Page 232 - Fundamentals of Light Microscopy and Electronic Imaging

CRITERIA DEFINING IMAGE QUALITY 215

The following additional advantages of CLSM should also be considered:

• Magnification can be adjusted electronically by varying the area scanned by the
laser (zoom factor) without having to change the objective lens. Since the display
area on the monitor remains unchanged, the image appears magnified or zoomed.
• Digitization by an analogue-to-digital converter transforms the continuous voltage
signal into discrete digital units that correspond to steps of light intensity, thus
allowing you to obtain quantitative measurements of fluorescence intensity.
• There are few restrictions on the objective lens. However, for accurate color focus
and proper spatial resolution of light intensities, high-NA plan-fluorite or apochro-
matic oil immersion objectives should be employed.
• Epi-illumination and point scanning are ideal for confocal reflection microscopy, in
which the laser beam scans the three-dimensional surface of a reflective object. Fluo-
rescence filter sets are not used for this mode of imaging; rather, the focused spot of
the laser is reflected off the surface. Reflections from features lying in the focal plane
pass through the confocal pinhole aperture, whereas light from reflections above and
below the focal plane is largely excluded just as in confocal fluorescence microscopy.

CRITERIA DEFINING IMAGE QUALITY AND THE PERFORMANCE
OF AN ELECTRONIC IMAGING SYSTEM

The quality of a confocal image or any image is determined by four principal factors:

1. Spatial resolution
2. Resolution of light intensity (dynamic range)
3. Signal-to-noise ratio
4. Temporal resolution

Spatial resolution describes the smallest resolvable distance between two points in an
image. Resolution between two points in the image plane and along the z-axis depends
on the excitation and fluorescence wavelengths and the numerical aperture of the objec-
tive lens and settings in the confocal scan head. The numerical aperture of the objective
is crucial, since it determines the size of the diffraction-limited scanning spot on the
specimen and the size of the focused fluorescent spot at the pinhole. The role of the
numerical aperture in determining spatial resolution has already been discussed (see
Chapter 6). In wide-field fluorescence optics, spatial resolution is determined by the
wavelength of the emitted fluorescent light; in confocal mode, both the excitation and
emission wavelengths are important, because the size of the scanning diffraction spot
inducing fluorescence in the specimen depends directly on the excitation wavelength.
(See Chapter 5 for the dependence of the size of the diffraction spot on the wavelength
of light.) Thus, unlike wide-field fluorescence optics, the smallest distance that can be
resolved using confocal optics is proportional to (1/ 1/ ), and the parameters of
1
2
wavelength and numerical aperture figure twice into the calculation for spatial resolu-
tion (Pawley, 1995; Shotton, 1993; Wilhelm et al., 2000).
In the confocal microscope, spatial resolution also depends on the size of the pin-
hole aperture at the detector, the zoom factor, and the scan rate, which are adjusted using