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

Abstract

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 are based on calculation of the actual resolution distance, information entropy, and signal-to-noise ratio (SNR). We apply the three measures to study the effect of photobleaching in cell nuclei stained with propidium iodide (PI) and chromomycin A3 (CA3) 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.

Adaptative, signal-preserving compression of microscopic images using noise modeling in the wavelet domain and JPEG2000 coding

“…The noise level was calculated using a previously described algorithm [11,12]. First, a set of 8 directional high-pass filters (3x3) was applied to an image.…”

Label free cell tracking in 3D tissue engineering constructs with high resolution imaging

“…This permits the identification and measurement of cell structure such as smooth muscle cells [2]. The disadvantage of the fluorescence and confocal techniques is that the duration of the experiment is limited to the amount of excitation that the markers used may undergo before photobleaching degrades them [1]. In addition, the usage of flurophores results in currently unquantifiable effects on the cells behavior and other characteristics, e.g.…”

“…The sigma value is based upon the distance of the object from the lens, which determines the degree to which the psf projects the defocussed image. Each pixel has a corresponding DL calculated by (1), and the minimum DL value is sought for each x, y location along the z-axis. These (x, y, z) locations form a depth map, which is used to reconstruct an all-in-focus image from the original z-stack of images.…”

Excitation-scanning hyperspectral imaging microscope

“…We estimated SNR in each image stack using the method initially described by Amer et al 30 that was further demonstrated for fluorescence microscopy applications by our group and Bernas et al 9,31 Briefly, unmixed images were analyzed to detect the fluorescence intensity in each pixel. Pixels within regions of homogeneous intensity were identified using an 8-way high-pass filter.…”