Using CMOS Cameras for Light Microscopy
a result of differences in response among the column readout amplifiers across the chip. In such CMOS cameras, this behavior can be clearly visualized by saturating the image sensor with light, and then the variations in the maximal column gray levels become quite apparent. It should be noted that the QImaging Rolera Bolt scientific CMOS camera used for the data collected in this paper does not exhibit such behavior and has much more uniform pixel response. Te non-uniform response seen in other CMOS cameras is mainly an issue in low-light, high-end imaging, so for these situations it may be more advantageous to use a CCD or EMCCD [2]. It should also be noted that fixed pattern noise can be removed through data post-processing by subtracting the pattern. Te SNR is a useful figure of merit to compare standard
CCD cameras with up-and-coming scientific-grade CMOS cameras. Two general approaches can be taken in calculating SNR. One approach uses parameters obtained from datasheets provided by the more reputable scientific imaging camera companies and known user parameters, such as magnification, numerical aperture (NA), pixel size, and specimen dye label concentration. Tus, the signal can be calculated along with the SNR using a somewhat complicated method outlined elsewhere [3]. Te second approach uses a more empirical method by extracting the noise and the signal from the acquired sample and bias images (these are images acquired with the camera in the absence of light). Tis is the method used in this paper and laid out in Figure 1. Based on the flow diagram in Figure 1 and images captured on a system with both a CCD and a scientific- grade CMOS attached, SNR can be measured as a function of exposure time, a common parameter in light microscopy for varying captured signal levels. In Figure 2, comparison images of fluorescently labeled
bovine pulmonary artery cells are shown, along with their respective SNR values, at various exposure times. Te epifluorescence images were taken using a Photometrics DC2 dual camera system with a 50/50 beam-splitter cube to simultaneously split the light equally between the two cameras to be compared [4]. Te images in the top row of Figure 3 were acquired with a standard front-illuminated CCD microscope camera using a 60× magnification, 1.35 NA, oil immersion objective such that its 6.45-µm pixel size is optimized for this magnification. Te bottom row of Figure 2 shows images acquired with the Rolera Bolt, a new QImaging scientific- grade front-illuminated CMOS camera using a 1.35 NA, 40× magnification oil immersion objective with smaller 3.63-µm pixels optimized for this lower magnification. A stack of 10 images was acquired for each camera and exposure time. Te standard deviation and mean of each pixel in the stack was found. Te average bias value was subtracted from the mean image to obtain a signal at each pixel. Tis image was then divided by the standard deviation image, or noise, to obtain the SNR at each pixel. Te mean and standard deviation of a background area was measured. A threshold was set at two of these background standard deviations above the background mean. In the final step, the mean value of the SNR in the pixels above this background threshold was measured and shown in Figure 2. Te SNR values demonstrate that the 3.63-µm pixel scientific-grade CMOS at 40× has similar SNR performance at short exposures compared to the 6.45-µm pixel CCD at 60×.
24
Figure 1: Outline of the process used for measuring SNR for an image with a given camera under a given set of conditions.
Again, both cameras are able to appropriately sample under these conditions. It could be proposed that the 6.45-µm-pixel CCD could also be used with a 40× objective or with a 60× objective and a 0.5× coupler, but at these smaller magnifications
Figure 2: Images acquired with a CCD and a CMOS at 60× and 40×, respectively, over three different exposure times. Image quality clearly improves with exposure time, and the two technologies produce images of comparable quality under these conditions.
www.microscopy-today.com • 2011 July
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