Mapping the number of molecules and brightness in the laser scanning microscope - PubMed (original) (raw)
Mapping the number of molecules and brightness in the laser scanning microscope
Michelle A Digman et al. Biophys J. 2008.
Abstract
We describe a technique based on moment-analysis for the measurement of the average number of molecules and brightness in each pixel in fluorescence microscopy images. The average brightness of the particle is obtained from the ratio of the variance to the average intensity at each pixel. To obtain the average number of fluctuating particles, we divide the average intensity at one pixel by the brightness. This analysis can be used in a wide range of concentrations. In cells, the intensity at any given pixel may be due to bright immobile structures, dim fast diffusing particles, and to autofluorescence or scattering. The total variance is given by the variance of each of the above components in addition to the variance due to detector noise. Assuming that all sources of variance are independent, the total variance is the sum of the variances of the individual components. The variance due to the particles fluctuating in the observation volume is proportional to the square of the particle brightness while the variance of the immobile fraction, the autofluorescence, scattering, and that of the detector is proportional to the intensity of these components. Only the fluctuations that depend on the square of the brightness (the mobile particles) will have a ratio of the variance to the intensity >1. Furthermore, changing the fluorescence intensity by increasing the illumination power, distinguishes between these possible contributions. We show maps of molecular brightness and number of cell migration proteins obtained using a two-photon scanning microscope operating with a photon-counting detector. These brightness maps reveal binding dynamics at the focal adhesions with pixel resolution and provide a picture of the binding and unbinding process in which dim molecules attach to the adhesions or large molecular aggregates dissociate from adhesion.
Figures
FIGURE 1
At pixels k (A) and j (B) the average intensity is the same but the standard deviation is much larger at pixel k than at pixel j. This is due to few bright particles fluctuating at pixel k while at pixel j there are a large number of dim particles. Panels C and D show schematically the histogram of counts corresponding to the situation in panels A and B, respectively.
FIGURE 2
Autocorrelation function of fluorescein number fluctuations. Below 10−6 s, the amplitude of the fluctuations is large. At 1 ms, the fluctuations have been eliminated.
FIGURE 3
Results of simulations. (A) Recovered brightness as a function of the laser power. (B) Recovered n as a function of the number of particles in the box. (C) B as a function of the number of particles. (D) B as a function of the number of frames.
FIGURE 4
Simulation of fixed beads. (A) Average intensity image. (B) B image. (C) Selecting regions of the two-dimensional _B_-versus-intensity plot at low intensity and large B give the regions colored in red in panel D where the beads are absent but fluorescein molecules are present. (E) Selecting regions of the two-dimensional _B_-versus-intensity plot at large intensity and _B_-value to ∼1 give the regions colored in red (F) where the beads are present. Fifty frames were simulated for this figure.
FIGURE 5
B versus intensity for constant illumination (•) and for solutions of EGFP at different concentrations (▪). Twenty-five frames were collected for each point in this figure.
FIGURE 6
(A) Typical two-dimensional histogram of B versus intensity. The spread of the points in the histogram shows the pixel variance. (B) The recovered brightness of a solution of 120 nM EGFP as a function of the laser intensity. The laser intensity was evaluated by the increase in counts of the overall image. (C) Recovered number of particles as a function of the EGFP concentration. (D) Recovered brightness as a function of protein concentration.
FIGURE 7
Fluorescent beads in a sea of 100 nM fluorescein. (A) The average image. (B) The B image. (C) Selecting points in the two-dimensional _B_-versus-intensity plot at low intensity and large B give the region in panel D red color where the beads are absent. (E) Selecting regions of the two-dimensional _B_-versus-intensity plot at large intensity and _B_-value at ∼1 give the regions in panel F red color where the beads are present. One-hundred frames were collected for this measurement. Bleaching artifacts were corrected using the high-pass filter procedure. The image size is 11.2 _μ_m × 11.2 _μ_m.
FIGURE 8
Fluorescent beads (fixed) in solution of 100 nM fluorescein. The entire slide was slowly moved using a piezo stage. The beads appear as elongated in the direction of the stage motion (A). The B image shown in panel C shows that motion produces value of B at the pixels of the beads larger than the value at the pixels where only the fluorescein is present. After immobile fraction removal using the high-pass filter method, the average intensity still shows a shadow of the beads (B). However, this shadow is very weak and the B image (D) appears uniform after the removal of the motion artifact. The image size is 11.2 _μ_m × 11.2 _μ_m.
FIGURE 9
(A) Average image of a CHO-K1 cell transfect with EGFP. (B) The B image. (C) Two-dimensional histogram of the _B_-values versus intensity. A threshold at 1.5 counts was applied to this image to delete all points outside the cell from the analysis. The histogram in panel C is elongated due to the change in cell thickness. However, the _B_-value is relatively constant and >1. The brightness calculated for this cell is 3810 count/s/m. Calibration done at the same laser power for EGFP in solution shows brightness of 3960 counts/s/molecule. The histogram of the _B_-values (D) shows that the pixel distribution is relatively well defined. Forty frames were acquired for this measurement. The image size is 7.8 _μ_m × 7.8 _μ_m.
FIGURE 10
Paxillin-EGFP in CHO-K1 cells. (A) Average intensity. Paxillin accumulates at focal adhesions. (B) The B image shows that the larger values of B are seen at the borders of some adhesions. (C) Using the two-dimensional histogram of B versus intensity, all points with brightness of 1150 counts/s/molecules (corresponding to EGFP monomers were selected. These points accumulate in the cytosol. In panel D, all pixels with brightness of 11,500 counts/s/molecule were selected. These pixels accumulate at the border of the adhesions. Four-hundred frames were collected for this measurement. Motion artifacts were corrected using the high-pass filter procedure. The image size is 31 _μ_m × 31 _μ_m.
References
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