If you are interested in stomata size, opening or frequency you could take footprints using, for example, nail polish. Later these footprints can be analysed by microscope.

In my lab, we also apply a thin layer of nail polish on the leaf surface and wait for a few minutes, then pill the thin layer from the leaf surface, put on a glass slide under a cover slip before analyzing by microscope.

It is also possible to use a see through shine/glaze spray, which can be found in art shops, to receive an epidermis and stomata imprint. Just spray your leaf until a continuous wet layer is formed, let the glazing material dry, then peel the thin layer off and fix it on a glass slide with a see through adhesive tape.

Dear Anamika

In the following papers you can find four ways that we have used for studying stomatal opening/closing patterns, in three of them the leaf samples are fixed:

1- gas exchange measurements through measuring stomatal conductance

2-studying stomatal aperture under microscope

3-chlorophyll fluorescence under non-photorespiratory conditions (indicative of stomatal patchiness and closure)

4-studying leaf RWC in response to changes in transpiration rate

Aliniaeifard S, van Meeteren U. 2016. Stomatal characteristics and desiccation response of leaves of cut chrysanthemum (Chrysanthemum morifolium) flowers grown at high air humidity. Scientia Horticulturae 205, 84-89.

Aliniaeifard S, Malcolm Matamoros P, van Meeteren U. 2014. Stomatal malfunctioning under low Vapor Pressure Deficit (VPD) conditions: Induced by alterations in stomatal morphology and leaf anatomy or in the ABA signaling? Physiologia Plantarum 152, 688-699.

I also agree with the previous comments. We take stomatal impressions using thin layer of colourless nail polish. The method is briefly explained in the following paper.

Wijeratne, T. L., Mohotti, A. J. and Nissanka, S. P. (2008). Impact of long term shade on physiological, anatomical and biochemical changes in tea (Camellia sinensis (L.) O. Kuntz). Proceedings of the 20th Annual Congress of the Postgraduate Institute of Agriculture, Gannoruwa, Sri Lanka. 20 -21-November 2008. pp. 38.

You can also use microscope with image analyzer software to easily get different measurements on stomata using these impressions later.

Dear all,

thanks a lot for your responses. After reading all your suggestions, I have decided to take leaf impressions, using nail polish. Israel Oren Your technique is quite interesting, I will definitely try and see if it works for me :)

By the way, while searching I found this protocol

https://bio-protocol.org/e2347#biaoti14880

Its worth trying as well.

Thank you all once again, and good luck with your respective projects/experiments.

Hi Anamika,

I agree with the others that nail varnish impressions can offer a window into the state of the stomatal pore aperture and I have been able to get meaningful quantitative data from these in the past. It depends on the plant tissue you are studying, but generally it is useful to take negative impressions with dental impression material (e.g. 3M ESPE Imprint3 types) and then you have a permanent mold to use the nail varnish on to prepare your microscope slide. Some leaves will work fine with the nail polish applied directly, but others won't. This may be the same with Israel Oren's method, but I have not tried this way yet. The impression method is also only possible with non-cryptic or non-occluded stomata and may be more sensitive for some species than others. If you can test with plant leaves under darkness versus different light intensities, this may be a good way to see if you can detect clear differences in aperture across your plants.

Aperture measurements on dead and fixed plant material, such as Lloyd's method, have been VERY misleading in the past (Francis Darwin explains this in a paper from 1911: https://www.jstor.org/stable/pdf/80430.pdf), but are occasionally used, albeit with certain caveats!

Cheers,

Caspar

We used nail polish in wheat very well. but in some plant like chickpea or other with hairy leaf, we didn't have clear pictures. It's best to test a sample before using it.

We also use nail polish to get the footprint of stomatal aperature and density and observe under microscope. You can not fix the leaf as such but you can preserve the thin layer of nail polish after removing from leaf and take the observation under microscope later.

Perhaps you wanna try this method for monocots (Article A previously unknown zinc finger protein, DST, regulates dro...

Hope it helps.