Sep 30, 2021
PSI Open Fluor CAM script for measuring qE component of NPQ in Chlorella vulgaris
- 1University of Technology Sydney
- Climate Change Cluster
Protocol Citation: Andrei Herdean 2021. PSI Open Fluor CAM script for measuring qE component of NPQ in Chlorella vulgaris. protocols.io https://dx.doi.org/10.17504/protocols.io.byn9pvh6
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it’s working
Created: September 30, 2021
Last Modified: September 30, 2021
Protocol Integer ID: 53697
Keywords: NPQ, qE, FluorCAM,
Abstract
This is a simple protocol that consists of
1) 10 minutes preillumination with far red light
2) 5 minutes of illumination with actinic light
3) 5 minutes of dark adaptation with far red light
qE is calculated as the differe between NPQ_Lss and NPQ_D5
qE=NPQ_Lss-NPQ_D5
qI=NPQ_D5
Protocol to be used with FluorCAM 7.0 on a PSI Open FC 800-O/1010-S.
Act 2 - are the white light LED arrays
ADD2 - is the far red LED array
Camera is placed at ~20 cm above the measured sample. Light intensity uniformity across the 96 well plate was measured according to manufacturer instructions.
!Important - protocol only works under weak far red light. Intense far red will interfere with the fluorescence measurement.
Script for FluorCAM software
Script for FluorCAM software
; Quenching protocol with Actinic2
;with FilterWheel
;version November 11, 2020
;high-resolution CCD TOMI-2
;optimized number of measured frames
;Protocol duration 183s
;
ADD1=0
ADD2=10
Act1=0
TS=50ms
include default.inc ;Includes standard options, do not remove it !
include light.inc ;Includes standard options, do not remove it !
include FW.inc ;Includes standard options, do not remove it !
;
Shutter=2
Sensitivity=29.3
Act2=18
Super=69.6
LightA=29.069
LightB=-34.732
;_________________________________________________________________________________________________________
<0s>=>SET_FILTER(CHL)
Preillumination=600s
<0s>=>add2(Preillumination)
start = Preillumination;
;-------------------------------------------------------------------------------------------------------------
;*** Fo Measurement ******************************************************************************************
;-------------------------------------------------------------------------------------------------------------
F0duration=5s
F0period=1s
start + <0,F0period…F0duration>=>mfmsub
;
;Fo definition
start + <0s>=>checkPoint,“startFo”
start + <F0duration-F0period>=>checkPoint,“endFo”
;
;-------------------------------------------------------------------------------------------------------------
;*** Saturating Pulse & Fm Measurement ***********************************************************************
;-------------------------------------------------------------------------------------------------------------
PulseDuration=800ms
a1 = start + F0duration+2mfmsub_length
;
<a1>=>SatPulse(PulseDuration)
<a1>=>act2(PulseDuration)
;
<a1>=>mpulse2
;
;Fm definition
<a1+PulseDuration/2>=>checkPoint,“startFm”
<a1+PulseDuration-mfmsub_length>=>checkPoint,“endFm”
;
;Visual frame definition; Imagee shown in pre-processing window
<a1+PulseDuration/2+TS>=>checkPoint,“timeVisual”
;
;-------------------------------------------------------------------------------------------------------------
;****** Dark Relaxation Measurement *************************************************************************
;-------------------------------------------------------------------------------------------------------------
DarkRelaxation1=17s
b1= a1+PulseDuration+2mfmsub_length
b2=2s
<b1, b1+2mfmsub_length… b1+b2>=>mfmsub
<b1+2b2, b1+4b2… b1+DarkRelaxation1>=>mfmsub
;
;-------------------------------------------------------------------------------------------------------------
;******* Kautsky Effect Measurement **************************************************************************
;-------------------------------------------------------------------------------------------------------------
;
;******* Actinic light Exposure **************************************
ALPeriod=300s
c1=a1+PulseDuration+DarkRelaxation1+mfmsub_length
<c1>=>act2(ALPeriod)
;
;Fast Kautsky kinetics*********************************
c2=2s
;
<c1+TS, c1+TS+2TS… c1+c2>=>mfmsub;
;
;Slow Kautsky kinetics********************************
;
c3=4s
;
<c1+c2+c3/10, c1+c2+c3/5, c1+c2+c3/2>=>mfmsub
;
<c1+c2+c3, c1+c2+2c3… c1+ALPeriod>=>mfmsub
;
;Fp definition
<c1>=>checkPoint,“startFp”
<c1+c2+c3>=>checkPoint,“endFp”
;
;-------------------------------------------------------------------------------------------------------------
;****** Saturating Pulses - Fm’ Quenching Analysis **********************************************************
;-------------------------------------------------------------------------------------------------------------
;
;******* Saturating Pulses - Fm_L1 ***********************************
f1=c1+<58s>
f11=f1#<mfmsub_length, 2mfmsub_length… PulseDuration-mfmsub_length>
f11=>mfmsub
f1+mfmsub_length=>checkPoint,“startFt_L1”
f1+PulseDuration-mfmsub_length=>checkPoint,“endFt_L1”
;
f2=f1+PulseDuration
f2=>SatPulse(PulseDuration)
f2=>mpulse2
f2+PulseDuration/2=>checkPoint,“startFm_L1”
f2+PulseDuration-mfmsub_length=>checkPoint,“endFm_L1”
;
;****** Saturating Pulses - Fm_L2 **********************************
f3=c1+<118s>
f31=f3#<mfmsub_length, 2mfmsub_length… PulseDuration-mfmsub_length>
f31=>mfmsub
f3+mfmsub_length=>checkPoint,“startFt_L2”
f3+PulseDuration-mfmsub_length=>checkPoint,“endFt_L2”
;
f4=f3+PulseDuration
f4=>SatPulse(PulseDuration)
f4=>mpulse2
f4+PulseDuration/2=>checkPoint,“startFm_L2”
f4+PulseDuration-mfmsub_length=>checkPoint,“endFm_L2”
;
;****** Saturating Pulses - Fm_L3 ***********************************
f5=c1+<178s>
f51=f5#<mfmsub_length, 2mfmsub_length… PulseDuration-mfmsub_length>
f51=>mfmsub
f5+mfmsub_length=>checkPoint,“startFt_L3”
f5+PulseDuration-mfmsub_length=>checkPoint,“endFt_L3”
;
f6=f5+PulseDuration
f6=>SatPulse(PulseDuration)
f6=>mpulse2
f6+PulseDuration/2=>checkPoint,“startFm_L3”
f6+PulseDuration-mfmsub_length=>checkPoint,“endFm_L3”
;****** Saturating Pulses - Fm_L4 ***********************************
f7=c1+<238s>
f71=f7#<mfmsub_length, 2mfmsub_length… PulseDuration-mfmsub_length>
f71=>mfmsub
f7+mfmsub_length=>checkPoint,“startFt_L4”
f7+PulseDuration-mfmsub_length=>checkPoint,“endFt_L4”
;
f8=f7+PulseDuration
f8=>SatPulse(PulseDuration)
f8=>mpulse2
f8+PulseDuration/2=>checkPoint,“startFm_L4”
f8+PulseDuration-mfmsub_length=>checkPoint,“endFm_L4”
;
;****** Saturating Pulses - Fm_Lss **********************************
f9=c1+<298s>
f91=f9#<mfmsub_length, 2mfmsub_length… PulseDuration-mfmsub_length>
f91=>mfmsub
f9+mfmsub_length=>checkPoint,“startFt_Lss”
f9+PulseDuration-mfmsub_length=>checkPoint,“endFt_Lss”
;
f10=f9+PulseDuration
f10=>SatPulse(PulseDuration)
f10=>mpulse2
f10+PulseDuration/2=>checkPoint,“startFm_Lss”
f10+PulseDuration-mfmsub_length=>checkPoint,“endFm_Lss”
;-------------------------------------------------------------------------------------------------------------
;** Dark relaxation after actinic light period **************************************************************
;-------------------------------------------------------------------------------------------------------------
DarkRelaxation2=300s
h1=c1+ALPeriod
<h1>=>add2(DarkRelaxation2)
h2=mfmsub_length
h3=10s
;
;******** Relaxation measurement**************************************
;
<h1+h2, h1+2h2, h1+4h2, h1+8h2, h1+16h2>=>mfmsub;
h1+h3/2+<0s, h3… DarkRelaxation2>=>mfmsub
;
;******* Saturating Pulses - Fm_D1 ***********************************
g1=h1+<58s>
g11=g1#<mfmsub_length, 2mfmsub_length… PulseDuration-mfmsub_length>
g11=>mfmsub
g1+mfmsub_length=>checkPoint,“startFt_D1”
g1+PulseDuration - mfmsub_length=>checkPoint,“endFt_D1”
;
g2=g1+PulseDuration
g2=>SatPulse(PulseDuration)
g2=>act2(PulseDuration)
g2=>mpulse2
g2+PulseDuration/2=>checkPoint,“startFm_D1”
g2+PulseDuration-mfmsub_length=>checkPoint,“endFm_D1”
;
;****** Saturating Pulses - Fm_D2 ***********************************
g3=h1+<118s>
g31=g3#<mfmsub_length, 2mfmsub_length… PulseDuration-mfmsub_length>
g31=>mfmsub
g3+mfmsub_length=>checkPoint,“startFt_D2”
g3+PulseDuration-mfmsub_length=>checkPoint,“endFt_D2”
;
g4=g3+PulseDuration
g4=>SatPulse(PulseDuration)
g4=>act2(PulseDuration)
g4=>mpulse2
g4+PulseDuration/2=>checkPoint,“startFm_D2”
g4+PulseDuration-mfmsub_length=>checkPoint,“endFm_D2”
;
;****** Saturating Pulses - Fm_D3 ***********************************
g5=h1+<178s>
g51=g5#<mfmsub_length, 2mfmsub_length… PulseDuration-mfmsub_length>
g51=>mfmsub
g5+mfmsub_length=>checkPoint,“startFt_D3”
g5+PulseDuration-mfmsub_length=>checkPoint,“endFt_D3”
;
g6=g5+PulseDuration
g6=>SatPulse(PulseDuration)
g6=>act2(PulseDuration)
g6=>mpulse2
g6+PulseDuration/2=>checkPoint,“startFm_D3”
g6+PulseDuration-mfmsub_length=>checkPoint,“endFm_D3”
;
;****** Saturating Pulses - Fm_D4 ***********************************
g7=h1+<238s>
g71=g7#<mfmsub_length, 2mfmsub_length… PulseDuration-mfmsub_length>
g71=>mfmsub
g7+mfmsub_length=>checkPoint,“startFt_D4”
g7+PulseDuration-mfmsub_length=>checkPoint,“endFt_D4”
;
g8=g7+PulseDuration
g8=>SatPulse(PulseDuration)
g8=>act2(PulseDuration)
g8=>mpulse2
g8+PulseDuration/2=>checkPoint,“startFm_D4”
g8+PulseDuration-mfmsub_length=>checkPoint,“endFm_D4”
;
;****** Saturating Pulses - Fm_D5 ***********************************
g9=h1+<298s>
g91=g9#<mfmsub_length, 2*mfmsub_length… PulseDuration-mfmsub_length>
g91=>mfmsub
g9+mfmsub_length=>checkPoint,“startFt_D5”
g9+PulseDuration-mfmsub_length=>checkPoint,“endFt_D5”
;
g10=g9+PulseDuration
g10=>SatPulse(PulseDuration)
g10=>act2(PulseDuration)
g10=>mpulse2
g10+PulseDuration/2=>checkPoint,“startFm_D5”
g10+PulseDuration-mfmsub_length=>checkPoint,“endFm_D5”
;
;END ******************************************************************