If my leaves are too small to cover the light guide, and I am trying to measure ECSt, should I be using a mask or not?
Hi Sean, I understand this is a common question so I will try to give a general answer.
What happens when the leaf does not fully cover the window (aperture)?
This is not a big worry with calculated fluorescence measurements, which should be pretty much independent of the whether the leaf completely fills the aperture. It will decrease the fluorescence amplitude, and this can lead to poor signal-to-noise ratios, but unless the signal is really small, the fluorescence parameters should be relatively unaffected.
However, its a big problem for absorbance signals (ECS, P700, SPAD) which measure the changes in absorbance of light by the leaf. Even a small gap will decrease the measured changes in absorbance. (If you want more details, see technical explanation, in my follow up post).
What can be done to avoid this problem? Make a mask out of a black (opaque) material, like paper or thin sheets of plastic. We laser cut them out of very thin plastic. We make a pair of identical masks and tape them together so that the edges form a hinge. We fold this over the small leaves so that only leaf material shows though the hole. Then we place the entire mask+leaf in the MultispeQ.
If anyone is interested, we can take some photos of the process and even make the laser cut template available.
With a completely covered window, the absorbance should approximately follow "Beer's law"*:
deltaC ~ deltaA ~ -log(I/I0)
where deltaC is the change in concentration of species in leaf ~ means proportional to, I is the light detected by the sensor after the sample and I0 is the light intensity before the sample.
With a gap in the system, part of the light simply bypasses the leaf altogether, so the apparent deltaA becomes:
deltaA(apparent) = -log(I/(I0 + bypassed light))
so deltaA(apparent) < deltaA.
With a big gap,
deltaA(apparent) <<< deltaA.
One more comment. While the amplitudes of absorbance changes are strongly affected by gaps, for the most part the kinetics of changes should be unaffected. So, we are really always interested in parameters that are measured by kinetics rather than amplitudes, for example gH+ or kP700, because they are likely to be free of these kinds of artifacts.
Here is a link to masking intstructions: https://photosynqprod.s3.amazonaws.com/files/photosynq.org/specialfeatures/measuring-small-leaves.pdf
Hi Dr. Kramer,
I am interested in learning more about this mask. I made one following the directions given here (https://photosynqprod.s3.amazonaws.com/files/photosynq.org/specialfeatures/measuring-small-leaves.pdf). The mask you are describing seems different. Is there a benefit to the mask you use over the one given in the pdf?
Also, is there a way to measure ECS signals (ECSt, vH+, and gH+) using a mask? The protocol designed to be used with the mask does not seem to collect these data. It would be mighty helpful to me if I could measure these parameters with small leaves.
If it makes any difference my research is in wheat. The leaf is long, but narrow. Is surface area an issue in getting a decent reading for these parameters? Would a rectangular mask as opposed to a circular hole make any difference?
Thank you. Very impressed by the capabilities of the MultispeQ so far!
I can answer a couple of your questions:
The mask from the pdf is installed on the MultispeQ, so it allows you to measure a many small leaves rapidly. The mask that Dr. Kramer mentioned goes on the leaf, so you would need to mask each leaf prior to taking the measurement.
If you have long narrow leaves, then a rectangular hole that maximizes the surface area of the leaf that gets measured would be more appropriate than the circular holes shown in the masking pdf.
There are some issues with the method for determining ECS curves in the "Leaf Photosynthesis MultispeQ V1.0" protocol, for that reason we have removed the ECS portion of the measurement from the masking version of the protocol. However, you can use the "Photosynthesis RIDES" protocol on masked leaves, which will measure ECS parameters. However, to use this protocol you will need to update your firmware to 1.22 or greater (https://www.photosynq.org/help/instruments_How_to_Install_Firmware_Updates).
I want to confirm your comment about varying the mask light guide to suit the measurement leaves. The pdf guide for creating the mask makes clear that the light guide can range from 2-4mm in diameter. Will creating a custom light guide for my mask somehow corrupt my data?
You can make a light guide of any custom size as long as you recalibrate your MultispeQ after you have masked it.
Excellent. Thank you.
Thanks for the response. And great to hear that the RIDES protocol supports masked ECS measurements! The only difficulty I'm having is that my device seems to think that it has the most updated firmware (1.17). In the android app when I click "Check for Firmware Updates," I get a message saying "No new updates available."
Any thoughts on how to proceed?