Faculty Opinions recommendation of Measuring tubulin content in Toxoplasma gondii: a comparison of laser-scanning confocal and wide-field fluorescence microscopy.

ABSTRACTWe describe the subcellular location of chitin synthase 1 (CHS-1), one of seven chitin synthases inNeurospora crassa. Laser scanning confocal microscopy of growing hyphae showed CHS-1–green fluorescent protein (GFP) localized conspicuously in regions of active wall synthesis, namely, the core of the Spitzenkörper (Spk), the apical cell surface, and developing septa. It was also present in numerous fine particles throughout the cytoplasm plus some large vacuoles in distal hyphal regions. Although the same general subcellular distribution was observed previously for CHS-3 and CHS-6, they did not fully colocalize. Dual labeling showed that the three different chitin synthases were contained in different vesicular compartments, suggesting the existence of a different subpopulation of chitosomes for each CHS. CHS-1–GFP persisted in the Spk during hyphal elongation but disappeared from the septum after its development was completed. Wide-field fluorescence microscopy and total internal reflection fluorescence microscopy revealed subapical clouds of particles, suggestive of chitosomes moving continuously toward the Spk. Benomyl had no effect on CHS-1–GFP localization, indicating that microtubules are not strictly required for CHS trafficking to the hyphal apex. Conversely, actin inhibitors caused severe mislocalization of CHS-1–GFP, indicating that actin plays a major role in the orderly traffic and localization of CHS-1 at the apex.

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High Resolution 3-D Fluorescence Microscopy: A Comparison of Confocal Laser Scanning Microscopy and a wide-Field Deconvolution Technique

Microscopy Today ◽

10.1017/s1551929500056054 ◽

1997 ◽

Vol 5 (6) ◽

pp. 10-13 ◽

Cited By ~ 1

Author(s):

Keyword(s):

Biological Sciences ◽

Fluorescence Microscopy ◽

Light Microscopy ◽

Laser Scanning ◽

Laser Scanning Microscopy ◽

Specific Structure ◽

Confocal Laser ◽

Wide Field ◽

Deconvolution Technique ◽

Transmitted Light Microscopy

Advances in the biological sciences have given rise for the need to visualize microscopic structures of interest. As the requirement to see specific structure and sub-resolution details arise, investigators have turned to fluorescent probes to label and observe these details, which are often undetectable using conventional methods in light microscopy. Fluorescence microscopy offers many advantages for visualizing specific structure and sub-resolution details, which are often undetectable using transmitted light microscopy. Recent advances in fluorescent probe and microscope design, as well as imaging instrumentation designed for fluorescence applications, are now permitting life-science researchers to view details in regions of interest with increasing precision, accuracy, and resolution.

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Recurrent neural network-based volumetric fluorescence microscopy

Light Science & Applications ◽

10.1038/s41377-021-00506-9 ◽

2021 ◽

Vol 10 (1) ◽

Author(s):

Luzhe Huang ◽

Hanlong Chen ◽

Yilin Luo ◽

Yair Rivenson ◽

Aydogan Ozcan

Keyword(s):

Neural Network ◽

Image Reconstruction ◽

Fluorescence Microscopy ◽

Recurrent Network ◽

Sample Volume ◽

Depth Of Field ◽

Objective Lens ◽

Wide Field ◽

Volumetric Imaging ◽

3D Image Reconstruction

AbstractVolumetric imaging of samples using fluorescence microscopy plays an important role in various fields including physical, medical and life sciences. Here we report a deep learning-based volumetric image inference framework that uses 2D images that are sparsely captured by a standard wide-field fluorescence microscope at arbitrary axial positions within the sample volume. Through a recurrent convolutional neural network, which we term as Recurrent-MZ, 2D fluorescence information from a few axial planes within the sample is explicitly incorporated to digitally reconstruct the sample volume over an extended depth-of-field. Using experiments on C. elegans and nanobead samples, Recurrent-MZ is demonstrated to significantly increase the depth-of-field of a 63×/1.4NA objective lens, also providing a 30-fold reduction in the number of axial scans required to image the same sample volume. We further illustrated the generalization of this recurrent network for 3D imaging by showing its resilience to varying imaging conditions, including e.g., different sequences of input images, covering various axial permutations and unknown axial positioning errors. We also demonstrated wide-field to confocal cross-modality image transformations using Recurrent-MZ framework and performed 3D image reconstruction of a sample using a few wide-field 2D fluorescence images as input, matching confocal microscopy images of the same sample volume. Recurrent-MZ demonstrates the first application of recurrent neural networks in microscopic image reconstruction and provides a flexible and rapid volumetric imaging framework, overcoming the limitations of current 3D scanning microscopy tools.

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Choline and phosphatidylcholine fluorescent derivatives localization in carcinoma cells studied by laser scanning confocal fluorescence microscopy

European Journal of Cancer ◽

10.1016/j.ejca.2005.02.028 ◽

2005 ◽

Vol 41 (10) ◽

pp. 1453-1459 ◽

Cited By ~ 11

Author(s):

Anna M. Villa ◽

Elvira Caporizzo ◽

Antonio Papagni ◽

Luciano Miozzo ◽

Paola Del Buttero ◽

...

Keyword(s):

Fluorescence Microscopy ◽

Laser Scanning ◽

Carcinoma Cells ◽

Confocal Fluorescence Microscopy ◽

Confocal Fluorescence ◽

Fluorescent Derivatives

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Advanced Static and Dynamic Fluorescence Microscopy Techniques to Investigate Drug Delivery Systems

Pharmaceutics ◽

10.3390/pharmaceutics13060861 ◽

2021 ◽

Vol 13 (6) ◽

pp. 861

Author(s):

Jacopo Cardellini ◽

Arianna Balestri ◽

Costanza Montis ◽

Debora Berti

Keyword(s):

Drug Delivery ◽

Fluorescence Microscopy ◽

Drug Delivery Systems ◽

Laser Scanning ◽

Super Resolution ◽

Laser Scanning Confocal Microscopy ◽

Delivery Systems ◽

Scanning Confocal Microscopy ◽

Biological Phenomena

In the past decade(s), fluorescence microscopy and laser scanning confocal microscopy (LSCM) have been widely employed to investigate biological and biomimetic systems for pharmaceutical applications, to determine the localization of drugs in tissues or entire organisms or the extent of their cellular uptake (in vitro). However, the diffraction limit of light, which limits the resolution to hundreds of nanometers, has for long time restricted the extent and quality of information and insight achievable through these techniques. The advent of super-resolution microscopic techniques, recognized with the 2014 Nobel prize in Chemistry, revolutionized the field thanks to the possibility to achieve nanometric resolution, i.e., the typical scale length of chemical and biological phenomena. Since then, fluorescence microscopy-related techniques have acquired renewed interest for the scientific community, both from the perspective of instrument/techniques development and from the perspective of the advanced scientific applications. In this contribution we will review the application of these techniques to the field of drug delivery, discussing how the latest advancements of static and dynamic methodologies have tremendously expanded the experimental opportunities for the characterization of drug delivery systems and for the understanding of their behaviour in biologically relevant environments.

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Challenges in 3D Live Cell Imaging

Photonics ◽

10.3390/photonics8070275 ◽

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).

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Low-Light Photodetectors for Fluorescence Microscopy

Applied Sciences ◽

10.3390/app11062773 ◽

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.

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