Background rejection and signal-to-noise optimization in confocal and alternative fluorescence microscopes
- Sandia National Laboratories, Albuquerque, New Mexico 87185 (United States)
- Department of Physics, Cornell University, Ithaca, New York 14853 (United States)
In the confocal microscope, tightly focused illumination and spatially filtered detection are combined to reduce out-of-focus background and to produce high-quality images that display thin optical sections within thick fluorescent specimens. We define background as the detected light that originates outside a resolution volume and signal as the detected light that originates within the same volume. Background rejection is measured by the signal-to-background ratio (S/B) and is calculated for confocal, spinning-disk, line-illumination, slit-detection, and conventional fluorescence microscopes as a function of both the spatial filter size and the specimen thickness. Spatial filter sizes that reject background and optimize the signal-to-noise ratio (S/N) are calculated for each microscope. These calculations are normalized so that the time-averaged illumination at each point in the specimen is the same for each microscope. For thick specimens, we show that the S/B obtained with a confocal microscope can be more than 100 times greater than the S/B available with a conventional microscope, and we find that the optimal confocal S/N can be a factor of 10 greater than the S/N in the conventional microscope.
- OSTI ID:
- 5037433
- Journal Information:
- Applied Optics; (United States), Vol. 33:4; ISSN 0003-6935
- Country of Publication:
- United States
- Language:
- English
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