Loss of image quality in photobleaching during microscopic imaging of fluorescent probes bound to chromatin

Tytus Bernas, J. Paul Robinson, Elikplimi K. Asem, Bartek Rajwa
2005 Journal of Biomedical Optics  
Prolonged excitation of fluorescent probes leads eventually to loss of their capacity to emit light. A decrease in the number of detected photons reduces subsequently the resolving power of a fluorescence microscope. Adverse effects of fluorescence intensity loss on the quality of microscopic images of biological specimens have been recognized, but not determined quantitatively. We propose three human-independent methods of quality determination. These techniques require no reference images and
more » ... eference images and are based on calculation of the actual resolution distance, information entropy, and signal-tonoise ratio ͑SNR͒. We apply the three measures to study the effect of photobleaching in cell nuclei stained with propidium iodide ͑PI͒ and chromomycin A3 ͑CA 3 ͒ and imaged with fluorescence confocal microscopy. We conclude that the relative loss of image quality is smaller than the corresponding decrease in fluorescence intensity. Furthermore, the extent of quality loss is related to the optical properties of the imaging system and the noise characteristics of the detector. We discuss the importance of these findings for optimal registration and compression of biological images. Materials and Methods Reagents Ribonuclease A ͑RNase͒, propidium iodide ͑PI͒, chromomycin A3 ͑CA 3 ͒, and N-propyl gallate ͑NPG͒ were all from
doi:10.1117/1.2136313 pmid:16409080 fatcat:sd6ctlxrxbgtrf5hmcgymupyde