Olympus Fluoview-1000 User Manual - Page 34

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V.M. Bloedel Hearing Research Center, Core for Communication Research

Center on Human Development and Disability, Digital Microscopy Center

b. optimize the laser and channel parameters

4. Repeat step 3 with each negative control, adjust laser power and channel settings;

a. negative labeling controls should not display a specific signal

5. Image each positive control specimen in the other channels without changing their optimized

parameters, after completing step 4;

6. If a positive control appears in another channel, adjust to laser power and the channel settings to

reduce the bleedthrough to acceptable levels

7. Rescan the positive controls using the current settings, if anything changed in steps 5, 6 or 7;

8. Evaluate image quality for acceptable signal strength, noise and other parameters;

a. do you have to make compromises to obtain acceptable signal strength, noise levels, etc?

9. Examine an experimental specimen with all labels;

a. do you have to make compromises to obtain acceptable signal strength, noise levels, etc?

10. Capture images.

6.6

Noise.

Noise reduces spatial resolution and contrast. The 2 major noise sources for confocal microscopy are the

statistical variation in the number of photons emitted from each sample point (shot noise) and the

detector noise. Noise will occur in all pixels and in the signals from label as well as from background.

It is generally reduced by avoiding low signal intensities, high PMT HV settings, use of Kalman

averaging and by deconvolution.

The number of photons recorded by the detector from each sample point in the specimen varies by +/-

the square root of the number photons from the mean of the population. This variation represents 1

standard deviation on each side of the mean number of photons. This translates into the reality that the

signal will lie somewhere within this range 68% of the time. Shot noise results in greater variability for

low intensity pixels representing fewer photons.

Shot noise is reduced by increasing the number of photons contributing to the signal either by increasing

signal or by averaging multiple images. Signal strength might be increased, with significant tradeoffs,

by using higher laser power, longer dwell times, high NA objectives or increased staining levels. The

amount of signal obtained by increased application of excitation decreases as the fluorophores begin to

saturate and undergo photobleaching. Averaging is discussed in section 6.6.3.

The maximum intensity in a typical confocal image represents only 25 photons. The shot noise in the

brightest pixels will be 25+/- 5 photons, or variation of +/- 20%. If the HV is set so that a maximum

signal of 25 photons creates an intensity of 4,000, then the signal will vary between 3,000 and 5,000

(saturated) for 68% of the times that the specimen is scanned and fall outside of this range 32% of the

time. Dark areas display greater variations in pixel values due to shot noise representing a larger portion

of the signal. If a moderate intensity pixel represents 9 photons, its shot noise will result in a 33%

variation in signal.

May 11, 2011

Olympus Fluoview-1000 User's Guide