Fig. 4: Combination of TPE with the photon counting techniques FCS and FLIM.

a The pulsed excitation and resulting fluorescence in TPE microscopy can be combined with FCS and FLIM. In FCS the fluorescence time trace at a fixed point in space is analysed by means of temporal autocorrelation. Fitting the resulting autocorrelation curve by an appropriate model gives information on the diffusion coefficient, concentration and oligomeric state (brightness) of moving particles in the sample. Histogramming the photon arrival times after laser pulses allows for the investigation of the fluorescence lifetime of the observed fluorophores. b The pixels in FLIM imaging contain lifetime values in addition to the intensity values. For every pixel a lifetime decay (photon counting histogram can be calculated). A convenient way to compress the data is to map the decay curves via Fourier transform onto the phasor space. Mono-exponential lifetimes lie on the universal circle. Combination of lifetimes (or multi-exponential decays) map within the circle. c Application of Phasor-FLIM imaging to investigate metabolic state of cells by exploiting the autofluorescence of bound and unbound NADH. The more NADH is bound, the more oxidative phosphorylation (Oxphos) is performed revealing the metabolic phenotype (i.e., Oxphos versus glycolysis). Lifetimes can be false-colored using the phasor plot and remapped onto the FLIM image to identify spatial patterns. d Example of metabolic imaging of NIH3T3 cells in low or high glucose media (images on the left) and corresponding phasor coordinates of the pixel containing fluorescence (right). NADH was imaged using 740 nm excitation. False colouring using a magenta to blue look up table is applied to the phasor cloud remapping the pixels from phasor to image space. This allows the identification of pixels with more free NADH (meaning more glycolytic cells, short NADH lifetime) and pixels with more bound NADH (meaning more oxidative phosphorylation, long NADH lifetime). This panel was adapted from reference¹¹⁷ Stringari et al. (2012), PLOS ONE https://doi.org/10.1371/journal.pone.0048014 published under CC BY 4.0 https://creativecommons.org/licenses/by/4.0/.