Abstract
Two-Photon Microscopy has become an invaluable tool for biological and medical research, providing high sensitivity, molecular specificity, inherent three-dimensional sub-cellular resolution and deep tissue penetration. In terms of imaging speeds, however, mechanical scanners still limit the acquisition rates to typically 10–100 frames per second. Here we present a high-speed non-linear microscope achieving kilohertz frame rates by employing pulse-modulated, rapidly wavelength-swept lasers and inertia-free beam steering through angular dispersion. In combination with a high bandwidth, single-photon sensitive detector, this enables recording of fluorescent lifetimes at speeds of 88 million pixels per second. We show high resolution, multi-modal - two-photon fluorescence and fluorescence lifetime (FLIM) – microscopy and imaging flow cytometry with a digitally reconfigurable laser, imaging system and data acquisition system. These high speeds should enable high-speed and high-throughput image-assisted cell sorting.
Highlights
Two-Photon Microscopy has become an invaluable tool for biological and medical research, providing high sensitivity, molecular specificity, inherent three-dimensional sub-cellular resolution and deep tissue penetration
Even when averaging is desired, e.g., for doubleexponential lifetime fits, it may be advantageous to average 2000 frames obtained with spectro-temporal laser imaging by diffracted excitation (SLIDE) within 1 s, as opposed to pixel averaging by applying 2000 pulses per pixel with a pixel dwell time of 23 μs and raster-scanning the image in 1 s. (ii) Longer pulses lead to reduced pulse peak powers at same SNR, having the advantage of avoiding higher-than-quadratic effects like photobleaching[32,33] and photodamage[34,35,36]
The picosecond pulses are shorter than timescales for intersystem crossing (ISC), so no further excitation from the triplet state should occur within the same pulse. (iii) The longer pulses are generated by digitally synthesized EO modulation, which renders the excitation pattern freely programmable
Summary
Two-Photon Microscopy has become an invaluable tool for biological and medical research, providing high sensitivity, molecular specificity, inherent three-dimensional sub-cellular resolution and deep tissue penetration. Multi-modal - two-photon fluorescence and fluorescence lifetime (FLIM) – microscopy and imaging flow cytometry with a digitally reconfigurable laser, imaging system and data acquisition system. These high speeds should enable high-speed and high-throughput image-assisted cell sorting. We present a solution to this problem by further employing temporal encoding from a wavelength swept laser This concept achieves spectro-temporal encoded imaging, where the wavelength is used for high-speed inertia-free point scanning and the temporal encoding for one-to-one mapping of the signal to the imaging pixels. Spectrotemporal encoded imaging has unique advantages over other high-speed non-linear imaging approaches[6,7,8,9,11,12,13] in terms of resolution, lifetime modality, compactness, flexibility, and fibrebased setup
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