Abstract
Widefield frequency-domain fluorescence lifetime imaging microscopy (FD-FLIM) measures the fluorescence lifetime of entire images in a fast and efficient manner. We report a widefield FD-FLIM system based on a complementary metal-oxide semiconductor camera equipped with two-tap true correlated double sampling lock-in pixels and lateral electric field charge modulators. Owing to the fast intrinsic response and modulation of the camera, our system allows parallel multifrequency FLIM in one measurement via fast Fourier transform. We demonstrate that at a fundamental frequency of 20 MHz, 31-harmonics can be measured with 64 phase images per laser repetition period. As a proof of principle, we analyzed cells transfected with Cerulean and with a construct of Cerulean-Venus that shows Förster Resonance Energy Transfer at different modulation frequencies. We also tracked the temperature change of living cells via the fluorescence lifetime of Rhodamine B at different frequencies. These results indicate that our widefield multifrequency FD-FLIM system is a valuable tool in the biomedical field.
Highlights
Fluorescence lifetime imaging microscopy (FLIM) is becoming increasingly attractive in bioimaging and potentially in medical diagnostics
Our results demonstrate that FLIM can be done in the widefield and multifrequency configuration, which can be combined with any widefield type fluorescence microscope, including total internal reflection fluorescence (TIRF) and single plane illumination microscope (SPIM)
One advantage of FLIM systems based on complementary metal-oxide semiconductor (CMOS) sensors is the easy configuration with existing widefield microscopes
Summary
Fluorescence lifetime imaging microscopy (FLIM) is becoming increasingly attractive in bioimaging and potentially in medical diagnostics. Using a proper sample which has a known lifetime as the reference, the fluorescence lifetime of an unknown sample at each pixel can be determined accurately and rapidly Such a time-resolved widefield system for frequency-domain FLIM involves an image intensifier and a camera with appropriate control, as we reported in detail earlier in [11]. It has a faster gating speed, and the very high harmonic content can be obtained even when a fundamental frequency of 20 MHz is used It is ideal for frequency-domain fluorescence lifetime imaging microscopy (FD-FLIM) measurement. Multiple molecular species contributing to each of these Fourier components can be extracted and displayed by the phasor approach [22] This technique requires certain hardware support in modulation and phase control, but it results in very high time resolution based on the harmonic content over entire images. Our results demonstrate that FLIM can be done in the widefield and multifrequency configuration, which can be combined with any widefield type fluorescence microscope, including total internal reflection fluorescence (TIRF) and single plane illumination microscope (SPIM)
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