Overview
ABSTRACT
Fluorescence microscopy is a photonic technique that requires a specific instrument, and fluorescent substances. This article outlines both aspects. This technique is mainly used on biological samples. We describe a battery of probes targeting biological molecules, along with the use of fluorescent fusion proteins such as GFP. A wide range of techniques is described related to the benefits for ease of observation: wide field, confocal, super-resolution optical sections, sample protection, and video microscopy of living material. There is a dynamic industrial base, composed of large instrumentation companies, manufacturers of devices (cameras, optomechanics, chemistry), optical and biomedical research laboratories, and pharmaceutical companies.
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Read the articleAUTHORS
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Léon ESPINOSA: Doctor of Science - CNRS Research Engineer - Head of the microscopy and screening department at LCB (Laboratoire de Chimie Bactérienne) UMR CNRS 7283 Aix Marseille Université
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Yves TOURNEUR: Engineer École Centrale de Lyon - Doctor of Science, CNRS researcher, INSERM U1060 laboratory - Platform Manager, Centre de quantimétrie, Université Claude Bernard Lyon 1
INTRODUCTION
This article describes the main techniques of fluorescence microscopy, existing commercial devices, and technologies under development. The main field of use for fluorescence microscopy is biomedical, with applications in fundamental and applied research, diagnostics, quality control and more. Other applications, particularly in chemistry and materials science, use the same principles described here from the point of view of life science applications. Today, fluorescence microscopy is used to study biological structures, their functioning and interactions (cell division, motility, transport, secretion, neuronal communication, etc.) at cellular and molecular levels.
Fluorescence microscopy explores areas from the nanometer range, with new super-resolution techniques, to millimeter sizes and, in the spectral range, from ultraviolet (350 nm) to near infrared (1 μm). With current sensors, recording times range from milliseconds to a few seconds.
From an industrial standpoint, the sector's development depends on dynamic interaction between fundamental research laboratories (users), academic instrumentation research laboratories, and manufacturers. Fluorescence microscopy is at the crossroads of several rapidly evolving techniques. Evolutions are appearing in the fields of probe chemistry, light sources, lasers, optomechanical devices, light detectors, signal processing and computer possibilities. Local developments can end up as a new commercial product within a year or two. Interaction with all these fields enables fluorescence microscopy to extend into new fields, from molecular studies to the living animal. We have chosen a multidisciplinary approach in the presentation of this article.
This technique is fairly universal and generally quick to implement. One of its major advantages is the extreme specificity offered by immunofluorescence and fusion proteins. Its limitations lie in the difficulty of obtaining a specific probe and exploring thick objects (> 0.5 mm).
It is now routinely applied in medical diagnostics, biomedical and pharmaceutical research, surgery, and instrumentation research.
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KEYWORDS
fluorescent molecules | fluorescence microscope | super-resolution | | Biomedical research | biomedical industry | Two-photon fluorescence microscopy | optical microscopy | fluorescence
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Bibliography
Software tools
Image capture and peripheral control
Metamorph http://www.moleculardevices.comcom/Products/Software/Meta-Imaging-Series/MetaMorph.html
Peripheral control
Websites
A wealth of information about microscopy can be found on the website published by the Federal University of Florida. http://www.micro.magnet.fsu.edu/
Historical and review articles on light microscopy from the journal Nature http://www.nature.com/milestones/light-microscopy
...Events
Thematic school on functional microscopy in biology http://www.mifobio.fr/
Directory
Manufacturers – Microscopes (non-exhaustive list)
Leica http://www.leica-microsystems.com/
Nikon http://www.nikoninstruments.com/
Olympus http://www.olympusmicro.com/
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