Article

Intensity calibration of a laser scanning confocal microscope based on concentrated dyes.

Department of Biological Sciences, Kent State University, Kent, Ohio, USA.
Analytical and quantitative cytology and histology / the International Academy of Cytology [and] American Society of Cytology (impact factor: 0.41). 11/2006; 28(5):253-61.
Source: PubMed

ABSTRACT To find water-soluble fluorescent dyes with absorption in various regions of the spectrum and investigate their utility as standards for laser scanning confocal microscopy.
Several dyes were found to have characteristics required for fluorescence microscopy standards. The intensity of biological fluorescent specimens was measured against the emission of concentrated dyes. Results using different optics and different microscopes were compared.
Slides based on concentrated dyes can be prepared in a highly reproducible manner and are stable under laser scanning. Normalized fluorescence of biological specimens remains consistent with different objective lenses and is tolerant to some mismatch in optical filters or imperfect pinhole alignment. Careful choice of scanning parameters is necessary to ensure linearity of intensity measurements.
Concentrated dyes provide a robust and inexpensive intensity standard that can be used in basic research or clinical studies.

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    Article: Calibrating excitation light fluxes for quantitative light microscopy in cell biology.
    David Grünwald, Shailesh M Shenoy, Sean Burke, Robert H Singer
    [show abstract] [hide abstract]
    ABSTRACT: Power output of light bulbs changes over time and the total energy delivered will depend on the optical beam path of the microscope, filter sets and objectives used, thus making comparison between experiments performed on different microscopes complicated. Using a thermocoupled power meter, it is possible to measure the exact amount of light applied to a specimen in fluorescence microscopy, regardless of the light source, as the light power measured can be translated into a power density at the sample. This widely used and simple tool forms the basis of a new degree of calibration precision and comparability of results among experiments and setups. Here we describe an easy-to-follow protocol that allows researchers to precisely estimate excitation intensities in the object plane, using commercially available opto-mechanical components. The total duration of this protocol for one objective and six filter cubes is 75 min including start-up time for the lamp.
    Nature Protocol 02/2008; 3(11):1809-14. · 8.36 Impact Factor
Data provided are for informational purposes only. Although carefully collected, accuracy cannot be guaranteed. The impact factor represents a rough estimation of the journal's impact factor and does not reflect the actual current impact factor. Publisher conditions are provided by RoMEO. Differing provisions from the publisher's actual policy or licence agreement may be applicable.

Keywords

biological fluorescent specimens
 
biological specimens
 
Careful choice
 
characteristics
 
clinical studies
 
concentrated dyes
 
different microscopes
 
different objective lenses
 
different optics
 
dyes
 
imperfect pinhole alignment
 
inexpensive intensity standard
 
intensity measurements
 
laser scanning
 
laser scanning confocal microscopy
 
optical filters
 
scanning parameters
 
stable
 
various regions
 
water-soluble fluorescent dyes