Development of time-integrated multipoint moment analysis for spatially resolved fluctuation spectroscopy with high time resolution

Abstract

Spatial gradients in the behaviors of soluble proteins are thought to underlie many phenomena in cell and developmental biology, but the nature and even the existence of these gradients are often unclear because few techniques can adequately characterize them. Methods with sufficient temporal resolution to study the dynamics of diffusing molecules can only sample relatively small regions, whereas methods that are capable of imaging larger areas cannot probe fast timescales. To overcome these limitations, we developed and implemented time-integrated multipoint moment analysis (TIMMA), a form of fluorescence fluctuation spectroscopy that is capable of probing timescales down to 20 μs at hundreds of different locations simultaneously in a sample. We show that TIMMA can be used to measure the diffusion of small-molecule dyes and fluorescent colloids, and that it can create spatial maps of the behavior of soluble fluorescent proteins throughout mammalian tissue culture cells. We also demonstrate that TIMMA can characterize internal gradients in the diffusion of freely moving proteins in single cells.

Figure 1

Mean and covariance curves of fluorescence intensities of Alexa 488 in aqueous solution as a function of exposure time. Top: Mean curve (blue dots) with a best fit to Eq. 3 (red line). Bottom: Covariance curve (blue dots) with a best fit to Eq. 4 (red line).

Figure 2

Analysis of data obtained with colloid sample. (A) Mean and covariance curves from an individual pinhole (blue dots) with fits to Eqs. 3 and 4 (red lines). (B) Histograms of fit parameters from 50 different observation volumes showing the measured concentration, diffusion coefficient, and particle brightness. (C) Measured concentration and diffusion coefficient as a function of inverse dilution from the stock solution.

Figure 3

Analysis of EGFP diffusion in HeLa cells. (A) Image of three HeLa cells taken with spinning-disk confocal in imaging mode. Scale bar: 10 μm. (B) Mean and covariance curves with associated fits for two different locations (indicated in A and C–E): cell 1 and cell 2. (C) Map of concentration measured at the each location corresponding to an observation volume. (D) Map of diffusion coefficient. (E) Map of single fluorophore brightness.

Figure 4

Analysis of EGFP diffusion in HeLa cells subjected to an osmotic gradient. (A) Schematics of the experimental setup. (B) Top: An image of a cell in the microfluidic device without exposure to PEG, with the PDMS walls (blue lines) and cell boundary (red dotted line) indicated. Bottom: A map of the measurements of EGFP diffusion coefficient at different locations. (C) Top: An image of a cell in the microfluidics device exposed to PEG from one end, with the PDMS walls (blue lines) and cell boundary (red dotted line) indicated. Bottom: A map of the measurements of EGFP diffusion coefficient at different locations. Scale bar: 10 μm. (D) The average diffusion coefficient in these two cells as a function of position along the cell. The red lines are guides to the eye.

Figure 5

Analysis of EGFP-Ran diffusion in U2OS cells. Upper left: Confocal image of an EGFP-Ran-transfected U2OS cell. Scale bar: 10 μm. Top: Three maps of concentration, diffusion coefficient, and brightness are shown (the dashed blue line encircles the nucleus). Bottom: Covariance curves from EGFP-Ran in the nucleus and cytoplasm.