High Speed Two-Photon Lifetime Imaging

Nonlinear optical techniques and in particular two photon excited fluorescence imaging have emerged as powerful tools for deep tissue imaging with subcellular resolution, brain mapping and 3D printing. At the same time, fluorescent lifetime imaging probes internal biochemical interactions and external environment of a molecule useful for DNA sequencing, detection of tumour margins necessary for successful surgical removal, and quantifying cellular energy metabolism in living cells. To image fast dynamic processes such as biological cells in flow or neural activities, these methods must provide frame rates beyond 1000Hz. However, achieving high speed is challenged by lower efficiency of nonlinear vs. linear processes requiring illumination with a high-intensity tightly-focused beam that is scanned over sample area. The scanning is typically done with mechanical scanners the speed of which limits the frame rate. Acousto-optic scanners provide an intermediate solution however the frame rate is limited by the acoustic velocity leading to a trade-off between resolution and speed. Here we report on a new tool for high-speed non-linear imaging and demonstrate its utility in two photon fluorescence and fluorescence lifetime imaging (FLIM). A rapidly wavelength-swept Fourier-Domain Mode-Locked (FDML) laser with digitally synthesized electro-optic modulation in a master oscillator-power amplifier configuration is combined with spectral encoding to eliminate the speed limitations of inertial scanning and achieve single-shot imaging. We demonstrate lifetime imaging with 2kHz frame-rate (88MHz pixel rate) – a record for both FLIM and two-photon FLIM. To the best of our knowledge, this is the first report of spectrally encoded multiphoton imaging. We also show optical image compression via spatiallywarped two photon excitation and scale invariant digital zoom. This method allows nonlinear imaging flow cytometry, rapid recording of neuronal activity and mapping non-repetitive biomolecular dynamics at a sub-cellular optical resolution. Being fibre based this method has the potential for endoscopic medical applications.