Technical Reference #1688

1688.

Mapping the Number of Molecules and Brightness in the Laser Scanning Microscope Michelle A. Digman; Rooshin Dalal; y Alan F. Horwitz; y and Enrico Gratton, University of California, Biophysical Journal, 94(1688), (2008)
Link To Paper

Abstract:
Wedescribe 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.

Materials & Methods:
Cell culture CHO-K1 and mouse embryo fibroblast (MEF) cell lines were cultured in minimum essential Dulbecco’s Modified Eagle Medium low glucose (13) (Invitrogen Carlsbad CA) supplemented with heat-inactivated 10% (vol/vol) of certified fetal bovine serum (Invitrogen) 50 U/ml penicillin/ streptomycin with the exception of the addition of nonessential amino acids added to the CHO-K1 medium. Cells were maintained at 37 C in a humidified chamber with 8.5% or 5% CO2 for the CHO-K1 and MEF cell lines respectively. Cells were transfected with plasmids containing GFP and paxillin-GFP using lipofectamine 2000 (Invitrogen) according to manufacturer’s protocol. Cells were removed from tissue culture dishes by washing with Dulbecco’s phosphate-buffered saline (Invitrogen) without calcium and magnesium followed by 3–5 min incubation with 0.5 mL of trypsin-ethylenediaminetetracetic acid and plated on 35-mm glass bottom dishes (MatTek Ashland MA) coated with 1 or 2 mg/ml of fibronectin. Cells were maintained in CCM1 medium (HyClone Logan UT) and were imaged 1–2 h after plating. During imaging cells were kept at 37 C with a Harvard Apparatus chamber (Holliston MA).

Microscopic Technique
Fluorescence Microscopy

Cell Type(s)
CHO-K1