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How does a confocal microscope work?
This web page explains how a confocal microscope works; I've tried to make this
explanation not too technical, although for certain parts I've included some details for
people who know more optics. If you like this web page, it would be great if you'd
consider citing our article:

"Confocal microscopy of colloids"
V Prasad, D Semwogerere, ER Weeks, J. Phys.: Cond. Mat. 19, 113102 (2007)
o View abstract / PDF version / Journal web page
I realize many people reading this page aren't interested in colloids per se, but part of this
article covers the same material as this web page.
To start with, you need to understand fluorescence:
What is fluorescence?
If you shine light on some
molecules, you may see light
of a different color emitted
from that molecules. This is
known as fluorescence. The
molecules absorbs high energy
light (blue, for example). This
increases the energy of the
molecules, represented as the
top black line in the diagram
(an "excited" molecules).
Some of the energy from the
blue photon is lost internally
(represented by the red
squiggly arrow in the picture).
The molecules then emits a
photon with less energy, green
in this example. Fluorescein is
a common dye that acts in
exactly this way, emitting
green light when hit with blue
excitation light. The color of
light emitted is material
dependent, and likewise the
excitation light wavelength
http://www.physics.emory.edu/~weeks/confocal/
depends on the material.
(There are other forms of
inelastic scattering;
fluorescence is particularly
strong.)
The advantage of fluorescence for microscopy is that you can often attach fluorescent dye
molecules to specific parts of your sample, so that only those parts are the ones seen in
the microscope. You can also use more than one type of dye. By changing the excitation
light, you can cause one type of dye to fluoresce, and then another, to distinguish two
different parts of your sample.
How does a fluorescence microscope work?
In the picture above, we are supposing the excitation light needs to be violet, and the
emitted light is red. The microscope uses a special dichroic mirror (or more properly, a
"dichromatic mirror", although this term only seems to be used by purists). This mirror
reflects light shorter than a certain wavelength, and passes light longer than that
wavelength. Thus your eye only sees the emitted red light from the fluorescent dye, rather
than seeing scattered purple light. The purple and red bars next to the dichroic mirror
represent additional filters to help prevent the different wavelengths of light from going
the wrong directions.
This particular style of fluorescence microscopy is known as epi-fluorescence, and uses
the microscope objective to illuminate the sample (rather than illuminating the sample
from the other side, which would be trans-fluorescence).
http://www.physics.emory.edu/~weeks/confocal/
What's this got to do with confocal microscopy?
Well, we're not there just yet, I have to explain one more idea.
Imagine we have some lenses inside the microscope, that focus light from the focal point
of one lens to another point. This is represented by the blue rays of light in the above
picture. The red rays of light represent light from another point in the sample, which is
not at the focal point of the lens, but which nonetheless get imaged by the lenses of the
microscope. (Note that the red and blue rays in the picture are meant to distinguish the
two sets of rays, but they aren't meant to be different wavelengths of light.) The image of
the red point is not at the same location as the image of the blue point. (You may
remember this from introductory optics, perhaps you have seen a formula such as 1/s +
1/s' = 1/f for locating the image formed by a lens. Points don't need to be at the focal
point of the lens in order for the lens to form an image.)
So, we want to just look at the blue point, that is, the point directly at the focus of the
lens. If we put a screen with a pinhole at the other side of the lens system, at the image of
the blue point, then all of the light from the original blue point will pass through this
pinhole. However, most of the light from the red point is still out of focus at this screen,
and gets blocked by the pinhole.
This solves one of the problems of regular fluorescence microscopy. Normally, the
sample is completely illuminated by the excitation light, so all of the sample is
fluorescing at the same time. Of course, the highest intensity of the excitation light is at
the focal point of the lens, but nonetheless, the other parts of the sample do get some of
this light and they do fluoresce. This contributes to a background haze in the resulting
image. Adding a pinhole/screen combination solves this problem. Because the focal point
of the objective lens of the microscope forms an image where the pinhole is, these two
points are known as "conjugate points" (or alternatively, the sample plane and the
pinhole/screen are conjugate planes). The pinhole is conjugate to the focal point of the
lens, thus it is a confocal pinhole.
How does a confocal microscope work?
http://www.physics.emory.edu/~weeks/confocal/
We put all these ingredients together:
A laser is used to provide the excitation light (in order to get very high intensities). The
laser light (blue) reflects off a dichroic mirror. From there, the laser hits two mirrors
which are mounted on motors; these mirrors scan the laser across the sample. Dye in the
sample fluoresces, and the emitted light (green) gets descanned by the same mirrors that
are used to scan the excitation light (blue) from the laser. The emitted light passes
through the dichroic and is focused onto the pinhole. The light that passes through the
pinhole is measured by a detector, ie., a photomultiplier tube.
So, there never is a complete image of the sample -- at any given instant, only one point
of the sample is observed. The detector is attached to a computer which builds up the
image, one pixel at a time. In practice, this can be done perhaps 3 times a second, for a
512x512 pixel image. The limitation is in the scanning mirrors. Our confocal microscope
(from Noran) uses a special Acoustic Optical Deflector in place of one of the mirrors, in
order to speed up the scanning. This uses a high-frequency sound wave in a special
crystal to create a diffraction grating, which deflects the laser light (actually, the first
diffraction peak is used, with the zeroth-order peak being thrown away). By varying the
frequency of the sound wave, the AOD changes the angle of the diffracted light, helping
scan the sample quickly, allowing us to take 512x480 pixel images 30 times per second.
If you want to look at a smaller field of view, our confocal microscope can go even faster
(up to 480 frames per second, although I personally find that 240 frames per second is a
good practical limit).
What is the advantage of using a confocal microscope?
By having a confocal pinhole, the microscope is really efficient at rejecting out of focus
fluorescent light. The practical effect of this is that your image comes from a thin section
http://www.physics.emory.edu/~weeks/confocal/
of your sample (you have a small depth of field). By scanning many thin sections through
your sample, you can build up a very clean three-dimensional image of the sample. Some
examples of this are given at this page, looking at emulsions.
Also, a similar effect happens with points of light in the focal plane, but not at the focal
point -- emitted light from these areas is blocked by the pinhole screen. So a confocal
microscope has slightly better resolution horizontally, as well as vertically. In practice,
the best horizontal resolution of a confocal microscope is about 0.2 microns, and the best
vertical resolution is about 0.5 microns. I wrote a brief discussion here of the difference
between resolution and magnification.
How big is a confocal microscope?
The left picture shows our inverted
microscope, with a big white box to the
left of it: the white box is the confocal
microscope attachment. As far as the
inverted microscope is concerned, the
confocal attachment is just some sort of
fancy camera. As far as the confocal is
concerned, the inverted microscope is
just some sort of fancy lens.
The right picture shows the confocal
microscope attachment mounted on top
of the upright microscope.
The confocal microscope attachment
shown in these pictures contains the
optics for scanning the laser beam, and
the pinhole. The confocal also includes a
very large box containing electronics,
which is not shown in the photographs;
there is also a laser, and an SGI
computer.
Anything else?
That's about it. The confocal microscope was invented by Marvin Minsky, who has
written a nice summary about this on the web. His original idea was to move the sample
in order to scan it, rather than to use scanning mirrors. With the invention of the laser,
confocal microscopes became practical.
http://www.physics.emory.edu/~weeks/confocal/
By the way, you
may recall that the
image of a point
source of light isn't
actually a point;
due to diffraction,
it's actually an
Airy disk. The
graph to the left
shows a plot of the
intensity of light as
a function of
radius; the image is
circularly
symmetric, as
shown at the right.
In an ideal world
the image of a
point would just be
a single intense
point right at
radius=0. The size
of the confocal
pinhole needs to be
matched to the size
of the Airy disk.
Any smaller, and
you are throwing
out useful light.
Any larger, and
you see more out
of focus light. For
the graph at the
left, the pinhole
would have a
radius of about
"4". The image
below shows an
overexposed
picture of an Airy
disk, where you
can see the
secondary ring.
http://www.physics.emory.edu/~weeks/confocal/
Further reading
Two articles we wrote that expand on this webpage:


"Confocal Microscopy", D Semwogerere & ER Weeks, published in the
Encyclopedia of Biomaterials and Biomedical Engineering, Taylor & Francis
(2005).
"Confocal microscopy of colloids", V Prasad, D Semwogerere, ER Weeks, J.
Phys.: Cond. Mat. 19, 113102 (2007)
A related webpage I wrote:

The difference between resolution and magnification.
Related links:


Wikipedia entry
Detailed explanation of confocal microscopy at "Molecular Expressions"
I like these two books:


"Video Microscopy," Shinya Inoue and Kenneth R. Spring, 2nd ed., Plenum
Press, 1997.
"Handbook of biological confocal microscopy," edited by James B. Pawley, 2nd
ed., Plenum Press, 1995.
Links


PSIgate - Physical Sciences Information Gateway has listed this page here.
This explanation was written by Eric Weeks
http://www.physics.emory.edu/~weeks/confocal/

Send me email: weeks (at) physics.emory.edu. Let me know if you have further
questions, or if there are parts of this explanation that are confusing.