Andor Technology iXon Ultra 897 User Manual page 158

TUTORIAL 3
increased (this may be more easily visualised by clicking the cursor in a
background region of the image, then selecting the 2D icon), it is likely that
background light is being leaked into the system. This can markedly reduce
the effectiveness of this technology, since leaked background or unwanted
background fluorescence is primarily contributing to the detection limit at
higher gain settings, rather than the camera readout noise. This principle
applies to all low-light microscopy techniques and efforts should be made
to eliminate as far as possible any sources of background light.
N.B. At higher gain settings, another source of spurious noise may be
observed, in the form of occasional and random single pixel spikes on top
of the readout noise floor which are typically of lower intensity than the
true signal (furthermore, the signal in techniques such as Single Molecule
Microscopy is normally dispersed over a few pixels). This is called Clock
Induced Charge and is due to the electrons being generated electronically
during the charge shifting process, then amplified within the gain register.
Care must be taken also since single photon amounts of background light
may also appear as single pixel spikes above the readout noise floor, upon
application of gain.
Record Immobilised Beads and Use ROI - Leave the slide to dry for a
while, in order to immobilise the beads. Running the camera in Video
Mode as described above, at a suitable exposure time and gain setting for
detection, focus on one or more immobilised beads within the camera field
of view. Record a Kinetic Series of images as described above, whilst
performing an action which will result in the intensity of the fluorescence
fluctuating periodically during the total acquisition time (such as cycling of
the excitation aperture, insertion/extraction of ND filter, sliding gain setting
up and down, tune in and out of focal plane etc.).
An alternative approach to recording a kinetic series of full images, is to
record only an image sub-area. This has the effect of both increasing the
frame rate (by decreasing the readout time required for each frame) and
decreasing the file size of each image. To do this, it is necessary to first
define the minimum dimensions of the sub-area. This can readily be
accomplished by clicking the cursor onto four pixels on the full image
Section 7. 3
Page 158