Measuring fast dynamics in solutions and cells with a laser scanning microscope

Abstract

Single-point fluorescence correlation spectroscopy (FCS) allows measurements of fast diffusion and dynamic processes in the microsecond-to-millisecond time range. For measurements on living cells, image correlation spectroscopy (ICS) and temporal ICS extend the FCS approach to diffusion times as long as seconds to minutes and simultaneously provide spatially resolved dynamic information. However, ICS is limited to very slow dynamics due to the frame acquisition rate. Here we develop novel extensions to ICS that probe spatial correlations in previously inaccessible temporal windows. We show that using standard laser confocal imaging techniques (raster-scan mode) not only can we reach the temporal scales of single-point FCS, but also have the advantages of ICS in providing spatial information. This novel method, called raster image correlation spectroscopy (RICS), rapidly measures during the scan many focal points within the cell providing the same concentration and dynamic information of FCS as well as information on the spatial correlation between points along the scanning path. Longer time dynamics are recovered from the information in successive lines and frames. We exploit the hidden time structure of the scan method in which adjacent pixels are a few microseconds apart thereby accurately measuring dynamic processes such as molecular diffusion in the microseconds-to-seconds timescale. In conjunction with simulated data, we show that a wide range of diffusion coefficients and concentrations can be measured by RICS. We used RICS to determine for the first time spatially resolved diffusions of paxillin-EGFP stably expressed in CHOK1 cells. This new type of data analysis has a broad application in biology and it provides a powerful tool for measuring fast as well as slower dynamic processes in cellular systems using any standard laser confocal microscope.

FIGURE 1

Simulated images of freely diffusing 10-nm beads (A) and EGFP (D) in a plane. The fast EGFP moving molecules appear as a streak in the image (D). B and E are the two-dimensional spatial correlation functions of images A and D, respectively. C and F are the fit of the autocorrelation functions B and E according to the equations in the text. Image parameters: frame 256 × 256, 16 μs pixel time, and 0.050 μm pixel size.

FIGURE 2

Diagram of the range of diffusion times accessible by different scanning techniques. Depending on the timescale of the process, pixel (microseconds), line (milliseconds), or frame (seconds) correlation methods can be used.

FIGURE 3

10-nm fluorescent beads freely diffusing in solution. (A) Single-point FCS. (B) Pseudo-image obtained with circular scan. Orbit period is 1 ms, there are 64 points per orbit, and 100 orbits are averaged together per each line of the pseudo-image. The orbit diameter was 1.5 μm. (C) Two-dimensional spatial correlation of the pseudo-image. (D) Fit of the two-dimensional correlation using the equations in the text.

FIGURE 4

EGFP freely diffusing in solution. (A) Image obtained with an Olympus LSM. (B) Two-dimensional spatial correlation of a stack of images. (C) Fit according to the equations in the text. The recovered diffusion coefficient is D = 89 μm²/s. Pixel dwell-time is 4 μs, line time is 3.176 ms, pixel size is 0.09207 μm, and image size is 128 × 128 cut from the original 512 × 512. The protein concentration was 20 nM.

FIGURE 5

(A) Image of a CHOK1 cell expressing paxillin-EGFP. (B) 64 × 64 subframe in the cytosolic part free of focal adhesion structures. (C and D) Spatial autocorrelation before (C) and after (D) subtraction of immobile structures. (E) Fit of the spatial correlation function in D. The diffusion coefficient in this cell region is 8.3 μm²/s. Pixel rate was 8 μs, pixel size is 0.09207 μm, line time is 5.048 ms, frame size 512 × 512, and a total of 23 frames were collected.

FIGURE 6

(A) Image of a CHOK1 cell expressing paxillin-EGFP. (B) 64 × 64 subframe in the cytosolic part on a focal adhesion structure. (C and D) Spatial autocorrelation before (C) and after (D) subtraction of immobile structures. (E) Fit of the spatial correlation function in D. The diffusion coefficient in this cell region is 0.49 μm²/s. Image parameters are the same as in Fig. 5.

FIGURE 7

(A) Pseudo-image of the line scan (green line in B) of a CHOK1 cell expressing paxillin-EGFP. The image has 512 × 512 pixels. The pixel size is 0.09207 μm. The line time is 5.04 ms. Data were processed according to the equations in the text. For each point along the line the temporal autocorrelation function was calculated and fit using Eq. 7. The recovered values of D (in μm²/s) are given at selected positions along the line. There is a clear correlation between low diffusion values and visible structures in the images corresponding to focal adhesion sites.