Biology Reference
In-Depth Information
mirrors in the scanhead that deflect the beam along a fast and slow axes perpen-
dicular to one another. Thus, during a 2D frame scan, the beam is first directed
along the horizontal axis, after which it ''jumps'' down one pixel and scans the next
line, and this process continues until a full frame has been scanned. This may then
be repeated for serial frame scanning, or the plane of focus may be moved along
the Z-axis for 3D imaging. The fluorescence emission returns along the same light
path (descanning), but is deflected by a dichroic mirror splitting the excitation and
emission lights, such that the emission light only is directed onto the confocal
aperture, where the in-focus light penetrates though the pinhole to reach the signal
detector.
Unfortunately, scanning is a rate-limiting step for gaining fast images, especially
in a 2D frame scan mode, because of the mechanical characteristics of the mirrors.
A typical 512
1 s, depending on the
settings under which the scan is performed. When scanspeed is of concern, several
approaches may be taken to increase this, for example, by linescanning instead of
framescanning. In this configuration, the same line is scanned sequentially for a
given period of time to allow detection of one or more events occurring within the
time frame of the scan. Although this provides a high temporal resolution, the
acquired information is limited to a one-pixel wide area of the cell, such that
information about events occurring elsewhere in the cell is missed. Furthermore,
reducing the pixel dwell time (the period over which each pixel is scanned) also
increases scanspeed, but this also reduces the SNR. Reduced SNR may partially be
compensated for by increasing the laser power, but this may present other problems
such as photodamage to the specimen; especially in live cells, and photobleaching.
Finally, the length of the line or the size of the frame to be scanned may also be
reduced, or fewer pixels may be scanned during 2D frame scanning to yield the same
e
512 pixel 2D frame may be scanned in
ect, and the scan may also be run in a bidirectional mode instead of unidirection-
al, though using two lines running in opposite directions may cause a slight o
V
V
set
between them, which tends to blur the signal. More recently, several approaches
have been taken to increase scanspeeds, in particular for 2D frame imaging, such as
utilizing resonant oscillating mirrors in the scanhead instead of the more conven-
tional galvanometer-driven mechanical mirrors. Other options include arranging
prisms and acousto-optical deflectors into the excitation light path to illuminate the
entire line simultaneously, instead of a pixel-by-pixel illumination applied by the
conventional laser scanning microscopes described above. This means that the
scanning in a 2D frame mode would only involve movement along one dimension
(X), since the other dimension (Y) would all be scanned at once (simultaneously),
and therefore, 2D frame scanning may be performed at linescan speeds or at speeds
approaching video rates, if the emitted fluorescence is deflected onto a linear CCD
camera, though PMT arrays may also be used. Single PMTs would, however, not be
able to construct the images if lines instead of single pixels are scanned. The caveat
with these approaches is that true confocality will be lost because the pinhole of the
confocal aperture must be replaced by one or more longitudinal slit openings to
accommodate the simultaneous scanning of lines (hence, this is also called slit
