6.6.2 Detector Noise.
The HV setting on the PMT controls the likelihood photo-electrons will be accumulated and the degree
of signal amplification. When the HV is raised, electrons will begin to spontaneously jump between
anode stages (amplification stages) in the PMT. These spontaneous electron movements create random
intensities that add to the signal output at random times. These appear as random intensity variations in
the image with no relationship to the specimen signal. Keeping the HV below 750 is highly
recommended to avoid detector noise.
6.6.3 Kalman Averaging.
Noise is a random event that can be reduced by averaging multiple images. The amount of noise
reduction in the final image is the square root of the number of scans. There is a diminishing return to
increasing the number of scans, for example, 2 scans will reduce noise by a factor of 1.41, but 9 scans
reduces noise by only a factor of 3. However, averaging only 2 or 3 images can have a dramatic impact
on images with high noise, regardless of origin. The tradeoff is that repeated scanning causes
photobleaching, which can be mitigated by using reduced laser power. The averaging method of
Kalman is commonly employed in microscopy. Averaging frames acquired with reduced laser power
and increased HV may be useful with weak or photosensitive samples. The FV-1000 generally does not
require averaging with relatively bright images.
6.7 Background
Background is an imprecise term for intensities that can not be attributed to specific (or desired) label
signal. Background reduces contrast and dynamic range, interferes with computerized feature
recognition and image processing. Sources of background signal include autofluorescence from tissue,
fixatives, specimen preparation or culture reagents, non-specific antibody binding, non-specific label
binding, reflections and bleedthrough. Background elevates signals and adds noise to the image as shot
noise and detector noise are superimposed on background signal.
6.8 Bright Images versus Scorching the Specimen
Everyone wants “bright” images with high intensity pixels that stand out against the non-specific
background intensities. Striving for “brighter” images through increased laser power or dwell time will
always result in photobleaching and usually give increased background due to autofluorescence and
bleedthrough. Raising the PMT’s HV will increase noise to reduce resolution and the SNR, unless
accompanied by lowered laser intensity and averaging.
The labeled structures in your images only need to display intensities that are high enough to be
distinguished from background and noise. Intensity of the image on the monitor or print can be adjusted
by histogram stretch, offset and gamma adjustments in software.
6.9 Images with Weak Signals
Images can be obtained from specimens that are weakly fluorescent or prone to photobleaching or
phototoxicity. Each approach has trade-offs that must balanced.
Olympus Fluoview-1000 User’s Guide
V.M. Bloedel Hearing Research Center, Core for Communication Research
Center on Human Development and Disability, Digital Microscopy Center
May 11, 2011 35
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