Part 1 Principles
1. Fluorescence microscope
3. How concocal differs?
What is confocal?
Resolution in confocal
7. Confocal image formation
8. SNR in
Variations of confocal
10. Special features from
Leica sp2 confocal
3. Three-D reconstruction
4. Thick specimen
5. Physiological study
3 Operation and
2. Settings in detail
Laser intensity and
PMT gain and offset
Scan format, Zoom
Frame average, and
Pinhole and Z-resolution
Emission collecting rang
and Sequential scan
you need confocal?
Confocal Microscopy tutorial
Part 1 Principles of Confocal microscopy
4. What is confocal?
As mentioned on previous section, a
point light soure illumination within the specimen will improve image quality by
eliminating stray lighe intererence. Laser is a good candidate for providing a
point-like light soure. Unlike a mixture of wavelength, laser provides light at
discreted band of wavelength and has very high intensity which are also advantages for fluorescence exitation. For
this reason, laser is widely usd in confocal microscope.
practical, a point-like light source is achieved by using a laser light
passing through a illumination pinhole. This point-like light source is
directed to the specimen by a beam splitter (or AOBS in Leica's BSP-free
system) to form a point-like
illumination in the specimen. The point-illumination move or scan on the
specimen by the help of a scanner. The reflected emission light from specimen's focal
plane passes through the detecting pinhole and form point-like image on
detector PMT (photon multiply tube).
PMT converts detected photon into
It is possible to amplify weak signal by manipulating the voltage (gain) on
the tube. It is also possible to cut off background signal by set certain
threshold (Offset) on the tube.
PMT has large active area to receive photons thus high saturate point, and PMT has
low dark current thus low background, together, which provide high dynamic range
that is defined as the ratio of maximum allowed intensity / dark current.
Besides, PMT has very high refresh rate since there is no charge accumulate
on it. It detects event at nano- seconds level.
The electrons convert from photons form analogue current which is then digitized
by analogue-digital converter (ADC) and send to the digital image processor.
Taking together, in confocal system:
A point light source for
A point light focus within the specimen
A pinhole at the image detecting plane
These three points are
together and aligned accurately to each other in the light
path of image formation, this is confocal. Confocal effects result in
supression of out-of-focal-plane light, supression of stray light in the
Confocal images have following
void of interference from
lateral stray light: higher contrast.
void of supperimpose of
out-of-focal-plane signal: less blur, sharper image.
images derived from optically
sectioned slices (depth discrimination)
(theoretically) due to better wave-optical performance.
Is confocal effect a free cake?
No, confocal effect is obtained at a cost of greatly
reduced detecting volume (total signal amount), increase vulnerability to
noise, reduced dynamic range, etc. For detailed discussion, refer section 6:
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