3D-resolved fluorescence and phosphorescence lifetime imaging using temporal focusing widefield two-photon excitation Fluorescence and phosphorescence lifetime imaging are powerful techniques for studying intracellular protein interactions and for diagnosing tissue pathophysiology. While lifetime-resolved microscopy has long been in the repertoire of the biophotonics community, current implementations fall short in terms of simultaneously providing 3D resolution, high throughput, and good tissue penetration. This report describes a new highly efficient lifetime-resolved imaging method that combines temporal focusing wide-field multiphoton excitation and simultaneous acquisition of lifetime information in frequency domain using a nanosecond gated imager from a 3D-resolved plane (Fig. 1). This approach is scalable allowing fast volumetric imaging limited only by the available laser peak power. Most importantly, the parallelism

of this approach may enhance the imaging speed of long lifetime processes such as phosphorescence by several orders of magnitude (Fig. 2). The accuracy and performance of the proposed method is demonstrated in several imaging studies and initial applications in studying peripheral nerve regeneration processes has been undertaken (Fig. 3).

Fig. 1. Temporal focusing wide-field (TFWF) FLIM/PLIM design. (a) Optical sub-system: temporal focusing widefield multiphoton microscopy, (b) Electronic sub-system: frequency domain lifetime measurement via heterodyne detection.

Fig. 2. (a) An intensity scaled mean lifetime image of fixed fibroblasts with vacuoles loaded with endocytosed conjugated polymer nanoparticles of high two-photon absorption cross section. Color scale represents pixel lifetime values corresponding to the color bar with units of seconds. Image brightness represents pixel intensity values. Black regions are ignored in analysis corresponding to locations with intensity below 500 photons that are mostly outside the boundary of this cell. (b) Representative polar plot of pixel lifetime values for 10 ms data acquisition time. (c) Tabulated mean lifetime values and their standard deviations are estimated from the modulation or the phase data for four different image acquisition rates.

Fig. 3. Fast 3D-resolved TFWF PLIM in sample consisting of human fibroblasts stained with Rhodamine DHPE, seeded inside a collagen matrix and treated, in PBS buffer containing 1 mM TDRT ruthenium-based oxygen sensor. PLIM Images were acquired at 300 kHz modulation frequency. In sequence from left to right: intensity image, phosphorescence lifetime-resolved image from phase data, phosphorescence lifetime-resolved image from modulation data. On the far right is the polar plot of the pixel phosphorescence lifetimeresolved measurements. The pixel lifetime data in the polar plot distributes between the ruthenium phosphorescence lifetime that lies close to the universal circle and the rhodamine fluorescence lifetime at the lower right hand corner corresponding to an effective zero lifetime for 300 kHz modulation frequency. PLIM Movie sequences corresponding to phase and modulation lifetime measurements throughout a 3D matrix are included.