Evaluating the performance of fluorescence microscopes

Abstract

SummaryA simple means of evaluating the performance of fluorescence microscopes is described. The proposed test gives an overall figure of merit that takes into account all of the important instrumental parameters that determine image quality. The essence of the test is to use a specimen whose photobleaching rate is a measure of the illumination timeintensity integral. When this time-intensity integral is controlled, the signal-to-noise ratio in the image of small subresolution objects becomes a system-independent measure of light throughput, resolution and intrinsic noise.

Accuracy and precision in quantitative fluorescence microscopy

“…Saturation should be avoided by using image acquisition software to monitor intensity values when setting up the acquisition parameters (Table II). (Keller, 2006) c (Goodwin, 2007) d (Waters, 2007) e (Inoué and Spring, 1997) f (Moomaw, 2007) g (Allan, 2000) h (Murray, 1998) In most live biological specimens, saturation is much less of a problem than collecting enough signal to get adequate SNR images for quantitation. Many research-grade cooled cameras allow binning of adjacent pixels on the CCD chip.…”

Potential of ultraviolet wide‐field imaging and multiphoton microscopy for analysis of dehydroergosterol in cellular membranes

“…The obtained difference image was squared, and the integrated intensity calculated and divided by the pixel number in the image to obtain the mean intensity (Murray, 1998). The square root of this mean difference in intensity between raw and median-filtered image gave the RMS noise, which was plotted as function of the number of averaged frames (see Fig.…”

“…Image fidelity can also be assessed by calculating the signal-tonoise ratio (SNR). The SNR can be approximated by dividing the estimated RMS noise image into the original image (Murray, 1998). To compare a reference image r(x, y) with a noisy test image t(x, y) one often uses the peak-SNR (PSNR) as a measure of restoration quality (Gonzalez and Woods, 2002 Kolin and Wiseman (2007), but with tools in ImageJ (developed at the U.S. National Institutes of Health and available on the Internet at http://rsb.info.nih.gov/ij).…”

Fluorescence Imaging

“…For example, a stretched exponential decay [25] and a single exponential function plus a constant term [35] provide much better fits to the photobleaching kinetics than a monoexponential function. For example, a stretched exponential decay [25] and a single exponential function plus a constant term [35] provide much better fits to the photobleaching kinetics than a monoexponential function.…”