2.3. Fluorescence Lifetime Imaging Microscopy, Data Acquisition, and Processing

Fluorescence lifetime images were acquired with a Zeiss LSM 710 microscope (Carl Zeiss, Jena, Germany). A Titanium:Sapphire MaiTai laser (Spectra-Physics, Mountain View, California) with 100 fs pulses and 80 MHz repetition rate was used as a two-photon excitation source at 740 nm. The laser light was focused through a 40 × , 1.2 N.A., water immersion objective (Carl Zeiss, Oberkochen, Germany). The autofluorescence was detected with a photomultiplier tube (H7422P-40, Hamamatsu, Japan) after passing through a bandpass 460/80  nm filter. A dichroic filter at 690 nm served to separate the excitation from the emission signals. Fluorescence lifetime data were acquired using an A320 FastFLIM FLIMbox (ISS, Champaign, Illinois). For each image, 60 frames were collected to obtain at least 100  photons/pixel at 3 mW of power in the sample plane. The scan speed was 25.21  μs/pixel, and the image size 256 × 256  pixels, which span across 154  μm. The lifetime of rhodamine 110 was measured to calibrate the FLIM system, as it is established at 4 ns.

The SimFCS software, developed at the Laboratory of Fluorescence Dynamics (UC Irvine), was used to collect and process these FLIM data. As previously described,^23,29 the fluorescence intensity decay associated to each pixel of the FLIM image is mapped onto a two coordinates (g, s) system, the so-called phasor plot. The phasor transformation is as follows:

where ω is the laser repetition rate, I(t) is the fluorescence intensity decay, and the indices i and j identify each pixel of the image. After the transformation, the data are represented as clusters of lifetime distributions in the phasor plot, also known as phasors. These lifetime distributions (or phasors) are either attributed to single exponential decays, which are located along the periphery of a semicircle, or to multiple exponential decays, placed within the semicircle. In the phasor space, a lifetime distribution attributed to multiple exponential decays is a linear combination of the lifetime distributions associated with the single exponential decays that constitute the combination.²³ This property simplifies the analysis and provides a fitting-free approach for identifying areas in the image with similar or distinct lifetimes.