Modulated CMOS camera for fluorescence lifetime microscopy

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 20MHz 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 non-uniform 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.

Characterisation of new gated optical image intensifiers for fluorescence lifetime imaging

“…21 Modulated CMOS cameras can enable frequency domain FLIM with a higher spatial resolution than intensifier-based systems and are now available with modulation frequencies up to 50 MHz. 22,23 With an increasing number of technologies being developed for FLIM, it is important to understand the state-of-theart for different approaches. This paper focusses on widefield time-gated FLIM, describing new developments in the GOI technology that improve performance.…”

Characterization of SPAD Array for Multifocal High-Content Screening Applications

“…There are a number of research groups actively pursuing integrated circuit detector arrays that are capable of creating hundreds to thousands of independent time-resolved pixels. A large number of these efforts involve the use of SPAD arrays using TCSPC [23][24][25][26], while there are a few on-board time-gating approaches [27][28][29].…”

Picosecond wide-field time-correlated single photon counting fluorescence microscopy with a delay line anode detector

“…The latest frequencydomain FLIM cameras are based on solid state CCD/CMOS detectors, 6,7 without an image intensifier, which avoid spatial resolution degradation and time lag artifacts due to the phosphor. 8,9 These are suitable for sufficiently bright samples so that modulation is practical, 10 where they work well with an appropriate calibration up to frame rates of $20 fps and a time resolution down to $100 ps. 9 For low light levels, e.g., for single molecule imaging, or low excitation intensities 11 photon counting approaches are preferable.…”

“…8,9 These are suitable for sufficiently bright samples so that modulation is practical, 10 where they work well with an appropriate calibration up to frame rates of $20 fps and a time resolution down to $100 ps. 9 For low light levels, e.g., for single molecule imaging, or low excitation intensities 11 photon counting approaches are preferable. 10 Wide-field TCSPC requires the position of the arriving photon to be measured and recorded at the same time as the arrival time.…”