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.

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)
3 comments:
Sounds very cool, I'd love to hear more about what you've got planned. The interactions of bacteria and fungi are often overlooked.
are you SURE you don't need a post-doc or technician to start next spring? :)
soil mama, you don't want to revert back to a technician, do you? Please say no!
As for a post-doc, I don't know. If I get retention, and my research leader thinks it's a good idea, I may apply for the funding for one. I'll let you know!
bioenergyrus.blogspot.com is very informative. The article is very professionally written. I enjoy reading bioenergyrus.blogspot.com every day.
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