snr
Confocal tutorial: Priniples: SNR in Confocal. AMU Dept Pathol Univ Hel
A:hover {color: #FF0000; font-weight: bold}
Part 1 Principles
1. Fluorescence microscope
2. Filterset
in FL-Mic
3. How concocal differs?
4.
What is confocal?
5.
Resolution in confocal
6. Optical
sectioning
7. Confocal image formation
and
time resolution
8. SNR in
confocal
9.
Variations of confocal
microscope
10. Special features from
Leica sp2 confocal
Part 2
Application
1. Introduction
2.
Tomographic view
(Microscopical CT)
3. Three-D reconstruction
4. Thick specimen
5. Physiological study
6.
Fluorescence detecting
General
consideration
Multi-channel detecting
Background correction
Cross-talk correction
Cross excitation
Cross emission
Unwanted FRET
Part
3 Operation and
Optimization
1.
Getting started
2. Settings in detail
Laser line
selection
Laser intensity and
AOTF control
Beam
splitter
PMT gain and offset
Scan
speed
Scan format, Zoom
and Resolution
Frame average, and
Frame accumulation
Pinhole and Z-resolution
Emission collecting rang
and Sequential scan
When Do
you need confocal?
FAQ
Are
you abusing
confocal?
Confocal Microscopy tutorial
Part 1 Principles of Confocal microscopy
8. SNR (Signal to Noise Ratio) in confocal microscopy
As mentioned in last section, PMT (photon multiplier tube) has a
large active area with high capacity for photons thus can detecting strong
signal with less saturation problem. PMT can multiply received photons thus has
high photon sensitivity suitable for detecting weak signal at very low noise
level. These features endow PMT with wide dynamic range and good
SNR.
But in fluorescence microscopy, the total photon number is very
small, less than 1000 photons per pixel time, and even smaller in confocal
microscope, about 10-30 photons/pixel/µs due to the
massive rejecting of signal by pinhole. The dynamic
range can not be calculated from the ratio of full photon capacity / noise
anymore, and solely affected by signal intensity.
It is determined by formula:
30 photon influx corresponds to only 5 bits grey level.
Similarly, the SNR is affected more by signal intensity (total
photon influx) than
by background noise level as mentioned in part 1
Optical section,
Formula 4 describes
different factors in SNR.
Where
N: photoelectron number per pixel time.
S: secondary noise from random variation of multiplication.
d: dark current of PMT.
q: laser noise.
When N is 1000 photoelectron per pixel time, if set S: 1.2, d:
100. q: 0.05, SNR is 25.
At 400 Hz scan speed, 512 format, pixel time is about 5 µs
and N is about 150 assuming photon influx is 30 photons/pixel/µs,
from formula above, with S, D, Q unchanged, SNR is 7.89 only.
In formula 4, photoelectron number on the numerator has to be squared,
it has more weight in the formula and results in a pronounced effect on SNR.
This makes confocal microscope very vulnerable to weak signal. The reduced photon number not only weakens signal
intensity, but also deteriorates image quality. Raising gain (voltage)
on PMT can amplify weak signal but also raise noise, raising offset on PMT (threshold) can cut off
background noise but signal is equally affected, the SNR and
image quality won't improve. In worst case, the structure details are buried in
the noise, you even
can't get usable data at all. To improve image quality, approaches which
increase photon number has to be used, such as average, accumulation, slow scan
speed, lower scan format, larger pinhole size, etc..
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This page was last updated
28.11.2005
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