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
5. Lateral and Axial Resolution in
Point Spread Function (PSF) and resolution
In optical lens system,
lens can magnify an object because of its geometric optical property. But because of the wave property of light, diffraction
occurs when light pass it like the depicted in the figure. The image formed by
a point light at
the focal plane is not a single point, instead, the point distributed in a
possible area on the image plane. This distribution has higher probability
(higher intensity) at center and lower probability (lower intensity) at
peripheral, i.e., the point is spread after lens. This is Point Spread Function
(PSF). PSF describes the probability for an area where a given point will appear.
The distribution pattern looks like
a disc with a central high intensity spot and many concentric rings, a pattern
called Airy disc. Airy pattern describes the light intensity distribution as a
function of distance from the optical axis.
The intensity distribution takes
about 84%, 91%, 94% total photons within the inner, the 2nd and the third ring,
and weaker further along distance.
The optical resolution of a lens is
determined by the size of the inner dark ring of its Airy disk.
The radius of the inner ring is related to the aperture of the lens. The
smaller the aperture, the larger the PSF and the bigger the inner ring, thus
the lower its optical resolution. When single point
resolution is concerned,
the radius of the inner ring, as shown in the figure, is used to define the
resolution, thus called resolution element (resel). When point-to-point
resolution is concerned, the resolution is defined as the distance of the
two points where the maximum of one point is just above the first minimum of
the second point (Rayleigh criterion).
Another frequently used definition
for resolution of a lens is the Full Width of Half Maximum (FWHM) of the airy
disk which is diameter of the central spot where the intensity drop to its half
maximum. FWHM is widely used because it is easy to measure.
depends on wavelength of the light in use, the numerical aperture (NA) of
the lens. Formula 1 is used for the lateral resolution:
|In confocal microscope, due to its improved contrast, there
is a slight gain of resolution but it is still diffraction limited. When pinhole is infinite close to zero, (in
practical, < 0.25 AU), lateral Res. is calculated by Formula 2:
Compare these two formula, you can see that, in theory, confocal system
provides higher resolution than conventional. It comes from the ratio of
roughly, about 1.4 times of conventional.
But the pinhole size can not be infinite to zero, when pinhole is
between 0.25-1 AU, a value between 0.37-0.51 is used to replace
0.37 in the formula. When pinhole is over 1 AU, 0.51 has to be used and the
resolution advantage over conventional fluorescence microscope is minimized to
the ratio of Em/Ex: in case of FITC, it is 510/488 = 1.05 times.
pinhole < 0,25 AU, the resolution advantage is often compromised by the
vulnerability to noise due to its reduced detecting volume
and general weaker signal and deteriorated SNR. For detailed discussion, see section
9, SNR in confocal.
function applies not only to the focal plane horizontally, but also to the
plane vertically. A perpendicular cut of the Airy disc shows a even
wide spread range. That is why the Z-resolution of a lens is
worse than its lateral resolution.
When pinhole is close to zero (or < 0,25 AU in practice):
Formula 3 : is used for
In general, the Z-Resolution
is twice of the x-y-Resolution. 0.64-0.88 is used to replace 0.64 when
pinhole is at 0.25-1.0 AU.
The effect of PSF makes image from
any diffraction-limited system blurred and distorted, or
convoluted (twisted). Deconvolution algorithm has to be used to correct this
distortion after data collection if high demanding of a true
representative of original is required.
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