Abstract
Traditional fluorescence microscopy is blind to molecular microenvironment information that is present in a fluorescence lifetime, which can be measured by fluorescence lifetime imaging microscopy (FLIM). However, most existing FLIM techniques are slow to acquire and process lifetime images, difficult to implement, and expensive. Here we present instant FLIM, an analog signal processing method that allows real-time streaming of fluorescence intensity, lifetime, and phasor imaging data through simultaneous image acquisition and instantaneous data processing. Instant FLIM can be easily implemented by upgrading an existing two-photon microscope using cost-effective components and our open-source software. We further improve the functionality, penetration depth, and resolution of instant FLIM using phasor segmentation, adaptive optics, and super-resolution techniques. We demonstrate through-skull intravital 3D FLIM of mouse brains to depths of 300 µm and present the first in vivo 4D FLIM of microglial dynamics in intact and injured zebrafish and mouse brains for up to 12 h.
Highlights
Imaging molecular contrast is essential to continued advances in cellular biology
Built upon our preliminary work presented in Ref. [46], instant fluorescence lifetime imaging microscopy (FLIM) uses a radio frequency (RF) analog signal processing approach, where the 2PEF signal is split four ways and mixed with the phase-shifted 80 MHz reference signals from the Ti:sapphire laser in a multiplexing manner
Since instant FLIM utilizes the intrinsic femtosecond laser pulses as the modulation source, the modulation frequency is fixed at 80 MHz; as shown in Fig. S4C of Supplement 1, the SNR performance in lifetime measurements, quantified by the F-value, will become worse for fluorophores with longer lifetimes
Summary
Imaging molecular contrast is essential to continued advances in cellular biology. Fluorescence microscopy has been a significant tool over the past decades in imaging cellular and subcellular molecular contrast [1]. Traditional fluorescence microscopy typically obtains molecular contrast by labeling parts of cells with different fluorophores and imaging emission intensity. By performing fluorescence lifetime imaging microscopy (FLIM), fluorophores with overlapping emission spectra can be differentiated as long as their lifetimes are different, and physiological parameters such as pH, refractive index, ion concentration, dissolved gas concentration, and Förster resonance energy transfer (FRET) can be measured [2,3,4,5]. Despite the biologically relevant information provided by fluorescence lifetime, the widespread use of FLIM in biomedical imaging has been limited due to its slow image
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