Page 233 - Fundamentals of Light Microscopy and Electronic Imaging

216 CONFOCAL LASER SCANNING MICROSCOPY

software that controls the scan head. Decreasing the size of the pinhole reduces the thick-
ness of the focal plane along the z-axis, thereby allowing higher resolution in optical sec-
tioning, which is essential for high-quality projection images and high-resolution
three-dimensional viewing. Reducing the size of the pinhole also improves contrast by
excluding out-of-focal-plane sources of light. Under certain conditions, the lateral spatial
resolution in the xy plane obtainable in a confocal fluorescence microscope can exceed that
obtainable with wide-field optics by a factor of 1.4, a condition sometimes called super-
resolution. Normally the detector pinhole is adjusted to accommodate the full diameter of
the diffraction disk. However, if the pinhole is stopped down to about one-quarter of the dif-
fraction spot diameter, the effective spot diameter is slimmed down so that the disk diame-
ter at one-half maximum amplitude is reduced by a factor of 1.4. Oldenbourg et al. (1993)
have shown that image contrast is also improved by stopping down the pinhole.
As given by R. Webb in Pawley (1995), the minimum resolvable distance d between
two points in the xy horizontal plane of the confocal microscope can be approximated as:
d x,y 0.4λ/NA.

Resolution along the z-axis is described:
2
d 1.4λn/NA .
z
The effect of pinhole diameter on section thickness is shown in Figure 12-9. A con-
stricted pinhole in confocal optics increases horizontal resolution in the x,y plane by a
factor of 1.4 but decreases the axial resolution by the same factor. The effects of zoom
factor and scan speed on resolution are discussed later in this chapter.
Dynamic range (DR) describes the resolution of light intensity in the image and is
defined as the number of gray levels that are assigned to an image by the analogue-to-

1.0

0.8

Z 1/2 ( m) 0.7

0.5

0.4

0.2
0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0
Pinhole diameter (mm)

Figure 12-9
Confocal pinhole diameter vs. optical section thickness. The thickness of the optical section
through the z-axis of the confocal image (Z) is plotted vs. the diameter of the pinhole
aperture in millimeters. Fluorescent beads (0.2 m) were examined with a Nikon Planapo
60 , 1.4 NA oil immersion lens. (From Pawley, 1995; Kluwer/Penum Publishers, with
permission)