How the Confocal Laser Scanning Microscope entered Biological Research

Amos, W. B.; White, John

doi:10.1016/s0248-4900(03)00078-9

Cited by 265 publications

(159 citation statements)

References 24 publications

(13 reference statements)

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“…Since then, many researchers and optical engineers step by step improved the technical realization (Brakenhoff et al 1979;Davidovi and Egger 1973;Egger and Petran 1967;Hamilton and Wilson 1986;Sheppard and Wilson 1979). The rapid developments in laser and detector technology, along with the onset of fiber optics certainly helped in the rapid dissemination of confocal microscopy into cell biology laboratories around the world (Amos and White 2003).…”

Section: Fluorescence To Study Lipid Dynamicsmentioning

confidence: 76%

Fluorescence Techniques to Study Lipid Dynamics

Sezgin¹,

Schwille²

2011

Cold Spring Harbor Perspectives in Biology

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Biological research has always tremendously benefited from the development of key methodology. In fact, it was the advent of microscopy that shaped our understanding of cells as the fundamental units of life. Microscopic techniques are still central to the elucidation of biological units and processes, but equally important are methods that allow access to the dimension of time, to investigate the dynamics of molecular functions and interactions. Here, fluorescence spectroscopy with its sensitivity to access the single-molecule level, and its large temporal resolution, has been opening up fully new perspectives for cell biology. Here we summarize the key fluorescent techniques used to study cellular dynamics, with the focus on lipid and membrane systems.T o elucidate cellular processes in their native dynamic environment has been one of the main issues in cell biology over the past decades. The lack of appropriate techniques has long been the main limiting step for the research on dynamic systems, because it was impossible to acquire real time information with the well-known biochemical techniques. The key challenge in dynamically observing biological systems is to combine the ability to resolve moderate to very low concentrations of molecules-because they are simply limited in living cells-on relevant timescales. Relevant timescales in cell biology can be minutes and hours, on a systemic level of cell metabolism, down to the microsecond and even nanosecond regime in which molecular and intramolecular rearrangements take place. With respect to lipidic systems, relevant dynamics range from the local movements of lipids by diffusion to the mechanical transformations of whole membranes, spanning several orders of magnitude in time to be covered. Like for other cellular processes, the investigation of lipids and membranes also in general benefited greatly from the introduction of fluorescence microscopy and spectroscopy to biology. After the 1960s, great technological inventions based on the phenomenon of fluorescence were made, such as confocal microscopy, fluorescence recovery after photobleaching (FRAP), fluorescence correlation spectroscopy (FCS), Förster resonance energy transfer (FRET), total internal reflection fluorescence (TIRF), and two-photon microscopy, that not only revolutionized imaging but also yielded access to dynamics on previously inaccessible timescales. Another very big step was certainly taken after the introduction of fluorescent proteins, which again accelerated the use of these techniques in living cells and organisms. Nowadays, the technical advancements of fluorescence-based methods allow us to explore systems as small as single molecules with temporal resolution down to the nanoseconds regime. Lately, even the resolution limit of optical microscopy, for a long time being one of the fundamental barriers in elucidating cellular processes, has been overcome by smart applications of the phenomenon of fluorescence.This article aims at giving a short overview on mainly fluorescence-based metho...

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Section: Fluorescence To Study Lipid Dynamicsmentioning

confidence: 76%

Fluorescence Techniques to Study Lipid Dynamics

Sezgin¹,

Schwille²

2011

Cold Spring Harbor Perspectives in Biology

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“…The disadvantage is its poor rejection of the fluorescence background signal from the cell. Confocal microscopy (middle panel) uses a focused light beam and spatial filtering techniques (pinholes in excitation and detection paths) to eliminate out-of-focus background fluorescence, but at the cost of signal reduction 82 . For the observation of fast dynamics in live cells, a spinning disk confocal setup is often preferred over the relatively slow scanning confocal microscope, which relies on rotating mirrors to scan a focused laser beam in the imaging plane and a point detector, such as avalanche photodiode or photomultiplier tubes, for signal acquisition 81 .…”

Section: Box 2 | Fluorescence Microscopes For Single-virus Imagingmentioning

confidence: 99%

Virus trafficking – learning from single-virus tracking

2007

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What could be a better way to study virus trafficking than 'miniaturizing oneself' and 'taking a ride with the virus particle' on its journey into the cell? Single-virus tracking in living cells potentially provides us with the means to visualize the virus journey. This approach allows us to follow the fate of individual virus particles and monitor dynamic interactions between viruses and cellular structures, revealing previously unobservable infection steps. The entry, trafficking and egress mechanisms of various animal viruses have been elucidated using this method. The combination of single-virus trafficking with systems approaches and state-of-the-art imaging technologies should prove exciting in the future.

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“…The enzyme-based method is sensitive, easy to automate, and allows a rudimentary analysis of intracellular localization. However, the fluorescent-based methods can be used for detailed subcellular analysis and three-dimensional reconstruction of the cell can be done using confocal microscopy (42). It is thus possible to discriminate not only between nuclear, cytoplasmic, and membrane-bound localization, but also to determine proteins expressed in mitochondria, nucleoli, nuclear membrane, etc.…”

Section: Analysis Using Tissue Microarrays (Tma)mentioning

confidence: 99%

Antibody-based Proteomics for Human Tissue Profiling

Uhlén

Pontén

2005

Molecular & Cellular Proteomics

263

161

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Here, we describe the use of antibody-based proteomics involving the generation of protein-specific antibodies to functionally explore the human proteome. The antibodies can be used for analysis of corresponding proteins in a wide range of assay platforms, including i) immunohistochemistry for detailed tissue profiling, ii) specific affinity reagents for various functional protein assays, and iii) capture ("pull-down") reagents for purification of specific proteins and their associated complexes for structural and biochemical analyses. In this review, the use of antibodies for such analysis will be discussed with focus on the possibility to create a descriptive and comprehensive protein atlas for tissue distribution and subcellular localization of human proteins in both normal and disease tissues.

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How the Confocal Laser Scanning Microscope entered Biological Research

Cited by 265 publications

References 24 publications

Fluorescence Techniques to Study Lipid Dynamics

Fluorescence Techniques to Study Lipid Dynamics

Virus trafficking – learning from single-virus tracking

Antibody-based Proteomics for Human Tissue Profiling

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