TITLE: PSI Open Fluor CAM script for measuring qE component of NPQ in Chlorella vulgaris
AUTHORS: Andrei Herdean

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This is a simple protocol that consists of </div><div class = "text-block">1) 10 minutes preillumination with far red light</div><div class = "text-block">2) 5 minutes of illumination with actinic light</div><div class = "text-block">3) 5 minutes of dark adaptation with far red light</div><br/><div class = "text-block">qE is calculated as the differe between NPQ_Lss and NPQ_D5</div><div class = "text-block">qE=NPQ_Lss-NPQ_D5</div><div class = "text-block">qI=NPQ_D5</div><br/><div class = "text-block">Protocol to be used with FluorCAM 7.0 on a PSI Open FC 800-O/1010-S. </div><div class = "text-block">Act 2 - are the white light LED arrays</div><div class = "text-block">ADD2 - is the far red LED array</div><br/><div class = "text-block">Camera is placed at ~20 cm above the measured sample. Light intensity uniformity across the 96 well plate was measured according to manufacturer instructions. </div><br/><div class = "text-block">!Important - protocol only works under weak far red light. Intense far red will interfere with the fluorescence measurement.</div></div>

[STEPS]
?. 
?. [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=69.6LightA=29.069
LightB=-34.732
 ;_________________________________________________________________________________________________________
 =>SET_FILTER(CHL)
 Preillumination=600s
 =>add2(Preillumination)
 start = Preillumination;
 ;-------------------------------------------------------------------------------------------------------------
 ;*** Fo Measurement ******************************************************************************************
 ;-------------------------------------------------------------------------------------------------------------
 F0duration=5s
 F0period=1s
 start + =>mfmsub
 ;
 ;Fo definition
 start + =>checkPoint,“startFo”
 start + =>checkPoint,“endFo”
 ;
 ;-------------------------------------------------------------------------------------------------------------
 ;*** Saturating Pulse & Fm Measurement ***********************************************************************
 ;-------------------------------------------------------------------------------------------------------------
 PulseDuration=800ms
 a1 = start + F0duration+2mfmsub_length
 ;
 =>SatPulse(PulseDuration)
 =>act2(PulseDuration)
 ;
 =>mpulse2
 ;
 ;Fm definition
 =>checkPoint,“startFm”
 =>checkPoint,“endFm”
 ;
 ;Visual frame definition;     Imagee shown in pre-processing window
 =>checkPoint,“timeVisual”
 ;
 ;-------------------------------------------------------------------------------------------------------------
 ;****** Dark Relaxation Measurement *************************************************************************
 ;-------------------------------------------------------------------------------------------------------------
 DarkRelaxation1=17s
 b1= a1+PulseDuration+2mfmsub_length
 b2=2s
 mfmsub_length… b1+b2>=>mfmsub
 b2, b1+4b2… b1+DarkRelaxation1>=>mfmsub
 ;
 ;-------------------------------------------------------------------------------------------------------------
 ;******* Kautsky Effect Measurement **************************************************************************
 ;-------------------------------------------------------------------------------------------------------------
 ;
 ;******* Actinic light Exposure **************************************
 ALPeriod=300s
 c1=a1+PulseDuration+DarkRelaxation1+mfmsub_length
 =>act2(ALPeriod)
 ;
 ;Fast Kautsky kinetics*********************************
 c2=2s
 ;
 TS… c1+c2>=>mfmsub;
 ;
 ;Slow Kautsky kinetics********************************
 ;
 c3=4s
 ;
 =>mfmsub
 ;
 c3… c1+ALPeriod>=>mfmsub
 ;
 ;Fp definition
 =>checkPoint,“startFp”
 =>checkPoint,“endFp”
 ;
 ;-------------------------------------------------------------------------------------------------------------
 ;****** Saturating Pulses - Fm’ Quenching Analysis **********************************************************
 ;-------------------------------------------------------------------------------------------------------------
 ;
 ;******* Saturating Pulses - Fm_L1 ***********************************
 f1=c1+
 f11=f1#mfmsub_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+
 f31=f3#mfmsub_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+
 f51=f5#mfmsub_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+
 f71=f7#mfmsub_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+
 f91=f9#mfmsub_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
 =>add2(DarkRelaxation2)
 h2=mfmsub_length
 h3=10s
 ;
 ;******** Relaxation measurement**************************************
 ;
 h2, h1+4h2, h1+8h2, h1+16h2>=>mfmsub;
 h1+h3/2+=>mfmsub
 ;
 ;******* Saturating Pulses - Fm_D1 ***********************************
 g1=h1+
 g11=g1#mfmsub_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+
 g31=g3#mfmsub_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+
 g51=g5#mfmsub_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+
 g71=g7#mfmsub_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+
 g91=g9#
 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 ******************************************************************