Instrumentation for quantitative fluorescence microscopy

摘要

The aim of this study is to demonstrate the technical requirements and performance of fluorescent quantization using image analysis. The quality of fluorescent measurements is largely dependent on the characteristics of instrumentation: fluorescence microscope, low light level camera, fluorescence standard reference, and protocols for image acquisition and measurements calibration. This study is focused on the problem of the efficiency of microscope and low light level cameras. Various types of cameras are tested (SIT C2400-08, intensified CCD C2400-80, and cooled CCD C4880 from Hamamatsu) with respect to stability, linearity, and shading properties. Results show that evaluation of fluorescent instrumentation cannot be reduced to the evaluation of camera properties, the intrinsic evaluation of the fluorescent sample has to be considered as well. Moreover, this work demonstrates the deficiency of usual corrective protocols to lead to reliable quantitative fluorescence analysis.