scholarly journals Breaking the diffraction limit of light-sheet fluorescence microscopy by RESOLFT

2016 ◽  
Vol 113 (13) ◽  
pp. 3442-3446 ◽  
Author(s):  
Patrick Hoyer ◽  
Gustavo de Medeiros ◽  
Bálint Balázs ◽  
Nils Norlin ◽  
Christina Besir ◽  
...  
Keyword(s):  
Focal Plane ◽  
Axial Direction ◽  
Light Sheet ◽  
Molecular State ◽  
Wide Field ◽  
Low Light ◽  
Optical Fluorescence ◽  
Plane Scanning ◽  
Diffraction Barrier ◽  
Light Sheet Fluorescence Microscopy

We present a plane-scanning RESOLFT [reversible saturable/switchable optical (fluorescence) transitions] light-sheet (LS) nanoscope, which fundamentally overcomes the diffraction barrier in the axial direction via confinement of the fluorescent molecular state to a sheet of subdiffraction thickness around the focal plane. To this end, reversibly switchable fluorophores located right above and below the focal plane are transferred to a nonfluorescent state at each scanning step. LS-RESOLFT nanoscopy offers wide-field 3D imaging of living biological specimens with low light dose and axial resolution far beyond the diffraction barrier. We demonstrate optical sections that are thinner by 5–12-fold compared with their conventional diffraction-limited LS analogs.

scholarly journals Light-Sheet Fluorescence Microscopy with Scanning Non-diffracting Beams

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Hosein Kafian ◽  
Meelad Lalenejad ◽  
Sahar Moradi-Mehr ◽  
Shiva Akbari Birgani ◽  
Daryoush Abdollahpour
Keyword(s):  
Fluorescence Microscopy ◽  
Spatial Coherence ◽  
Three Dimensional ◽  
Light Sheet ◽  
Tissue Imaging ◽  
Wide Field ◽  
Self Healing ◽  
Main Challenge ◽  
Cancer Tumor ◽  
Light Sheet Fluorescence Microscopy

Abstract Light-sheet fluorescence microscopy (LSFM) has now become a unique tool in different fields ranging from three-dimensional (3D) tissue imaging to real-time functional imaging of neuronal activities. Nevertheless, obtaining high-quality artifact-free images from large, dense and inhomogeneous samples is the main challenge of the method that still needs to be adequately addressed. Here, we demonstrate significant enhancement of LSFM image qualities by using scanning non-diffracting illuminating beams, both through experimental and numerical investigations. The effect of static and scanning illumination with several beams are analyzed and compared, and it is shown that scanning 2D Airy light-sheet is minimally affected by the inhomogeneities in the samples, and provides higher contrasts and uniform resolution over a wide field-of-view, due to its reduced spatial coherence, self-healing feature and longer penetration depth. Further, the capabilities of the illumination scheme is utilized for both single-and double-wavelength 3D imaging of large and dense mammospheres of cancer tumor cells as complex inhomogeneous biological samples.

scholarly journals Light-Sheet Fluorescence Microscopy with Scanning Non-diffracting Beams

2019 ◽  
Author(s):  
Hosein Kafian ◽  
Meelad Lalenejad ◽  
Sahar Moradi-Mehr ◽  
Shiva Akbari Birgani ◽  
Daryoush Abdollahpour
Keyword(s):  
Fluorescence Microscopy ◽  
Spatial Coherence ◽  
Three Dimensional ◽  
Light Sheet ◽  
Tissue Imaging ◽  
Wide Field ◽  
Self Healing ◽  
Main Challenge ◽  
Cancer Tumor ◽  
Light Sheet Fluorescence Microscopy

AbstractLight-sheet fluorescence microscopy (LSFM) has now become a unique technique in different fields ranging from three-dimensional (3D) tissue imaging to real-time functional imaging of neuronal activities. Nevertheless, obtaining high-quality artifact-free images from large, dense and inhomogeneous samples is the main challenge of the method that still needs to be adequately addressed. Here, we demonstrate significant enhancement of LSFM image qualities by using scanning non-diffracting illuminating beams, both through experimental and numerical investigations. The effect of static and scanning illumination with several beams are analyzed and compared, and it is shown that scanning 2D Airy light sheet is minimally affected by the artifacts, and provide higher contrasts and uniform resolution over a wide field-of-view, due to its reduced spatial coherence, self-healing feature and higher penetration depth. Further, the capabilities of the illumination scheme is utilized for both single and double wavelength 3D imaging of a large and dense mammospheres of cancer tumor cells as complex inhomogeneous biological 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 Low-Light Photodetectors for Fluorescence Microscopy

2021 ◽  
Vol 11 (6) ◽  
pp. 2773
Author(s):  
Hiroaki Yokota ◽  
Atsuhito Fukasawa ◽  
Minako Hirano ◽  
Toru Ide
Keyword(s):  
Fluorescence Microscopy ◽  
Fluorescence Imaging ◽  
Single Molecule ◽  
Science Studies ◽  
Single Photon ◽  
Laser Scanning Microscopy ◽  
Oxide Semiconductor ◽  
Wide Field ◽  
Single Molecule Fluorescence ◽  
Low Light

Over the years, fluorescence microscopy has evolved and has become a necessary element of life science studies. Microscopy has elucidated biological processes in live cells and organisms, and also enabled tracking of biomolecules in real time. Development of highly sensitive photodetectors and light sources, in addition to the evolution of various illumination methods and fluorophores, has helped microscopy acquire single-molecule fluorescence sensitivity, enabling single-molecule fluorescence imaging and detection. Low-light photodetectors used in microscopy are classified into two categories: point photodetectors and wide-field photodetectors. Although point photodetectors, notably photomultiplier tubes (PMTs), have been commonly used in laser scanning microscopy (LSM) with a confocal illumination setup, wide-field photodetectors, such as electron-multiplying charge-coupled devices (EMCCDs) and scientific complementary metal-oxide-semiconductor (sCMOS) cameras have been used in fluorescence imaging. This review focuses on the former low-light point photodetectors and presents their fluorescence microscopy applications and recent progress. These photodetectors include conventional PMTs, single photon avalanche diodes (SPADs), hybrid photodetectors (HPDs), in addition to newly emerging photodetectors, such as silicon photomultipliers (SiPMs) (also known as multi-pixel photon counters (MPPCs)) and superconducting nanowire single photon detectors (SSPDs). In particular, this review shows distinctive features of HPD and application of HPD to wide-field single-molecule fluorescence detection.

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.

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