Fast fluorescence lifetime imaging of calcium in living cells

Abstract

A fast fluorescence lifetime imaging (FLIM) system is developed that can acquire images at a rate of hundreds of frames per second. The FLIM system is based on a wide-field microscope equipped with a time-gated intensified CCD detector and a pulsed laser. The time-gated detector acquires the signals from two time gates simultaneously and is therefore insensitive to movements of the specimen and photo-bleaching. The system is well suited for quantitative biological FLIM experiments and its performance is evaluated in calcium imaging experiments on beating neonatal rat myocytes. Several calcium sensitive dyes are characterized and tested for their suitability for fast FLIM experiments: Oregon Green Bapta-1 (OGB1), Oregon Green Bapta-2 (OGB2), and Oregon Green Bapta-5N (OGB5N). Overall the sensitivity range of these dyes is shifted to low calcium concentrations when used as lifetime dyes. OGB1 and OGB2 behave very similarly and can be used for FLIM-based calcium imaging in the range 1 to approximately 500 nM and OGB5N can be used up to 3 microM. The fast FLIM experiments on the myocytes could be carried out at a 100-Hz frame rate. During the beating of the myocytes a lifetime change of about 20% is observed. From the lifetime images a rest calcium level of about 65 nM is found.

Single Photon Imaging in CMOS

“…Third, a better penetration in turbid media can be achieved, due to reduced scattering. Fourth, the large spectral difference between in-and out-going radiation simplifies the separation of response from excitation [5].…”

“…Such applications range from fluorescence-based imaging, such as Förster Resonance Energy Transfer (FRET), fluorescence lifetime imaging microscopy (FLIM) [5], and fluorescence correlation spectroscopy (FCS) [6], to voltage sensitive dye (VSD) based imaging [7], [8], particle image velocimetry (PIV) [9], instantaneous gas imaging [10], etc.…”

Highly Sensitive Arrays of Nano-sized Single-Photon Avalanche Diodes for Industrial and Bio Imaging

“…Thanks to these properties, CMOS SPAD arrays have been proposed for imaging where speed and/or event timing accuracy are critical. Such applications range from fluorescence-based imaging, such as Förster Resonance Energy Transfer (FRET), fluorescence lifetime imaging microscopy (FLIM) [13], and fluorescence correlation spectroscopy (FCS) [14], to voltage sensitive dye (VSD) based imaging [15,16], particle image velocimetry (PIV) [17], instantaneous gas imaging, [18,19], etc.…”

Towards large scale CMOS single-photon detector arrays for lab-on-chip applications

“…Fluorescence-based imaging (both single and multiphoton) is perhaps the research direction that has most influenced the development of fast and sensitive optical detectors [7][8][9]. Examples of techniques in this class include Förster resonance energy transfer (FRET) [10], fluorescence lifetime imaging microscopy (FLIM) [7,11], and fluorescence correlation spectroscopy (FCS) [12][13][14]. The success of these techniques, particularly FLIM, derives from the ability to characterize an environment based on the timedomain behaviour of certain fluorophores with high resolution in space domain.…”