Modulated CMOS camera for fluorescence lifetime microscopy. Issue 12 (24th October 2015)

• Record Type: Journal Article
• Authors: Chen, Hongtao; Holst, Gerhard; Gratton, Enrico
• Is Part Of: Microscopy research and technique. Volume 78:Issue 12(2015:Dec.)
• Page Start: 1075
• Page End: 1081
• Publication Date: 2015-10-24
• Subjects: FLIM -- fluorescence lifetime -- frequency‐domain -- wide‐field -- PCO FLIM camera -- CMOS -- phasor plot -- harmonic frequency; Electron microscopy -- Technique -- Periodicals; Microscopy -- Periodicals; Microscopy -- Technique -- Periodicals; 502.825
• DOI: 10.1002/jemt.22587
• Languages: English
• ISSNs: 1059-910X

Abstract

ABSTRACT: Widefield frequency‐domain fluorescence lifetime imaging microscopy (FD‐FLIM) is a fast and accurate method to measure the fluorescence lifetime of entire images. However, the complexity and high costs involved in construction of such a system limit the extensive use of this technique. PCO AG recently released the first luminescence lifetime imaging camera based on a high frequency modulated CMOS image sensor, QMFLIM2. Here we tested and provide operational procedures to calibrate the camera and to improve the accuracy using corrections necessary for image analysis. With its flexible input/output options, we are able to use a modulated laser diode or a 20 MHz pulsed white supercontinuum laser as the light source. The output of the camera consists of a stack of modulated images that can be analyzed by the SimFCS software using the phasor approach. The nonuniform system response across the image sensor must be calibrated at the pixel level. This pixel calibration is crucial and needed for every camera settings, e.g. modulation frequency and exposure time. A significant dependency of the modulation signal on the intensity was also observed and hence an additional calibration is needed for each pixel depending on the pixel intensity level. These corrections are important not only for the fundamental frequency, but also for the higher harmonics when using the pulsed supercontinuum laser. With these post data acquisition corrections, the PCO CMOS‐FLIM camera can be used for various biomedical applications requiring a large frame and high speed acquisition. Microsc. Res. Tech. 78:1075–1081, 2015 . © 2015 Wiley Periodicals, Inc.