Has anyone used Walz Imaging-PAM MAXI on microphytobenthos (MPB)?
I am using the Special SP-Routine on a petri dish of raw sediment sample, and I see patches of fluorescence, the most intense ones coming from large(er) gravels. As I increase the ML intensity, I see more fluorescence in previously black spaces, and I am not sure if those are from gravels too, or from MPB.
1) How do you define your AOI? Do you draw a polygon over a random fluorescent patch, taking care not to include any black part?
2) How do you distinguish fluorescence from gravels vs those from MPB?
3) Do you pre-treat your sample before viewing it with the Imaging-PAM?
4) Is there any setting that is crucial for this work that I need to pay attention to?
Thank you so much in advance!
Dear Yan Loo, I asked my colleague Oliver Meyerhoff if he could answer your question. A problem is that organisms growing in the light are exposed to quite different light intensities, from the Imaging instrument as well.
1. How do you define your AOI?
Look for a spot that is showing a robust signal for the measuring purpose needed
2. How do you distinguish fluorescence
The background fluorescence from the gravel is supposed to be steady – no variable fluorescence. In case of an imaging system a background fluorescence image is no option. The only possibility is that background value is taken as a constant value and as such is subtracted from the F-value and the Fm-value. This is only a rough approach – fluorescence parameters then have to be calculated manually.
On the other hand the unmodulated background fluorescence is a steady value related to a homogeneous gravel material and generally lowers variably fluorescence To determine the background unmodulated part alcohol washed gravel can be taken.
3. Do you pre-treat your sample before viewing it with the Imaging-PAM?
No, not necessarily. Samples can be measured in their native state. Depending on the parameters you would like to derive from your experiments it is potentially recommended to implement a pre-darkening phase to the experiment.
4. Is there any setting that is crucial
For such measurements, same settings are important as for “normal” PSII measurements on e.g. higher plants.
Starting parameters are listed in the manual. So please keep the measuring light as low as possible if Fo values are needed and adjust the Ft value mainly by using the Gain settings so that the illumination during dark phases can be kept on a minimum.
I hope this helps you,
Gert Schansker
- Badges
- Science topic
- Similar topics
- Physical Sciences
- Physical Phenomena
- Luminescence
- Fluorescence