Biology Reference
In-Depth Information
Light
detector
Confocal aperture
with pinhole
Laser
Dichroic
beamsplitter
Objective
Focal plane
Fig. 1
Overview of the optical pathway of a confocal microscope. The blue path illustrates the
excitation light, whereas the green path illustrates the emitted fluorescence light. Note that the confocal
aperture with the pinhole in front of the light detector (usually, a photomultiplier tube (PMT)) blocks
out-of-focus light.
cell volume will contribute to unwanted out-of-focus background signal, or noise
(
Lichtman and Conchello, 2005
), and this cannot be distinguished from in-focus
fluorescence. Thus, most of the cell will be out of focus, and with it, the vast majority
of the signal will come from out-of-focus areas. In contrast, setting the pinhole of
the confocal aperture to 1 airy unit to achieve true confocality will provide an
optical section or Z-resolution of 0.5-1
m
m with the same high NA objective as
described above. This will only allow a minimum of out-of-focus light to reach the
signal detector, without any other interference to the optical pathway or any
secondary digital processing of the signal at the time of recording apart from the
scanning and building of the image itself, which otherwise would have further
compromised the scanning speed.
The full 3D XYZ-resolution, or the ability to discern two points from each
other, will, however, be di
V
raction-limited as determined by the point spread
function (PSF) set by the optical performance of the microscope. With high NA
objectives (
>
1.2), this is typically
0.3
0.3
0.6
m
m(
Cox and Sheppard, 2004
).
Although Z-resolution is dramatically di
erent between conventional epifluores-
cence and confocal microscopes, the 2D spatial resolution in the XY-field is
not, though factors, such as excitation wavelength, objectives and the optical
V
