Dear Researchers,
I am seeking a method to assess the sterility (not viability) of pollen grains using fluorescence flow cytometry. I have gone through the relevant literature. The best match is impedence flow cytometry (Amphasys). There are certain bright-field microscopic methods such as I2/KI (estimation of starch content), Alexander's staining, etc. But I am interested in flow cytometry based method. I have gone through an old research gate article on the same subject. In that article, most are speaking about microscopic methods
Could someone please assist me?
Thank you very much in advance!
Himanshu.
You can collect the starch specific green fluorescence using the FL1 (FITC) detector of a flow cytometer after I2/KI staining and the autofluorescence using the other far red detectors.
Thank you Ashutosh!
I was not knowing that the binding of iodine with starch generates a fluorescent product.
Yes the unstained samples do not show the green fluorescence. This was observed in the cells of plant leaves so should hold true for the pollens too. Flow cytometry is more sensitive in measuring fluorescence than microscopy and with proper controls it is quantitative too. Good luck!
Thanks Ashutosh! I also came across this paper by Ovecka et al., 2012. They have visualized stained starch granules using fluorescence microscopy. Is the red autofluorescence because of chlorophyll? Will it be to a detectable level in the pollen grains?
Yes the red fluorescence is due to chlorophyll. Since I have not worked with pollen it is for you to find out.
- Badges
- Science method
- Similar topics
- Biological Science
- Histology
- Staining