2 Microscopy and Imaging Systems
61
What is the minimum CCD resolution that is needed to use the full
optical resolution of a good microscope objective? Do we need a mega
pixel CCD or will a standard CCD do? Let us consider two objective lenses
commonly used in cytogenetics: a 100x/1.3 Oil, and a 63x/1.4 Oil objective. The optical resolution limit is 240 nm and 230 nm, respectively (in the
object plane). This corresponds to 100 x 240 nm =24 /lm and 63x230
nm=14 /lm, respectively, on the CCD sensor (image plane), due to the
magnification between object and image plane. To fulfill the sampling theorem, the pixel size must be smaller than 12 /lm for the 100x objective,
and smaller than 7 /lm for the 63 x objective.
A standard 2/3" video CCD camera having 756x581 pixels and a pixel
size of II /lm will fulfill the sampling theorem when using the 100x objective. At 63 x magnification, the same camera will reduce the image resolution below the theoretical optical limit of the objective. The mega pixel
cameras with their pixel size of 6.7 /lm will make use of the full resolution
of the 63 x and 40 x objectives. We conclude that a standard video CCD
camera is appropriate to exploit the full theoretical optical resolution of a
100 x immersion objective lens.
The signal to noise ratio of the CCD defines the dynamic range. CCD
noise is mainly photon noise due to the statistical nature of the photonelectron conversion process, read-out noise and (particularly for low signal intensities requiring long exposure times) dark current. The digitization depth is directly related to the contrast resolution or number of gray
levels that are available in the digital image. Apparently, digitization to 8
bit or 256 gray levels reduces the camera dynamic range. Does this mean
that 10 bit or 12 bit digitization is necessary? In most applications 8-bit
digitization (256 gray levels) will be sufficient. However, if FISH signals
with very different intensities are to be captured in the same image (labeled with the same fluorochrome), some information will be lost. Depending on the exposure time selected, one of the signals will be correctly
exposed while the other will either be saturated or will disappear in the
background noise. The problem can be solved if two acquisitions are performed with appropriate integration times. Multiplied with the respective
integration times, the 256 image gray levels of two separate exposures allow even quantitative analysis of signals that differ by a factor of 1000 and
more.
61
What is the minimum CCD resolution that is needed to use the full
optical resolution of a good microscope objective? Do we need a mega
pixel CCD or will a standard CCD do? Let us consider two objective lenses
commonly used in cytogenetics: a 100x/1.3 Oil, and a 63x/1.4 Oil objective. The optical resolution limit is 240 nm and 230 nm, respectively (in the
object plane). This corresponds to 100 x 240 nm =24 /lm and 63x230
nm=14 /lm, respectively, on the CCD sensor (image plane), due to the
magnification between object and image plane. To fulfill the sampling theorem, the pixel size must be smaller than 12 /lm for the 100x objective,
and smaller than 7 /lm for the 63 x objective.
A standard 2/3" video CCD camera having 756x581 pixels and a pixel
size of II /lm will fulfill the sampling theorem when using the 100x objective. At 63 x magnification, the same camera will reduce the image resolution below the theoretical optical limit of the objective. The mega pixel
cameras with their pixel size of 6.7 /lm will make use of the full resolution
of the 63 x and 40 x objectives. We conclude that a standard video CCD
camera is appropriate to exploit the full theoretical optical resolution of a
100 x immersion objective lens.
The signal to noise ratio of the CCD defines the dynamic range. CCD
noise is mainly photon noise due to the statistical nature of the photonelectron conversion process, read-out noise and (particularly for low signal intensities requiring long exposure times) dark current. The digitization depth is directly related to the contrast resolution or number of gray
levels that are available in the digital image. Apparently, digitization to 8
bit or 256 gray levels reduces the camera dynamic range. Does this mean
that 10 bit or 12 bit digitization is necessary? In most applications 8-bit
digitization (256 gray levels) will be sufficient. However, if FISH signals
with very different intensities are to be captured in the same image (labeled with the same fluorochrome), some information will be lost. Depending on the exposure time selected, one of the signals will be correctly
exposed while the other will either be saturated or will disappear in the
background noise. The problem can be solved if two acquisitions are performed with appropriate integration times. Multiplied with the respective
integration times, the 256 image gray levels of two separate exposures allow even quantitative analysis of signals that differ by a factor of 1000 and
more.