Document Detail


Epi-fluorescence microscopy.
MedLine Citation:
PMID:  23026996     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
Epi-fluorescence microscopy is available in most life sciences research laboratories, and when optimized can be a central laboratory tool. In this chapter, the epi-fluorescence light path is introduced and the various components are discussed in detail. Recommendations are made for incident lamp light sources, excitation and emission filters, dichroic mirrors, objective lenses, and charge-coupled device (CCD) cameras in order to obtain the most sensitive epi-fluorescence microscope. The even illumination of metal-halide lamps combined with new "hard" coated filters and mirrors, a high resolution monochrome CCD camera, and a high NA objective lens are all recommended for high resolution and high sensitivity fluorescence imaging. Recommendations are also made for multicolor imaging with the use of monochrome cameras, motorized filter turrets, individual filter cubes, and corresponding dyes being the best choice for sensitive, high resolution multicolor imaging. Images should be collected using Nyquist sampling and images should be corrected for background intensity contributions and nonuniform illumination across the field of view. Photostable fluorescent probes and proteins that absorb a lot of light (i.e., high extinction co-efficients) and generate a lot of fluorescence signal (i.e., high quantum yields) are optimal. A neuronal immune-fluorescence labeling protocol is also presented. Finally, in order to maximize the utility of sensitive wide-field microscopes and generate the highest resolution images with high signal-to-noise, advice for combining wide-field epi-fluorescence imaging with restorative image deconvolution is presented.
Authors:
Donna J Webb; Claire M Brown
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Publication Detail:
Type:  Journal Article; Research Support, N.I.H., Extramural; Research Support, Non-U.S. Gov't    
Journal Detail:
Title:  Methods in molecular biology (Clifton, N.J.)     Volume:  931     ISSN:  1940-6029     ISO Abbreviation:  Methods Mol. Biol.     Publication Date:  2013  
Date Detail:
Created Date:  2012-10-02     Completed Date:  2013-02-11     Revised Date:  2014-01-10    
Medline Journal Info:
Nlm Unique ID:  9214969     Medline TA:  Methods Mol Biol     Country:  United States    
Other Details:
Languages:  eng     Pagination:  29-59     Citation Subset:  IM    
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MeSH Terms
Descriptor/Qualifier:
Cells, Cultured
Fluorescent Antibody Technique / methods
Fluorescent Dyes / chemistry
Green Fluorescent Proteins / metabolism
Humans
Image Processing, Computer-Assisted*
Lighting
Microscopy, Fluorescence / instrumentation,  methods
Phalloidine / metabolism
Staining and Labeling
Grant Support
ID/Acronym/Agency:
MH071674/MH/NIMH NIH HHS; R01 GM092914/GM/NIGMS NIH HHS; R01 MH071674/MH/NIMH NIH HHS
Chemical
Reg. No./Substance:
0/Fluorescent Dyes; 0/enhanced green fluorescent protein; 147336-22-9/Green Fluorescent Proteins; 17466-45-4/Phalloidine
Comments/Corrections

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