Chlorophyll in Pansy leaf

pwnell · Post by **pwnell** » Fri Dec 28, 2012 3:02 pm

Since I am still learning a lot about fluorescence microscopy, I'd appreciate some feedback on this. I have taken a small piece of a Pancy's leaf and submerged it in a drop of water. A GFP filter cube was used to excite the chlorophyll in the leaf. This GFP cube has a bandpass emitter of between 500nm and 555nm.

When I moved the leaf to a new position, I could see very faint green. After about 5 - 15 seconds the green became more intense, as if something got activated in the leaf. When I then turned up the intensity of the light a bit, I saw bubbles coming out from underneath the leaf and it appeared like the leaf got scorched? Visual inspection showed several tiny brown spots on the leaf suggesting the intensity of the blue light was too much for the living tissue and scarred it. Is this what happened? If so, how would I go about taking photos of something like chlorophyll's emissions without scorching the cells? ND filters and long exposure times?

Here is a photo I got at 40x magnification:

20121228-DSLR_IMG_0117.jpg by pwnell, on Flickr
Pansy Leaf, 40x, GFP FLUO

René · Post by **René** » Fri Dec 28, 2012 3:55 pm

Hi Waldo, chlorophyll fluorescence is a deep red. You will need long pass emitter filters to see that, or specific bandpass > 600nm.

What you see here is autofluorescence, and yes, it increases a great deal with exposure to short wave irradiation. ND filters and long exposures are indeed a good way of dealing with it.

Good luck, René

Carduus · Post by **Carduus** » Fri Dec 28, 2012 4:09 pm

You might do better working out your technique using something thin like the leaf of a moss (1 cell thick) or a waterweed (e.g. Elodea), so that the individual cells and chloroplasts are more visible and you can see exactly what structures are fluorescing.

pwnell · Post by **pwnell** » Fri Dec 28, 2012 4:33 pm

Brilliant responses, thanks. I was struggling with the thick leaf and wondering what I should do to get a thinner slice - a leaf from moss would do well.

Will try that.

curt0909 · Post by **curt0909** » Fri Dec 28, 2012 10:34 pm

Last year I used an FITC cube to capture chlorophyll fluorescence. I never had the scorching problem probably due to my weak light source. Elodea would likely require a short exposure to capture effectively as the Chloroplasts are constantly in cyclosis.

http://www.photomacrography.net/forum/v ... scence+led