Biology Reference
In-Depth Information
VII. Parallel Scanning Confocal Systems
The main limitation of the traditional laser scanning confocal microscope sys-
tems discussed so far is their low full frame temporal resolution. At typical rates of
just a few Hertz, an increase of 1-2 orders of magnitude in the speed of image
acquisition is necessary to decipher the fast Ca
2
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dynamics of systems such as
neuronal networks or cardiac and muscle tissues.
Recent developments in parallel multispot confocal illumination devices have
gone some way to addressing these concerns, providing high contrast optical
sectioning with typical rates in the 10-100 Hz domain. The multispot system uses
an aperture mask at an illumination plane conjugate with the sample, where
multiple illumination points with nonoverlapping airy disk profiles are projected
to the sample simultaneously. This illumination pattern is then changed in a
sequential fashion such that every image point is uniformly illuminated in a
given time interval. Two approaches have been used in recent years to successfully
achieve this fast pattern change: the Nipkow spinning disk where a patterned disk is
spun at high speeds, and programmable matrix systems (digital mirror and liquid
crystal arrays) where individual pixels can be switched ''on'' and ''o
V
'' at very high
speeds.
The parallel illumination approach has the obvious advantage that all image points
can be illuminated much faster than in a conventional single scan system, but the
drawback is that a 2D imaging detector is required to record the image, which
then becomes the limiting factor for capturing image frames. Limitations of the
detectors (typically CCD cameras) are further exacerbated by the requirement for
fast detection, as these devices have a noise floor below which the fluorescence signal
cannot be resolved. The fundamental problem is that in order to increase frame rates,
the exposure time of a frame has to be reduced, thus limiting the number of detectable
photons in an integration interval. Simultaneously, the transfer rate of data from
the camera has to be increased, a process by which the read noise of CCD cameras
increases. Many of these concerns are resolved by modern high-end electron multi-
plication (EM) CCD cameras such as the iXon 897 (Andor), Evolve (Photometrics),
or ImagEM (Hamamatsu). These EM-CCD cameras can amplify small photoelec-
tron signals above the read noise of the camera; providing the sensitivity and speed
required making these theoretical parallel approaches a practical reality.
VIII. Spinning Disk Confocal Microscopy
These systems illuminate the specimen simultaneously with a large number of
non-overlapping points of light by using multiple (hundreds to thousands) pinholes
arranged in a geometrically precise spiral pattern on a spinning disk (Nipkow disk).
The disk is placed at an image plane conjugate with the sample, and the illumination
is filtered by this disk. Thus, the specimen is raster scanned rather than single-spot
