scholarly journals Laser Scanning versus Wide-Field—Choosing the Appropriate Microscope in Life Sciences

2021 ◽  
Vol 11 (2) ◽  
pp. 733
Author(s):  
Herbert Schneckenburger ◽  
Verena Richter
Keyword(s):  
Laser Scanning ◽  
Light Exposure ◽  
Life Sciences ◽  
Super Resolution ◽  
Light Sheet ◽  
Laser Scanning Microscopy ◽  
Wide Field ◽  
Optimal Method ◽  
Recording Time ◽  
Light Sheet Microscopy

Methods and applications of light microscopy in the life sciences are compared with respect to 3D imaging, resolution, light exposure, sensitivity, and recording time. While conventional wide-field or laser scanning microscopy appear appropriate for smaller samples of only a few micrometers in size with a limited number of light exposures, light sheet microscopy appears to be an optimal method for larger 3D cell cultures, biopsies, or small organisms if multiple exposures or long measuring periods are desired. Super-resolution techniques should be considered in the context of high light exposure possibly causing photobleaching and photo-toxicity to living specimens.

scholarly journals Widefield light sheet microscopy using an Airy beam combined with deep-learning super-resolution

2020 ◽  
Author(s):  
Stella Corsetti ◽  
Philip Wijesinghe ◽  
Persephone B. Poulton ◽  
Shuzo Sakata ◽  
Khushi Vyas ◽  
...  
Keyword(s):  
Deep Learning ◽  
High Resolution ◽  
Dynamic Range ◽  
Tissue Sample ◽  
Super Resolution ◽  
Light Sheet ◽  
Wide Field ◽  
Light Sheet Microscopy ◽  
Airy Beam ◽  
Model Of Alzheimer’S Disease

AbstractImaging across length scales and in depth has been an important pursuit of widefield optical imaging. This promises to reveal fine cellular detail within a widefield snapshot of a tissue sample. Current advances often sacrifice resolution through selective sub-sampling to provide a wide field of view in a reasonable time scale. We demonstrate a new avenue for recovering high-resolution images from sub-sampled data in light-sheet microscopy using deep-learning super-resolution. We combine this with the use of a widefield Airy beam to achieve high-resolution imaging over extended fields of view and depths. We characterise our method on fluorescent beads as test targets. We then demonstrate improvements in imaging amyloid plaques in a cleared brain from a mouse model of Alzheimer’s disease, and in excised healthy and cancerous colon and breast tissues. This development can be widely applied in all forms of light sheet microscopy to provide a two-fold increase in the dynamic range of the imaged length scale. It has the potential to provide further insight into neuroscience, developmental biology and histopathology.

scholarly journals Fluorescence lifetime DNA-PAINT for multiplexed super-resolution imaging of cells

2022 ◽  
Vol 5 (1) ◽  
Author(s):  
Nazar Oleksiievets ◽  
Yelena Sargsyan ◽  
Jan Christoph Thiele ◽  
Nikolaos Mougios ◽  
Shama Sograte-Idrissi ◽  
...  
Keyword(s):  
Fluorescence Lifetime ◽  
Laser Scanning ◽  
Super Resolution ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Wide Field ◽  
Fluid Exchange ◽  
Multiplexed Imaging ◽  
Bio Imaging ◽  
A Cell

AbstractDNA point accumulation for imaging in nanoscale topography (DNA-PAINT) is a powerful super-resolution technique highly suitable for multi-target (multiplexing) bio-imaging. However, multiplexed imaging of cells is still challenging due to the dense and sticky environment inside a cell. Here, we combine fluorescence lifetime imaging microscopy (FLIM) with DNA-PAINT and use the lifetime information as a multiplexing parameter for targets identification. In contrast to Exchange-PAINT, fluorescence lifetime PAINT (FL-PAINT) can image multiple targets simultaneously and does not require any fluid exchange, thus leaving the sample undisturbed and making the use of flow chambers/microfluidic systems unnecessary. We demonstrate the potential of FL-PAINT by simultaneous imaging of up to three targets in a cell using both wide-field FLIM and 3D time-resolved confocal laser scanning microscopy (CLSM). FL-PAINT can be readily combined with other existing techniques of multiplexed imaging and is therefore a perfect candidate for high-throughput multi-target bio-imaging.

scholarly journals Challenges in 3D Live Cell Imaging

Photonics ◽  
2021 ◽  
Vol 8 (7) ◽  
pp. 275
Author(s):  
Herbert Schneckenburger ◽  
Verena Richter
Keyword(s):  
Live Cell Imaging ◽  
Laser Scanning ◽  
Cell Imaging ◽  
Live Cell ◽  
Laser Scanning Microscopy ◽  
Radiative Energy ◽  
Wide Field ◽  
Optical Sectioning ◽  
Continuous Increase ◽  
Radiative Energy Transfer

A short overview on 3D live cell imaging is given. Relevant samples are described and various problems and challenges—including 3D imaging by optical sectioning, light scattering and phototoxicity—are addressed. Furthermore, enhanced methods of wide-field or laser scanning microscopy together with some relevant examples and applications are summarized. In the future one may profit from a continuous increase in microscopic resolution, but also from molecular sensing techniques in the nanometer range using e.g., non-radiative energy transfer (FRET).

scholarly journals Circumvention of common labeling artifacts using secondary nanobodies

2019 ◽  
Author(s):  
Shama Sograte-Idrissi ◽  
Thomas Schlichthaerle ◽  
Carlos J. Duque-Afonso ◽  
Mihai Alevra ◽  
Sebastian Strauss ◽  
...  
Keyword(s):  
Super Resolution ◽  
Light Sheet ◽  
Common Procedure ◽  
Tissue Samples ◽  
Localization Accuracy ◽  
Light Sheet Microscopy ◽  
Large Size ◽  
Resolution Imaging ◽  
Single Domain Antibodies ◽  
Secondary Antibodies

AbstractThe most common procedure to reveal the location of specific (sub)cellular elements in biological samples is via immunostaining followed by optical imaging. This is typically performed with target-specific primary antibodies (1.Abs), which are revealed by fluorophore-conjugated secondary antibodies (2.Abs). However, at high resolution this methodology can induce a series of artifacts due to the large size of antibodies, their bivalency, and their polyclonality. Here we use STED and DNA-PAINT super-resolution microscopy or light sheet microscopy on cleared tissue to show how monovalent secondary reagents based on camelid single-domain antibodies (nanobodies; 2.Nbs) attenuate these artifacts. We demonstrate that monovalent 2.Nbs have four additional advantages: 1) they increase localization accuracy with respect to 2.Abs; 2) they allow direct pre-mixing with 1.Abs before staining, reducing experimental time, and enabling the use of multiple 1.Abs from the same species; 3) they penetrate thick tissues efficiently; and 4) they avoid the artificial clustering seen with 2.Abs both in live and in poorly fixed samples. Altogether, this suggests that 2.Nbs are a valuable alternative to 2.Abs, especially when super-resolution imaging or staining of thick tissue samples are involved.

scholarly journals Wide field intravital imaging by two-photon-excitation digital-scanned light-sheet microscopy (2p-DSLM) with a high-pulse energy laser

2014 ◽  
Vol 5 (10) ◽  
pp. 3311 ◽  
Author(s):  
Atsushi Maruyama ◽  
Yusuke Oshima ◽  
Hiroko Kajiura-Kobayashi ◽  
Shigenori Nonaka ◽  
Takeshi Imamura ◽  
...  
Keyword(s):  
Pulse Energy ◽  
Light Sheet ◽  
Intravital Imaging ◽  
Wide Field ◽  
High Pulse Energy ◽  
Two Photon ◽  
Light Sheet Microscopy ◽  
Photon Excitation ◽  
High Pulse ◽  
Energy Laser

scholarly journals Simultaneous multiview capture and fusion improves spatial resolution in wide-field and light-sheet microscopy

Optica ◽  
2016 ◽  
Vol 3 (8) ◽  
pp. 897 ◽  
Author(s):  
Yicong Wu ◽  
Panagiotis Chandris ◽  
Peter W. Winter ◽  
Edward Y. Kim ◽  
Valentin Jaumouillé ◽  
...  
Keyword(s):  
Spatial Resolution ◽  
Light Sheet ◽  
Wide Field ◽  
Light Sheet Microscopy

scholarly journals Multiview Deconvolution Two-photon Laser Scanning Microscopy

2020 ◽  
Author(s):  
Dimitrios Kapsokalyvas ◽  
Rodrigo Rosas ◽  
Rob Janssen ◽  
Jo Vanoevelen ◽  
Martin Strauch ◽  
...  
Keyword(s):  
Second Harmonic ◽  
Light Sheet ◽  
Laser Scanning Microscopy ◽  
Three Dimensions ◽  
Scanning Microscopy ◽  
Fluorescence Signal ◽  
Microscopy Imaging ◽  
Two Photon ◽  
Light Sheet Microscopy ◽  
3D Image Reconstruction

Abstract Imaging in three dimensions is necessary for thick tissues and small organisms. This is possible with tomographic optical microscopy techniques such as confocal, two-photon and light sheet microscopy. All these techniques suffer from anisotropic resolution and limited penetration depth. In the past, Multiview microscopy - imaging the sample from different angles followed by 3D image reconstruction - was developed to address this issue for light sheet microscopy based on fluorescence signal. In this study we applied this methodology to accomplish Multiview imaging with two-photon microscopy based on fluorescence and additionally second harmonic signal from myosin and collagen. It was shown that isotropic resolution was achieved, the entirety of the sample was visualized, and interference artifacts were suppressed allowing clear visualization of collagen fibrils and myofibrils. This method can be applied to any scanning microscopy technique without microscope modifications. It can be used for imaging tissue and whole mount small organisms such as heart tissue, and zebrafish larva in 3D, label-free or stained, with at least 3-fold axial resolution improvement which can be significant for the accurate quantification of small 3D structures.

scholarly journals A polymer gel index-matched to water enables diverse applications in fluorescence microscopy

2020 ◽  
Author(s):  
Xiaofei Han ◽  
Yijun Su ◽  
Hamilton White ◽  
Kate M. O’Neill ◽  
Nicole Y. Morgan ◽  
...  
Keyword(s):  
Surface Passivation ◽  
Super Resolution ◽  
Light Sheet ◽  
Polymer Gel ◽  
Structured Illumination ◽  
Structured Illumination Microscopy ◽  
Neural Structure ◽  
Uv Curable ◽  
Isotropic Resolution ◽  
Light Sheet Microscopy

AbstractWe demonstrate diffraction-limited and super-resolution imaging through thick layers (tens-hundreds of microns) of BIO-133, a biocompatible, UV-curable, commercially available polymer with a refractive index (RI) matched to water. We show that cells can be directly grown on BIO-133 substrates without the need for surface passivation and use this capability to perform extended time-lapse volumetric imaging of cellular dynamics 1) at isotropic resolution using dual-view light-sheet microscopy, and 2) at super-resolution using instant structured illumination microscopy. BIO-133 also enables immobilization of 1) Drosophila tissue, allowing us to track membrane puncta in pioneer neurons, and 2) Caenorhabditis elegans, which allows us to image and inspect fine neural structure and to track pan-neuronal calcium activity over hundreds of volumes. Finally, BIO-133 is compatible with other microfluidic materials, enabling optical and chemical perturbation of immobilized samples, as we demonstrate by performing drug and optogenetic stimulation on cells and C. elegans.

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