Confocals produce an image in quite a different way from wide-field fluorescence
scopes. Rather than exciting the entire field at once, the light
is focused into a very small spot and this is scanned across the
sample and an image is built up. This may sound like quite a strange
way of making an image, what is the point?
The key part of the confocal
is the pinhole. Notice that only the light from the focal
passes through the pinhole to the detector. The light from
just below the focal plane (follow the dotted red lines) is
mainly blocked. Same for the light from just above (follow
the blue dotted lines).
By blocking the light from outside the focal plane confocals
have good z-axis resolution and are great for imaging thick
samples because the haze from out of focus objects is mostly
Confocal microscopes are much more complex than widefield systems. They
consist of a normal microscope with the confocal bit stuck on the side.
This shows the basics of a system: Lasers
are used for excitation. The laser beam comes into the system, and
is reflected by the dichroic. Next, two scanning mirrors
move the beam in a raster (like writing on a page) across the sample.
The fluorescence light
than passes back through the objective and is descanned (ie reflects
off both scanning mirrors). The light then passes through the dichroic
and pinhole to the PMT (photomultiplier tube) detector. There are really
lens and mirrors involved and our systems all have 3 PMT for 3 different
The improved resolution in the z-axis makes 3D imaging with confocals very
successful. The basis of 3D imaging is to take several images at
different positions along the z-axis. . .
There are lots of things to adjust to get a good
confocal image . . .
These might make more sense when you have used a confocal a little bit,
but hopefully this explains some of the principles -
The power of each laser line can be controlled using AOTFs
(acousto-optical tunable filters). These let through between
0 and 100% of the light from a particular laser line. Rather
than changing how much light the lasers produce (many are either
on or off),
faster and gives finer control to adjust how much is let through
the AOTF to the sample.
Because a confocal microscope is imaging only a thin slice through your sample,
it is important to be imaging the correct thin slice.
the wrong word to use here as the confocal is always technically in focus.
It is a fine adjustment in the z-axis that is
You can make the scanning mirrors sweep a smaller area giving
you a zoomed-in image with the same number of pixels.
In many situations it is true zoom,
but only to a point. It is better to use the 100x objective
zoom in 10x with the 10x objective.
This effects the brightness (ie amount of light you collect),
slice thickness and resolution. A bigger pinhole increases
What is best? Try and use Airy Unit 1 if you can, if your sample
with a slightly larger pinhole.
|Gain and offset
These effect the sensitivity and background level of the
detectors (the PMTs).
Increasing the gain makes the PMT more sensitive and so your
sample look brighter. Reducing the offset reduces the background
Confocals usually have a special display mode (lookup
table) that helps you set these. Adjust the gain so just a
few pixels are the max colour, reduce the offset so the background
is about 50% the 0 colour. This ensures you have the full range
of brightness within your image.
Confocal images are often somewhat noisy and averaging helps
to reduce the noise. Line averaging of 4 means each line
is scanned 4 times and the 4 scans of each pixel are averaged.
Try and few values and see what the minimum
gives you a good image. Line averaging somewhere between 2
and 8 are the most common choices.
|Number of pixels
The same area can be scanned with a different number of lines
512 by 512 is a common starting point. This means the image is made up of 512 lines
and each line consists of 512 pixels.
Isn't more better? Sometimes.
At some point the number of pixels saturates the resolution
the system, and of course it is slower and makes the files