Figure 1.

Single-molecule detections in live cells can be clearly identified. (A) Oblique laser illumination fluorescence images of the wild-type ES cells (Brca2^WT/WT) and the heterozygous and homozygous Brca2-GFP knock-in cells. The nuclei of Brca2-GFP knock-in cells contain low-fluorescent diffusing and highly fluorescent bound particles. Brca2^WT/WT cell nuclei lack a distinct fluorescence signal. Cell nuclei were manually demarcated based on bright-field and fluorescence images and are indicated by white lines. Bar, 5 µm. (B) Each detected pixel area above background was fitted with a 2D Gaussian PSF characterized by a sigma and an intensity value, here based on 8-bit images. The intensity-sigma data pairs were plotted for all detections from one image stack. The data shown are representative of multiple experimental repeats (the number of individual stacks is >200). Camera noise contributes false detections (blue crosses), also found in Brca2^WT/WT cell nuclei and outside cell nuclei, which lie below an approximated exponential curve derived from this raw data (see Fig. S3 A). Remaining true detections are displayed as red circles. One pixel is 70 nm.