Figure 2. The importance of SNR in intensity and spatial measurements. (A) A digital image taken with a cooled CCD camera (ORCA-AG; Hamamatsu Photonics), with no light sent to the camera. Using MetaMorph software, a line (shown in red) was drawn across the bead and a line-scan graph was generated to show the intensity value of the pixels along the line. The graph shows line-scans of two similar images, taken in quick succession. The intensity values in the images fluctuate (range, 195–205) around the camera digital offset value of 200. Notice that the fluctuation in intensity values changes at each pixel from one image to the next. This variance is due primarily to thermal and readout noise from the CCD camera, and the extent of the variance will differ depending on the camera. This type of noise is superimposed on every fluorescence microscopy image. (B–E) Images of 6-µm beads that are fluorescently stained along their perimeter were collected with a wide-field microscope (model TE2000U; Nikon) using a Plan-Apochromat 60x 1.4 NA oil objective lens, the same camera as in A (ORCA-AG; Hamamatsu Photonics), and MetaMorph software. Line-scans generated as described for A. (B) An image of the bead taken with a 100-ms exposure time. The SNR is very low, making the bead indistinguishable from the noise in the line scan. (C) An image of the same bead as in B, taken with a longer (3 s) exposure time. The high SNR of this image would make quantitation of the intensity of the bead, or localization of the edge of the bead, highly precise. (D and E) The same bead images in B and C. Two images of the bead were taken, and one copy pseudo-colored red and one copy pseudo-colored green. The pseudo-colored images were shifted relative to one another by a few pixels and merged. (D) With low SNR images, it is nearly impossible to precisely locate the edges of the beads. (E) With high SNR images, the intensity line scan can be fit to Gaussian curves and the center located with nanometer precision. This allows the distance between objects of two wavelengths to be precisely determined, even if it is well below the resolution limit of the microscope (Churchman et al., 2005; Yildiz and Selvin, 2005; Huang et al., 2008; Manley et al., 2008). Bar = 5 µm.
Figure 2.

The importance of SNR in intensity and spatial measurements. (A) A digital image taken with a cooled CCD camera (ORCA-AG; Hamamatsu Photonics), with no light sent to the camera. Using MetaMorph software, a line (shown in red) was drawn across the bead and a line-scan graph was generated to show the intensity value of the pixels along the line. The graph shows line-scans of two similar images, taken in quick succession. The intensity values in the images fluctuate (range, 195–205) around the camera digital offset value of 200. Notice that the fluctuation in intensity values changes at each pixel from one image to the next. This variance is due primarily to thermal and readout noise from the CCD camera, and the extent of the variance will differ depending on the camera. This type of noise is superimposed on every fluorescence microscopy image. (B–E) Images of 6-µm beads that are fluorescently stained along their perimeter were collected with a wide-field microscope (model TE2000U; Nikon) using a Plan-Apochromat 60x 1.4 NA oil objective lens, the same camera as in A (ORCA-AG; Hamamatsu Photonics), and MetaMorph software. Line-scans generated as described for A. (B) An image of the bead taken with a 100-ms exposure time. The SNR is very low, making the bead indistinguishable from the noise in the line scan. (C) An image of the same bead as in B, taken with a longer (3 s) exposure time. The high SNR of this image would make quantitation of the intensity of the bead, or localization of the edge of the bead, highly precise. (D and E) The same bead images in B and C. Two images of the bead were taken, and one copy pseudo-colored red and one copy pseudo-colored green. The pseudo-colored images were shifted relative to one another by a few pixels and merged. (D) With low SNR images, it is nearly impossible to precisely locate the edges of the beads. (E) With high SNR images, the intensity line scan can be fit to Gaussian curves and the center located with nanometer precision. This allows the distance between objects of two wavelengths to be precisely determined, even if it is well below the resolution limit of the microscope (Churchman et al., 2005; Yildiz and Selvin, 2005; Huang et al., 2008; Manley et al., 2008). Bar = 5 µm.

This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal