically approved or agents in the advanced stage of clinical development. Candidate drugs were identified in vitro and then evaluated in vivo. Tumor growth was closely monitored by BLI. Vessel development was assessed by 3D light-sheet-fluorescence-microscopy. We identified the combination of gemcitabine and axitinib to be highly cytotoxic, requiring only low picomolar concentrations when used in combination. In the orthotopic model, gemcitabine and axitinib showed efficacy in terms of tumor control and survival. In both models, gemcitabine and axitinib were better tolerated than the standard regimen comprising of cisplatin and etoposide phosphate. 3D light-sheet-fluorescence-microscopy of intact tumors revealed thinning and rarefication of tumor vessels, providing one explanation for reduced tumor growth. Thus, the combination of the two drugs gemcitabine and axitinib has favorable effects on preventing tumor progression in an orthotopic group 3 medulloblastoma xenograft model while exhibiting a favorable toxicity profile. The combination merits further exploration as a new approach to treat high-risk group 3 medulloblastoma.

Investigating safe excitation in Light Sheet Fluorescence Microscopy

2021 ◽  
Vol 84 ◽  
pp. 296
Author(s):  
Gideon Oluniran ◽  
James Blackwell ◽  
Emmanuel Reynaud ◽  
Marcin Krasny ◽  
Niall Colgan ◽  
...  
Keyword(s):  
Fluorescence Microscopy ◽  
Light Sheet ◽  
Light Sheet Fluorescence Microscopy

scholarly journals Light sheet fluorescence microscopy of optically cleared brains for studying the glymphatic system

2020 ◽  
Vol 40 (10) ◽  
pp. 1975-1986
Author(s):  
Nicholas B Bèchet ◽  
Tekla M Kylkilahti ◽  
Bengt Mattsson ◽  
Martina Petrasova ◽  
Nagesh C Shanbhag ◽  
...  
Keyword(s):  
Fluorescence Microscopy ◽  
Fluid Transport ◽  
Light Sheet ◽  
Brain Parenchyma ◽  
Sleep State ◽  
Glymphatic System ◽  
Light Sheet Microscopy ◽  
Highly Active ◽  
Quantitative Analyses ◽  
Light Sheet Fluorescence Microscopy

Fluid transport in the perivascular space by the glia-lymphatic (glymphatic) system is important for the removal of solutes from the brain parenchyma, including peptides such as amyloid-beta which are implicated in the pathogenesis of Alzheimer’s disease. The glymphatic system is highly active in the sleep state and under the influence of certain of anaesthetics, while it is suppressed in the awake state and by other anaesthetics. Here we investigated whether light sheet fluorescence microscopy of whole optically cleared murine brains was capable of detecting glymphatic differences in sleep- and awake-mimicking anaesthesia, respectively. Using light-sheet imaging of whole brains, we found anaesthetic-dependent cerebrospinal fluid (CSF) influx differences, including reduced tracer influx along tertiary branches of the middle cerebral artery and reduced influx along dorsal and anterior penetrating arterioles, in the awake-mimicking anaesthesia. This study establishes that light sheet microscopy of optically cleared brains is feasible for quantitative analyses and can provide images of the entire glymphatic system in whole brains.

FEATURES

2016 ◽  
Vol 20 (12) ◽  
pp. 12-22
Keyword(s):  
Medical Imaging ◽  
Fluorescence Microscopy ◽  
Fluorescence Spectroscopy ◽  
Medical Images ◽  
Light Sheet ◽  
The Future ◽  
Light Sheet Fluorescence Microscopy

Scanning the Future of Medical Imaging Putting Numbers into Biology: The Combination of Light Sheet Fluorescence Microscopy and Fluorescence Spectroscopy Abyss Processing – Exploring the Deep in Medical Images

scholarly journals Spatial and Temporal Features of the Growth of a Bacterial Species Colonizing the Zebrafish Gut

mBio ◽  
2014 ◽  
Vol 5 (6) ◽  
Author(s):  
Matthew Jemielita ◽  
Michael J. Taormina ◽  
Adam R. Burns ◽  
Jennifer S. Hampton ◽  
Annah S. Rolig ◽  
...  
Keyword(s):  
Fluorescence Microscopy ◽  
Spatial Structure ◽  
Growth Kinetics ◽  
Spatial Organization ◽  
Temporal Dynamics ◽  
Bacterial Species ◽  
Bacterial Load ◽  
Light Sheet ◽  
Logistic Growth ◽  
Light Sheet Fluorescence Microscopy

ABSTRACTThe vertebrate intestine is home to microbial ecosystems that play key roles in host development and health. Little is known about the spatial and temporal dynamics of these microbial communities, limiting our understanding of fundamental properties, such as their mechanisms of growth, propagation, and persistence. To address this, we inoculated initially germ-free zebrafish larvae with fluorescently labeled strains of anAeromonasspecies, representing an abundant genus in the zebrafish gut. Using light sheet fluorescence microscopy to obtain three-dimensional images spanning the gut, we quantified the entire bacterial load, as founding populations grew from tens to tens of thousands of cells over several hours. The data yield the first ever measurements of the growth kinetics of a microbial species inside a live vertebrate intestine and show dynamics that robustly fit a logistic growth model. Intriguingly, bacteria were nonuniformly distributed throughout the gut, and bacterial aggregates showed considerably higher growth rates than did discrete individuals. The form of aggregate growth indicates intrinsically higher division rates for clustered bacteria, rather than surface-mediated agglomeration onto clusters. Thus, the spatial organization of gut bacteria both relative to the host and to each other impacts overall growth kinetics, suggesting that spatial characterizations will be an important input to predictive models of host-associated microbial community assembly.IMPORTANCEOur intestines are home to vast numbers of microbes that influence many aspects of health and disease. Though we now know a great deal about the constituents of the gut microbiota, we understand very little about their spatial structure and temporal dynamics in humans or in any animal: how microbial populations establish themselves, grow, fluctuate, and persist. To address this, we made use of a model organism, the zebrafish, and a new optical imaging technique, light sheet fluorescence microscopy, to visualize for the first time the colonization of a live, vertebrate gut by specific bacteria with sufficient resolution to quantify the population over a range from a few individuals to tens of thousands of bacterial cells. Our results provide unprecedented measures of bacterial growth kinetics and also show the influence of spatial structure on bacterial populations, which can be revealed only by direct imaging.

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