ECSt on Small Leaves
Measurements, Protocols & Macros
Sean
Feb 2018
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?
David M. Kramer
Feb 2018
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.
David M. Kramer
Feb 2018
Technical explanation:
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.
- absorbance changes in leaves do not truly follow Beer's law because their optical properties are not like a completely dispersed solution of a chemical, for which Beer's law applies. There are a number of interesting and complex effects of the optical structure of leaves on our measurements...but let's save that for another day.
Dave
David M. Kramer
Feb 2018
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.
Dave
Dan TerAvest
Mar 2018
David M. Kramer: 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 thi...
Here is a link to masking intstructions: https://photosynqprod.s3.amazonaws.com/files/photosynq.org/specialfeatures/measuring-small-leaves.pdf
Liam Dixon
Mar 2018
David M. Kramer: 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 thi...
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!
Liam
Dan TerAvest
Mar 2018
Liam,
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).
Liam Dixon
Mar 2018
Hi Dan,
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?
Thanks again, Liam
Liam Dixon
Mar 2018
Dan TerAvest: Liam, 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 th...
Hi Dan,
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?
Thanks, Liam
Dan TerAvest
Mar 2018
Liam,
You can make a light guide of any custom size as long as you recalibrate your MultispeQ after you have masked it.
Jie Yun
Apr 2019
David M. Kramer: 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 thi...
May I ask can we use the mask and program (https://photosynqprod.s3.amazonaws.com/files/photosynq.org/specialfeatures/measuring-small-leaves.pdf) to measure small leaves using MultispeQ v2.0? More specifically, (1)does the program work for both of them? (2)I guess the fluorescence measurements would be pretty much near to the true values. For absorbance signals, it probably need to be recalibrated, but there's no calibration program or SPAD cards come along with MultispeQ v2.0. What should we do?
David M. Kramer
Apr 2019
Hi Jie Yun!
As long as the mask does not allow light to go around the leaf, the absorbance measurements (e.g. ECSt) will be fine.
However, the older SPAD measurements will need to be recalibrated.
There are two ways to measure SPAD, the older (introduced some time ago by Greg Austic) and the newer (just developed). The older will need a complete recalibration using the cards. The new will be much easier, requiring only one (or at most 2) cards.
Dave
Jie Yun
Jun 2019
Great! Thank you for your reply! May I ask where I can find the protocol for SPAD calibration, as well as the calibration cards?