Page 289 - Fundamentals of Light Microscopy and Electronic Imaging

272 DIGITAL CCD MICROSCOPY

apply binning or increase the gain. The ability to perform digital imaging efficiently and
knowledgeably requires that the user become completely familiar with these terms and
gain experience in the art of balancing camera acquisition parameters in order to opti-
mize criteria of high priority. Although mentioned in previous chapters, we will now
examine these terms in greater detail and in the context of using a digital CCD camera.
This topic is also addressed in Chapter 15.

Temporal Resolution

Full-frame slow-scan CCD cameras do not perform high-resolution imaging at video
rates. Generally the readout time is 1 second per frame, though this depends on the
processing speed (given in MHz) of the camera electronics. Since exposures of bright
specimens can be made as short as 10 ms using judicious subarray selection and bin-
ning, cameras with electromechanical shutters can acquire images of limited size and
resolution at rates up to 10 frames/s. Interline and frame-transfer CCD cameras have
no shutters and operate at faster speeds. The latest interline transfer cameras now pro-
vide full-frame 12 bit imaging at close to video rates.

Spatial Resolution and Image Dimensions

The spatial resolution of a CCD is determined by the pixel size and can be excellent. The
pixels in cameras used for biological imaging are usually smaller than developed silver
grains in typical camera film (10 m). Even with 2- to 3-fold magnification during
printing on a dye sublimation printer, the pixels comprising the picture are essentially
invisible. With such small detector elements, CCD cameras usually meet the Nyquist
criterion for image sampling, thus preserving optical resolution and avoiding aliasing
(see Chapter 13 on aliasing and the Nyquist criterion). For example, for a chip with 6.8
m pixels, there are 4 pixels per diffraction spot radius produced by a 100 , 1.3 NA
objective lens (0.25 m 100 magnifications 6.8 m/pixel 3.7 pixels/radius).
This is double the Nyquist limit, so spatial resolution is very good. Even with binning at
2 2, the Nyquist sampling criterion is very nearly satisfied. The reader should refer to
Figure 14-11, which compares CCD pixel dimensions with diffraction spot radii made
by different objective lenses. Give this matter serious attention when selecting a CCD
camera. The figure shows that a CCD with 10 m or smaller pixels is ideal for images
produced by high-magnification, high-NA, oil immersion lenses as would typically be
encountered in fluorescence microscopy.
The number of pixels along the length and width dimensions of the CCD is also
important when considering the quality and size of a print made with a dye sublimation
printer. A megapixel CCD with 1,000 pixels along one edge of the CCD gives a 3.3 inch
print when printed on a 300 pixel-per-inch dye sublimation printer set at a printer magni-
fication of 1 . Pixelation in the image is not observed for printer magnifications under
2–3 (10 inches on a side) which is fine for most microscope imaging applications.

Quantum Efficiency and Spectral Range

Quantum efficiency refers to the efficiency of photon-to-electron conversion in the
CCD, whereas spectral range refers to the wavelengths that can be detected. Standard