Biology Reference
In-Depth Information
where
l
is the illumination wavelength) will substantially increase the SNR with
only minimal reduction in the Z-resolution (
Conchello et al., 1994
). Thus, this
would increase the quality of the signal with little degradation of the depth
discrimination.
III. Limitations in Speed of Confocal Imaging
0.6
m
m
thin sections either in spot, line, or frame modes that either may be repeated
sequentially, or combined with stepwise up- or down-focusing through the speci-
men in order to generate 3D reconstructions. 3D sectioning does not allow for
recording of cellular events in real time, but repeated 1D line imaging or even
repeated 2D imaging with reduced frame sizes or restricted pixel numbers in
contrast do allow for relatively fast recording with a temporal resolution of
approaching a microsecond scale. Although not as fast as regular widefield imag-
ing, even 2D frame imaging may still be acquired on fast time scales, usually within
hundreds of millisecond, though there will be a trade-o
The removal of out-of-focus light allows for relatively fast imaging of
between temporal and
spatial resolution. The reason for the lower temporal resolution compared to
widefield imaging is that conventional confocal imaging requires some form of
scanning, that is, sequential pixel sampling, in order to ''build'' an image, which
thus happens pixel-by-pixel. In contrast, the whole field during widefield imaging is
captured simultaneously either by PMTs or charge-coupled device (CCD) cameras
(
Ogden, 1994
). For some Ca
2
รพ
events, imaging with a temporal resolution in the
order of milliseconds may be satisfactory, but other events may occur considerably
faster than this. Likewise, some events allow the microscopist to sample images
with a low spatial resolution, whereas others require the opposite. Thus, the speed
of confocal image acquisition depends on the mode and the settings of the scanning
and how many pixels are scanned before returning to the same pixel again.
This will be discussed later.
V
IV. Laser Scanning Confocal Microscopy
Out-of-focus light rejection and image acquisition through a confocal aperture
with a pinhole is the common principle that constitutes confocal microscopes, but
the illumination and excitation principles may di
er between various systems.
First, confocal microscopy by laser scanning the specimen (laser scanning confocal
microscopy, often abbreviated to LSM or LSCM) is the most widely used illumi-
nation and excitation method today.
During LSCM, a laser beam is directed on to the specimen, whereupon it scans
the designated field, which may be a single spot (in reality rarely used for biological
imaging apart from fluorescence recovery after photobleaching (FRAP) applica-
tions), a 1D line, or a 2D frame. The laser is controlled by the use of two oscillating
V
