Controlling and online measurement of automatic dual-channel E-FRET microscope

Abstract

Based on the manual dual-channel E-FRET microscope we recently set up, we here develop an automatic dual-channel E-FRET microscope (AD-E-FRETM) with user-friendly interface. The software of AD-E-FRETM has been designed in two-grade mode: Administrator mode (Dual-channel system calibration interface) and user mode (Dual-channel E-FRET measurement interface). The first peak intensity in the pixel-to-pixel count-gray value plot of an image is automatically determined as background (IBG) for automatic subtraction from the original image and online quantitative FRET imaging (FRET efficiency (ED) and concentration ratio (RC) of acceptor to donor). After subtraction of the threshold (β×IBG)), the corrected image is binarized to obtain the mask image (Imask). The final binary mask (Bmask) is obtained by logical AND of the Imask(DD), Imask(DA) and Imask(AA), and is used to online filter the ED and RC images. After locating the cells expressing standard FRET construct (C4Y), clicking the capture button on the Dual-channel E-FRET measurement interface to trigger AD-E-FRETM to automatically implement quantitative online ED imaging with different threshold parameter (β) that was finally optimized to be 3. We implemented quantitative E-FRET imaging on AD-E-FRETM for living cells expressing different tandem constructs (C80Y, C40Y, C10Y and C4Y, respectively), and obtained consistent results for at least three months, demonstrating the stability of AD-E-FRETM. We also preformed time-lapse FRET imaging on AD-E-FRETM for living cells co-expressing CFP-Bax and YFP-Bax to monitor dynamic Bax translocation and oligomerization during STS-induced apoptosis.