Isolation of bright aggregate fluctuations in a multipopulation image correlation spectroscopy system using intensity subtraction

Abstract

Image correlation spectroscopy allows sensitive measurement of the spatial distribution and aggregation state of fluorescent membrane macro molecules. When studying a single population system (i.e., aggregates of similar brightness), an accurate measure can be made of the aggregate number per observation area, but this measurement becomes much more complex in a distributed population system (i.e., bright and faint aggregates). This article describes an alternate solution that involves extraction of the bright aggregate population information. This novel development for image correlation spectroscopy, termed intensity subtraction analysis, uses sequential uniform intensity subtraction from raw confocal images. Sequential intensity subtraction results in loss of faint aggregate fluctuations that are smaller in magnitude than fluctuations due to the brightest aggregates. The resulting image has correlatable fluctuations originating from only the brightest population, permitting quantification of this population's distribution and further cross-correlation measurements. The feasibility of this technique is demonstrated using fluorescent microsphere images and biological samples. The technique is further used to examine the spatial distribution of a plasma-membrane-labeled fluorescent synthetic ganglioside, and to cross-correlate this probe with various membrane markers. The evidence provided demonstrates that bright aggregates of the fluorescent ganglioside are associated with clathrin-coated pits, membrane microvilli, and detergent-resistant membranes.

FIGURE 1

An image composed of bright and faint fluorescent microspheres and its corresponding autocorrelation function. A 15.5 × 15.5 μm zoom-10 CLSM image of 0.44 μm diameter spheres was taken at ∼0.03 μm per pixel resolution. This image was then used to generate a second image with half-as-bright beads (Scion Image), but greater in number. This second image was merged with the first image to create the image shown (A). This image was sent for ICS analysis to obtain the correlation function shown (B). The front left and back quadrants show the raw correlation function, while the front right quadrant shows the fit to the Gaussian function.

FIGURE 2

Intensity subtraction analysis on mixed fluorescent microsphere images with varying contribution of intensity from the faint population. Images were created as in Fig. 1. All three images have the same number of bright microspheres (beads), but have varying contribution from the faint microsphere population: A contains faint microspheres one-eighth as bright as the bright microspheres; B contains the same number of faint microspheres as A, but they are only one-half as intense as the bright microspheres; and C has faint microspheres of the same intensity as B, but 3× in number. The corresponding calculated ICS parameters as a function of subtracted intensity (bottom axis) are shown: g(0,0)ω² (i–iii), intensity (iv–vi), and CD_obs (vii–ix). The intensity curves (iv–vi) show the mixed (▾), bright (○), and faint (•) microsphere contributions.

FIGURE 3

Intensity subtraction analysis on live cells labeled with cholera toxin B subunit. (A) A confocal image of a live CV1 cell at 10°C labeled with cholera toxin B subunit (Materials and Methods). The image is a 512 × 512 pixel intensity map with physical dimensions of 15.5 × 15.5 μm. The line drawn across the image indicates the region from which the line plots, shown in C–E, are generated. (B) An ISA intensity plot of g(0,0)ω² versus intensity subtracted. The two points indicated, D and E, are the subtraction levels that generated the images from which the intensity lines scans are shown in D and E. (C) Line plot measured from the original image from the line shown in A. (D) The same region plotted from the image with 26 Arb.U. subtracted from the original image. (E) The same region plotted from the image with 32 Arb.U. subtracted from the original image.

FIGURE 4

CLSM images of NBD-GD1a-labeled CV1 cells. CV1 cells were labeled with NBD-GD1a as described (Materials and Methods). Shown are images of a single cell (A), a z-section of a single cell (B), and a zoom-10 image used for ICS analysis (C). Bar in A is applicable to B.

FIGURE 5

Comparison of CD, SD, and CD_ISA values for three different labels that display multipopulation labeling. CV1 cells were labeled as described (Materials and Methods) and placed on the microscope at 10°C. Zoom-10 images were collected on NBD-GD1a (N = 180), NBD-PE (N = 170), DiQ (N = 110), and ConA-biotin (N = 120) labeled CV1 cells from at least three different sets of labeling for each label. The CD (black bar), SD (light gray bar), and CD_ISA was derived from these images as described (by ICS). N represents the individual number cells used to calculate the mean and standard error of the mean (error bars).

FIGURE 6

Analysis of NBD-PE and NBD-GD1a bright aggregate cross correlation with membrane structural markers. Cells were dual-labeled with NBD-PE or NBD-GD1a. NBD-PE samples were dual-labeled with (A) WGA-TRX, (B) ConA-biotin, (C) transferrin-biotin, or (D) DiQ. NBD-GD1a samples were dual-labeled with (A) WGA-TRX, (B) ConA-biotin, (C) Transferrin-biotin, or (D) DiQ. Also shown is dual-labeling of ConA-biotin and DiQ (E). Zoom-10 images were collected for each of these samples in two detection channels and the images were modified using ISA to isolate the bright aggregate fluctuations. Shown are the ISA-CD_g (black bar), ISA-CD_r (light gray bar), and ISA-CD_rg (dark gray bar).

FIGURE 7

NBD-GD1a aggregates bleach and then recover. CV-1 cells were labeled with NBD-GD1a and imaged on a microscope at 10°C as described (Materials and Methods). Shown is a representative image bleach and recovery. An initial image is collected (A). This area was then bleached by six scans of 0% attenuated laser, after which the zoom-10 image was collected (B). Four min was allowed to pass before collection of the recovered image (C). The image in B has a large contrast stretch to verify the removal of the fluorescence in this area. The pseudocolor overlap of A and C is shown (D).