PSI Open Fluor CAM script for measuring qE component of NPQ in Chlorella vulgaris

Andrei Herdean

Published: 2021-09-30 DOI: 10.17504/protocols.io.byn9pvh6

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 + =>checkPoint,“endFo”

;

;-------------------------------------------------------------------------------------------------------------

;*** Saturating Pulse & Fm Measurement ***********************************************************************

;-------------------------------------------------------------------------------------------------------------

PulseDuration=800ms

a1 mfmsub_length u ; t =>SatPulse(PulseDuration) > =>act2(PulseDuration) < ; > =>mpulse2 r ; i ;Fm definition > <a1+PulseDuration/2>=>checkPoint,“startFm” a <a1+PulseDuration-mfmsub_length>=>checkPoint,“endFm” s ; _ ;Visual frame definition; Imagee shown in pre-processing window e <a1+PulseDuration/2+TS>=>checkPoint,“timeVisual” i ; / ;------------------------------------------------------------------------------------------------------------- - ; ---------------------------------

;****** 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

=>act2(ALPeriod)

;

;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 ; =>checkPoint,“startFp” + <c1+c2+c3>=>checkPoint,“endFp” i ; t ;------------------------------------------------------------------------------------------------------------- - ; --------------------------------------------------------------------

;****** 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”