Confocals

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?

duke lmcf confocal pinhole basis

The key part of the confocal is the pinhole. Notice that only the light from the focal plane 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 eliminated.

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 lots more lens and mirrors involved and our systems all have 3 PMT for 3 different colours.

duke lmcf confocal miroscope schematic basis

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. . .

A stack of images . . .
duke lmcf confocal z -stack gallery display
. . . made into a 3D projection

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 -

Laser power
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), it is faster and gives finer control to adjust how much is let through the AOTF to the sample.
Focus
Because a confocal microscope is imaging only a thin slice through your sample, it is important to be imaging the correct thin slice.

(Focus is actually 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 required).
duke lmcf fine focus of confocal image
Zoom (optional)
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 than to zoom in 10x with the 10x objective.
duke lmcf 1x and 2x confocal zoom
Pinhole
This effects the brightness (ie amount of light you collect), slice thickness and resolution. A bigger pinhole increases brightness but reduces resolution.

What is best? Try and use Airy Unit 1 if you can, if your sample isn't bright you will probably be better off with a slightly larger pinhole.
duke lmcf pinhole size brightness vs resolution 1 airy unit
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 level.

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.
duke lmcf image display lookup tables for adjusting gain and offset
Averaging
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 that gives you a good image. Line averaging somewhere between 2 and 8 are the most common choices.
duke lmcf confocal line average noise improvement
Number of pixels
The same area can be scanned with a different number of lines and pixels.

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 of the system, and of course it is slower and makes the files larger.
duke lmcf number of pixels in a confocal image 512 by 512 or 1024 by 1024