PSI Open Fluor CAM script for measuring qE component of NPQ in Chlorella vulgaris
Andrei Herdean
Abstract
This is a simple protocol that consists of
-
10 minutes preillumination with far red light
-
5 minutes of illumination with actinic light
-
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.
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=69.6
LightA=29.069 LightB=-34.732 2 ; ;_________________________________________________________________________________________________________
<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 +
;
;-------------------------------------------------------------------------------------------------------------
;*** Saturating Pulse & Fm Measurement ***********************************************************************
;-------------------------------------------------------------------------------------------------------------
PulseDuration=800ms
a1 mfmsub_length u ; t
;****** Dark Relaxation Measurement *************************************************************************
;-------------------------------------------------------------------------------------------------------------
Dark mfmsub_length 7 b2=2s = <b1, b1+2 uration+2mfmsub_length
b2=2s
<b1 b2, b1+4 msub_length… b1+b2>=>mfmsub
<b1+2b2, b1+4b2… b1+DarkRelaxation1>=>mfmsub
;
;-------------------------------------------------------------------------------------------------------------
;******* Kautsky Effect Measurement **************************************************************************
;-------------------------------------------------------------------------------------------------------------
;
;******* Actinic light Exposure **************************************
ALPeriod=300s
c1=a1+PulseDuration+DarkRe Fast Kautsky kinetics th
;
;Fast Kautsky kinetics****** TS… c1+c2>=>mfmsub; * ; * ;Slow Kautsky kinetics Slow Kautsky kinetics TS, c1+TS+2TS… c1+c2>=>mfmsub;
;
;Slow Kautsky kinetics********************************
;
c3=4s
;
<c1+c c3… c1+ALPeriod>=>mfmsub c ; c ;Fp definition ;
;****** Saturating Pulses - Fm’ Quenching Analysis **********************************************************
;-------------------------------------------------------------------------------------------------------------
;
;******* Saturating Pulses - Fm_L1 mfmsub_length… PulseDuration-mfmsub_length> 1 f11=>mfmsub = f1+mfmsub_length=>checkPoint,“startFt_L1” r f1+PulseDuration-mfmsub_length=>checkPoint,“endFt_L1” > ; e f2=f1+PulseDuration f2=>SatPulse(PulseDuration) n f2=>mpulse2 i f2+PulseDuration/2=>checkPoint,“startFm_L1” = f2+PulseDuration-mfmsub_length=>checkPoint,“endFm_L1” i ; / ; >checkPoint,“startFm_L1”
f2+PulseDuration-mfmsub_length=>checkPoint,“endFm_L1”
;
;****** Saturating P mfmsub_length… PulseDuration-mfmsub_length> * f31=>mfmsub + f3+mfmsub_length=>checkPoint,“startFt_L2” n f3+PulseDuration-mfmsub_length=>checkPoint,“endFt_L2” f ; u f4=f3+PulseDuration , f4=>SatPulse(PulseDuration) i f4=>mpulse2 n f4+PulseDuration/2=>checkPoint,“startFm_L2” D f4+PulseDuration-mfmsub_length=>checkPoint,“endFm_L2” f ; P ; seDuration/2=>checkPoint,“startFm_L2”
f4+PulseDuration-mfmsub_length=>checkPoint,“endFm_L2”
;
;****** mfmsub_length… PulseDuration-mfmsub_length> * f51=>mfmsub * f5+mfmsub_length=>checkPoint,“startFt_L3” h f5+PulseDuration-mfmsub_length=>checkPoint,“endFt_L3” m ; s f6=f5+PulseDuration h f6=>SatPulse(PulseDuration) f f6=>mpulse2 i f6+PulseDuration/2=>checkPoint,“startFm_L3” f6+PulseDuration-mfmsub_length=>checkPoint,“endFm_L3” 6 ; mpulse2
f6+PulseDuration/2=>checkPoint,“startFm_L3”
f6+PulseDuration-mfmsub_length=>checkPoint,“endFm_L3 mfmsub_length… PulseDuration-mfmsub_length> * f71=>mfmsub * f7+mfmsub_length=>checkPoint,“startFt_L4” m f7+PulseDuration-mfmsub_length=>checkPoint,“endFt_L4” g ; > f8=f7+PulseDuration m f8=>SatPulse(PulseDuration) a f8=>mpulse2 7 f8+PulseDuration/2=>checkPoint,“startFm_L4” d f8+PulseDuration-mfmsub_length=>checkPoint,“endFm_L4” u ; t ; n)
f8=>mpulse2
f8+PulseDuration/2=>checkPoint,“startFm_L4”
f8+PulseDuration-mfmsub_length=>checkPoint, mfmsub_length… PulseDuration-mfmsub_length> - f91=>mfmsub * f9+mfmsub_length=>checkPoint,“startFt_Lss” > f9+PulseDuration-mfmsub_length=>checkPoint,“endFt_Lss” - ; m f10=f9+PulseDuration m f10=>SatPulse(PulseDuration) P f10=>mpulse2 _ f10+PulseDuration/2=>checkPoint,“startFm_Lss” i f10+PulseDuration-mfmsub_length=>checkPoint,“endFm_Lss” e ;------------------------------------------------------------------------------------------------------------- h ; checkPoint,“endFm_Lss”
;-------------------------------------------------------------------------------------------------------------
;** Dark relaxation after actinic light period **************************************************************
;-------------------------------------------------------------------------------------------------------------
DarkRelaxation2=300s
h1=c1+ALPeriod
<h h2, h1+4 (DarkRel h2, h1+16
h2=mfmsub_length
h3=10s
;
;******** Relaxation measurement**************************************
;
<h1+h2, h1+2h2, h1+4h2, h1+8h2, h1+16h2>=>mfmsub;
h1+h3 mfmsub_length… PulseDuration-mfmsub_length> ; g11=>mfmsub r g1+mfmsub_length=>checkPoint,“startFt_D1” * g1+PulseDuration - mfmsub_length=>checkPoint,“endFt_D1” m ; b g2=g1+PulseDuration o g2=>SatPulse(PulseDuration) u g2=>act2(PulseDuration) e g2=>mpulse2 r g2+PulseDuration/2=>checkPoint,“startFm_D1” c g2+PulseDuration-mfmsub_length=>checkPoint,“endFm_D1” a ; u ; e(PulseDuration)
g2=>act2(PulseDuration)
g2=>mpulse2
g2+PulseDuration/2=>checkPoint,“startFm_D1”
g2+ mfmsub_length… PulseDuration-mfmsub_length> d g31=>mfmsub g3+mfmsub_length=>checkPoint,“startFt_D2” * g3+PulseDuration-mfmsub_length=>checkPoint,“endFt_D2” m ; b g4=g3+PulseDuration g g4=>SatPulse(PulseDuration) g g4=>act2(PulseDuration) f g4=>mpulse2 > g4+PulseDuration/2=>checkPoint,“startFm_D2” f g4+PulseDuration-mfmsub_length=>checkPoint,“endFm_D2” r ; i ;
g4=>SatPulse(PulseDuration)
g4=>act2(PulseDuration)
g4=>mpulse2
g4+PulseDuration/2=>checkPoint,“sta mfmsub_length… PulseDuration-mfmsub_length> c g51=>mfmsub n g5+mfmsub_length=>checkPoint,“startFt_D3” F g5+PulseDuration-mfmsub_length=>checkPoint,“endFt_D3” > ; g6=g5+PulseDuration h g6=>SatPulse(PulseDuration) i g6=>act2(PulseDuration) > g6=>mpulse2 m g6+PulseDuration/2=>checkPoint,“startFm_D3” u g6+PulseDuration-mfmsub_length=>checkPoint,“endFm_D3” ; = ; +PulseDuration
g6=>SatPulse(PulseDuration)
g6=>act2(PulseDuration)
g6=>mpulse2
g6+PulseDuration/2=>c mfmsub_length… PulseDuration-mfmsub_length> f g71=>mfmsub > g7+mfmsub_length=>checkPoint,“startFt_D4” t g7+PulseDuration-mfmsub_length=>checkPoint,“endFt_D4” ; g g8=g7+PulseDuration # g8=>SatPulse(PulseDuration) t g8=>act2(PulseDuration) l g8=>mpulse2 = g8+PulseDuration/2=>checkPoint,“startFm_D4” t g8+PulseDuration-mfmsub_length=>checkPoint,“endFm_D4” n ; t ; 4”
;
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 ******************************************************************
