Fluctuation Imaging Spiced Up with a Piece of PIE

Abstract

In recent years there have been many new imaging-based extensions of fluorescence fluctuation spectroscopy introduced including one- and two-color variants of spatio-temporal image correlation spectroscopy (STICS and STICCS) (1,2) and raster image correlation spectroscopy (RICS and ccRICS) (3,4) as well as the number and brightness (N&B) method (5–7). The broad promise of these approaches hinges on the application of routine fluorescence microscopy imaging acquisition and live-cell imaging using cells expressing fluorescent protein constructs to measure transport, cotransport, and protein interaction stoichiometries. However, for the two-color (cross-correlation) variants of these methods, spectral cross-talk between channels is a significant problem, because this masquerades as an interaction between detection channels. This becomes more problematic in the case of fluorescent proteins where limited choice exists for fluorescent protein pairs that can be cleanly detected on standard microscopes. This entails that cross-talk controls have to be run for every cellular system to be studied. In their article in this issue of Biophysical Journal, Hendrix et al. (8) significantly extend the capabilities of fluctuation imaging methods by combining these with pulsed interleaved excitation (PIE) (9) and time-correlated single-photon counting detection. PIE was previously applied in single point measurements and involves rapid switching of pulsed laser excitation between the two channels with the fluorescence photons detected in a time-resolved manner that allows measurements without cross-talk. However, such an approach goes beyond simple removal of cross-talk because the time-correlated detection adds multiplexing of these methods with fluorescence lifetime imaging. Using this approach, the authors are able to register different fluorophores in different PIE channels rather than detection channels. This creates novel possibilities for lifetime weighting and gating and the extension of the image fluctuation methods to the lifetime domain is particularly exciting. The combination should be especially beneficial for measurements of molecular interactions because cross-correlation only detects species interacting within a common complex while the lifetime domain allows simultaneous fluorescence lifetime imaging microscopy/Forster resonance energy transfer measurements to assess direct interactions over the Forster distance scale. The authors demonstrate the wide versatility of the combination approach by using cells expressing standard fluorescent protein constructs (e.g., EGFP, mCherry, Venus FP) to perform PIE-RICS, PIE-ccRICS, PIE-N&B, and raster lifetime image correlation spectroscopy. One critique that might be considered is that the combination of methods moves this into the realm of the biophysics/optics specialist and out of the comfort zone of biomedical researchers equipped with standard confocal microscopes. To address this, the authors do provide a detailed description of the method, with an extensive Supporting Material section describing their home-built microscope as well as available software for those interested in implementing the techniques in their labs. One can envisage future instruments implementing this combination of methods with a broad landscape of possible PIE-FI measurements offered as a veritable dessert menu for the discerning experimentalist.