Confocal Microscopy

My lab has a confocal microscope. Actually, we have a pretty damn good microscope. It's a Nikon Eclipse C1 Plus. We're in the process of ramping up experiments for the confocal. Our hope is that we'll be able to see microbial (both bacterial and fungal) interactions -- primarily physical associations for now -- in soil aggregates which may explain how soil aggregates form, and hopefully shed some light on how these organisms obtain nutrients (from both each other and from the nutrients in the soil).

Using a confocal microscope however is not like using your light microscope. There are a number of additional factors which must be taken into account when setting up your experiments.

A couple of issues to consider:
1. Does my sample autofluoresce? Plants are notoriously bad autofluorescers (I don't think that's even a word), especially in the 488 excitation range. You want to keep this in mind when using fluors such as Alexa-488, FITC, and GFP. Soil, and even animal tissues, also suffer from autofluorescence. Sometimes this can be helpful, but often times it can be problematic.

2. What are you going to do with your samples? Are they dead or alive? If they are alive, you may want to do your experiments on a spinning disk confocal microscope (if one is available) which are less damaging to live cells. Also, are you going to try to determine colocalization? If so, you really need to know what objective lens you are going to use for your imaging.

Why? Well, when fluors are excited, they emit light. When you are using more than one color fluor, these fluors are emitting light at different wavelengths. When they pass through the objective lens, if it is not corrected for those different wavelengths, some of the light will hit the confocal microscopes pinhole (iris) out of focus. This is known as chromatic aberration. So, for example, if you look at the image to the right, you see that when the blue wavelength comes into focus (when it comes to a point), if that is where your pinhole is, then your red and green light are out of focus. What this is means then is that when you're doing your Z-stack (3rd dimension), this will result in a "shift" of your image between your in focus light and your out of focus wavelengths. There are a couple of ways to correct for this shift if it occurs. You can compress your Z-stack (but you lose your 3rd dimension, and are stuck with only XY ... and the Z is part of the whole point of confocal microscopy in the first place), you can scan your fluors consecutively and then computationally merge the images (hard to do with live, moving cells, and is problematic for other purposes), or you can make sure you have the appropriate set of lenses.

Types of lenses:
1. Plan - these lenses are corrected to provide a flat field of view. Your objective lens should be plan lenses.

2. Achromats* - corrects chromatic aberrations for blue and red wavelengths. This means you can colocalize with blue and red fluors.

3. Fluorites* - Also corrects chromatic aberrations for blue and red wavelengths.

4. Apochromats* - corrects chromatic aberrations for blue, green, and red wavelengths. Newer apochromats can correct for up to 6 wavelengths, but they're extremely expensive.

So, if you're doing a colocalization experiment with a blue, green, and red fluor, you're obviously going to want to your scope to have an objective lens which is a plan apochromat. Not all of them need to be plan apochromats (seems overkill on the 20X if you're not collecting your data at 20X), but the one you collect your data with (be it 40X, 63X, or 100X depending upon your sample) should be.

*These lenses correct for chromatic aberration, and to some degree spherical aberration as well.

Resources
Microscopy Society of America
Nikon - Microscopy U
Olympus Microscopy Resource Center

Reference
Ying Li, Warren A. Dick, and Olli H. Tuovinen. 2003. Evaluation of fluorochromes for imaging bacteria in soil. Soil Biology and Biochemistry. 35: 737-744 (PDF, 8 pages)