Dynamic Cellular Cartography: Mapping the Local Determinants of Oligodendrocyte Transcription Factor 2 (OLIG2) Function in Live Cells Using Massively Parallel Fluorescence Correlation Spectroscopy Integrated with Fluorescence Lifetime Imaging Microscopy (mpFCS/FLIM)

Abstract

Compartmentalization and integration of molecular processes through diffusion are basic mechanisms through which cells perform biological functions. To characterize these mechanisms in live cells, quantitative and ultrasensitive analytical methods with high spatial and temporal resolution are needed. Here, we present quantitative scanning-free confocal microscopy with singlemolecule sensitivity, high temporal resolution (∼10 μs/frame), and fluorescence lifetime imaging capacity, developed by integrating massively parallel fluorescence correlation spectroscopy with fluorescence lifetime imaging microscopy (mpFCS/FLIM); we validate the method, use it to map in live cell location-specific variations in the concentration, diffusion, homodimerization, DNA binding, and local environment of the oligodendrocyte transcription factor 2 fused with the enhanced Green Fluorescent Protein (OLIG2-eGFP), and characterize the effects of an allosteric inhibitor of OLIG2 dimerization on these determinants of OLIG2 function. In particular, we show that cytoplasmic OLIG2-eGFP is largely monomeric and freely diffusing, with the fraction of freely diffusing OLIG2-eGFP molecules being f D,free cyt

Increased intracellular crowding during hyperosmotic stress

“…We measured eGFP fluorescence lifetime (τ FL,eGFP ) in live N2a cells under normal growth conditions (Figure 1B, left) and hyperosmotic stress (Figure 1B, right) using a home-built massively parallel FLIM system (mpFLIM) without laser scanning in which 16×16 confocal foci were generated using a Diffractive Optical Element (DOE) to split the incident laser beam into a 2-dimensional (2D) array of beams and a 2D Single Photon Avalanche Diodes (SPAD) array camera with matching geometry 11 . Subcellular positions where FLIM measurements were performed were identified using bright field and fluorescence imaging (Figure 1B).…”

“…The instrumental design and construction of the massively parallel imaging system without laser scanning is described previously 10, 11 . Briefly, simultaneous excitation of fluorescent molecules across the sample is achieved by passing a single pulsed laser beam through a DOE, which transforms it into a rectangular illumination matrix that consists of 16×16 foci.…”

“…Fluorescence and bright field images are captured by the 18.0 megapixel digital single-lens reflex camera Canon EOS 600D (Canon Inc., Tokyo, Japan) to identify GFP localization and visualize cell morphology. FLIM data acquisition and analysis was carried out using the MPD SPC3 software (http://www.micro-photon-devices.com/Software) and the homebuilt MP-FLIM software 10, 11 . Acquisition of a single FLIM curve lasted 3.7 s. τ FL,eGFP was determined by fitting a single-exponential decay function to the experimental FLIM curves.…”

“…τ FL can be measured in either the frequency or the time domain. Here, time domain measurements were used 10, 11 to probe the local cellular environment 12 ; FRET-donor and -acceptor ratiometric imaging using the GimRET sensor was deployed to monitor the changes in MMC after cell exposure to hyperosmotic stress; and diffusion of eGFP was measured using fluorescence correlation spectroscopy (FCS) 6, 13, 14 , to acquire information about local viscosity 15 .…”

Mapping the Direction of Nucleocytoplasmic Transport of Glucocorticoid Receptor (GR) in Live Cells Using Two-Foci Cross-Correlation in Massively Parallel Fluorescence Correlation Spectroscopy (mpFCS)

“…Explanation of the multitau procedure can be found in the literature. 31,32,47,48 For in vitro mpFCS measurements in dilute suspensions of fluospheres or quantum dots, the multitau algorithm was used to analyze the raw photon counts directly, and the following formula was used for the calculation of the tfCCCs:…”