HANSATECH FMS1 Bedienungsanleitung

Fluorescence Monitoring System Manual

by Hansatech Instruments Ltd

in any form or by any means - graphic, electronic, or

mechanical, including photocopying, recording, taping, or

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permission of the publisher.

FMS 1

3Contents

Table of Contents

Introduction 6

Section 1: Measurement Principles and Hardware 7

Introduction .................................................................................................................... 8

What is chlorophyll fluorescence? ............................................................................................................. 8

How does the FMS measure chlorophyll fluorescence? ....................................................................... 9

Hardware ...................................................................................................................... 12

Connections to the FMS Control Unit ....................................................................................................... 12

Electrical Connections .............................................................................................................................. 12

Optical Connections ................................................................................................................................. 13

FMS Leaf-Clips ......................................................................................................................................... 14

The PAR / temperature leaf-clip ......................................................................................................... 14

The Dark-Adaptation Leaf-Clip and Fibre-Optic Adapter ................................................................... 17

Hardware Maintenance ...................................................................................................................... 18

Bulb Replacement ....................................................................................................................... 18

The External User Interface ..................................................................................................................... 19

Section 2: Software Installation, System Operation,

Data Handling 21

FMS Software ............................................................................................................... 22

Installing FMS Software .............................................................................................................................. 22

Executing “Modfluor” and “Parview” ....................................................................................................... 22

Troubleshooting Communications Problems ....................................................................................... 23

Operating the FMS ....................................................................................................... 24

“PC Mode” Operation Under Modfluor .................................................................................................... 24

The Recording Screen ................................................................................................................................. 24

Testing the FMS ............................................................................................................................................ 27

Drop-Down” Menu Functions ........................................................................................ 30

Configuration of Hardware and Software ................................................................................................ 31

Hardware_2 ............................................................................................................................................. 31

Instrument Status ..................................................................................................................................... 31

Clear memory ........................................................................................................................................... 32

Set Time / Date ......................................................................................................................................... 32

Config settings ......................................................................................................................................... 33

View Functions ............................................................................................................................................. 33

Graph Functions ........................................................................................................................................... 34

Clear ......................................................................................................................................................... 34

Autoscale ................................................................................................................................................. 35

Zoom ........................................................................................................................................................ 35

Set Axis .................................................................................................................................................... 35

Tools ............................................................................................................................. 36

Tabulate .......................................................................................................................................................... 36

Event marker colour .................................................................................................................................... 37

Data Handling and Export ............................................................................................ 38

FMS 1 - Fluorescence Monitoring System4

File Management With the Files Menu ..................................................................................................... 38

Save Data ................................................................................................................................................. 38

Load Data ................................................................................................................................................. 39

Text search .............................................................................................................................................. 40

Exporting Data to Other Windows® Applications ................................................................................... 41

Params to clipboard and Params to ASCII ................................................................................................ 41

Data to Clipboard and Data to ASCII ......................................................................................................... 43

Printing ........................................................................................................................................................... 46

Instrument Programming Via User-Defined Scripts ...................................................... 47

The Script Recorder ..................................................................................................................................... 47

Running a Script Under Windows .............................................................................................................. 48

The Script Editor ........................................................................................................................................... 49

Creating an Example Script ...................................................................................................................... 50

Script Editor “Drop-Down” Menu .............................................................................................................. 55

Edit ...................................................................................................................................................... 55

Files .................................................................................................................................................... 56

Download ........................................................................................................................................... 56

The Instrument Toolbar ................................................................................................ 57

Recording Functions .................................................................................................................................... 57

Light Source Controls ................................................................................................................................. 58

The Modulation Beam ............................................................................................................................... 58

The Actinic Light Source .......................................................................................................................... 58

The Saturating Light Source ..................................................................................................................... 59

The Far-Red Light Source ........................................................................................................................ 59

Measurement Functions ............................................................................................................................ 59

Dark-adapted Measurement Routines ...................................................................................................... 59

Fo (fluorescence origin) ..................................................................................................................... 60

Fm(fluorescence maximum) .............................................................................................................. 60

Fv/Fm(Maximumquantumefficiency of PSIIphotochemistry) ........................................................... 60

Light-adapted measurement routines ....................................................................................................... 62

The phi PSIIfunction ........................................................................................................................... 62

The phi PSIIRfunction ......................................................................................................................... 64

Fs ....................................................................................................................................................... 65

Fo ....................................................................................................................................................... 65

Logging PARand Temperature .......................................................................................................... 65

External Device Interface and Event Markers ......................................................................................... 66

External Control Interface ......................................................................................................................... 66

Event Markers .......................................................................................................................................... 66

Hansatech Scripting Language Elements ..................................................................... 67

Section 3: Stand-Alone Instrument Operation and Data

Upload 71

Local Mode” Operation Without a PC. ......................................................................... 72

The "Run" Menu ............................................................................................................................................ 72

Running the script .................................................................................................................................... 73

Selecting a different script to run ............................................................................................................. 74

Checking instrument status ...................................................................................................................... 75

Data Upload to PC .......................................................................................................................................... 76

Uploading Trace Data to “Modfluor" ......................................................................................................... 76

Upload to Screen ................................................................................................................................ 76

Upload to File ...................................................................................................................................... 77

Uploading Parameter Data To “Parview” .................................................................................................. 78

5Contents

The File Menu ..................................................................................................................................... 78

Upload params ................................................................................................................................... 79

Configuring the Parameters Display ................................................................................................... 80

Parameter Data Export From“Parview” ............................................................................................. 81

Other “Parview” Functions ................................................................................................................. 81

“Local Mode” Error Codes ............................................................................................ 82

"Local Mode” Menu Structure ...................................................................................... 84

References 85

6FMS 1 - Fluorescence Monitoring System

1 Introduction

The FMS 1 fluorescence monitoring system is a versatile pulse-modulated instrument, designed to

measure chlorophyll fluorescence emission from samples engaged in photosynthesis under

ambient or artificial light conditions in the laboratory.

It consists of a control unit housing all of the electronics, optics and light sources necessary to

derive most common fluorescence parameters. These are optically linked to the sample by a

statistically randomised fibre optic that is suitable for insertion into a range of sample containers

such as oxygen electrodes, gas analysis chambers, petri dishes and microtitre plates.

The system may be operated in several different modes: serial connection to a Windows PC

enables real-time instrument control and data presentation. Captured data is simultaneously

presented as a real-time chart recorder emulation and parameters-only format for easy

identification of key experimental events. This “PC” mode of operation is suitable for development

of complex protocols which may be programmed into the instrument using the simple “drag and

drop” editor to generate user-defined scripts. These scripts automate the execution of

experiments, allowing complex protocols involving many control events to be operated with the

same ease as single control event measurement such as Fv/Fm.

Once programmed, the FMS 1 can be operated as a stand-alone fluorometer inside the laboratory

or outside (via connection to an optional external battery). All measurement data and calculated

parameters are saved to integral protected memory. The unit can store up to six experimental

protocols, any one of which may be accessed and executed using the built-in menu system. When

data collection is complete the FMS can be down-loaded to the Windows software for full

analysis. This document aims to briefly cover the measurement principles used by FMS 1, optimum

hardware configuration, system operation and data handling with the “Modfluor” and “Parview”

software packages. The document is separated into three Sections:

·Section 1: Measurement principles and hardware.

·Section 2: Software installation, instrument operation and data handling.

·Section 3: Stand-alone instrument operation and data upload.

Section 1: Measurement

Principles and Hardware

8FMS 1 - Fluorescence Monitoring System

2Section 1: Measurement Principles and Hardware

2.1 Introduction

2.1.1 What is chlorophyll fluorescence?

These processes utilise absorbed energy for photochemistry, during which electron donation from

pigment to an acceptor molecule occurs. Such processes direct energy for the chemical work

involved in photosynthesis.

Non-Photochemical Processes:

These processes dissipate energy from the photosynthetic apparatus in a manner which does not

drive photosynthesis. Energy is usually re-emitted from the sample in the form of infra-red

radiation (heat) and red / far-red radiation which is known as chlorophyll fluorescence.

The competition between these processes for absorbed energy ensures that a reduction in the

rate of one process will be associated with a corresponding increase in the rates of competing

processes e.g. a reduction in the dissipation of energy by

photochemistry will be reflected in an increase in energy dissipation by nonphotochemical

processes such as heat production and chlorophyll fluorescence.

9Section 1: Measurement Principles and Hardware

Consequently measurement of changes in the extent of fluorescence emission with FMS can be

used to infer information about changes in the efficiency of light use for photochemistry

(Reviewed in Krause and Weis 1991 and Govindjee 1995)

Since the work of Emerson et al. (1932) it has been widely accepted that there are two separate

photochemical steps during photosynthesis which are associated with different groups of

pigments; these groups have become known as photosystem I and photosystem II. In practice

differences in the functionality of photosystem I and II mean that at least 95% of the chlorophyll

fluorescence signal observed under physiological temperatures is derived from chlorophyll

molecules associated with

photosystem II (PSII). Hence the fluorescence signal detected by FMS reflects changes in the

efficiency with which absorbed light is used for PSII photochemistry.

2.1.2 How does the FMS measure chlorophyll fluorescence?

At physiological temperatures peak fluorescence emission occurs in the red region of the

spectrum ( 685 nm) and extends into the infra-red region to around 800 nm (Krause and Weiss

1984) (Fig ure 1-1).

10 FMS 1 - Fluorescence Monitoring System

Fig. 1-1. Room temperature chlorophyll fluorescence emission spectra. (Adapted from Krause and

Weis 1984)

Accurate determination of fluorescence requires a detection system which is sensitive to

fluorescence wavelengths yet blind to the actinic (or excitation) light which drives photochemistry.

In continuous excitation instruments such as the Plant Efficiency Analyser signal discrimination is

achieved using a custom designed 650 nm actinic light source to drive photochemistry; optical

filtering of the detector prevents detection of non-fluorescence wavelengths of light.

Unfortunately such systems must be shielded from ambient light during use, otherwise the red /

far-red component of day-light will be super-imposed on the fluorescence signal.

This problem is avoided by the FMS 1 as it uses an electronic method, commonly referred to as

modulated fluorometry, to separate actinic light from the fluorescence signal (Ögren and Baker

1985). During measurement the tissue sample is exposed to a pulsed amber (or optional blue) LED

light source (the modulating beam) which induces a pulsed fluorescence signal from the sample

under conditions where ambient light is excluded (A, Figure 2). When ambient light is applied the

optical filtering of the FMS 1 allows three types of light signal to reach the detector:

·ambient light of fluorescence wavelengths,

·non-pulsed fluorescence signal induced by the ambient light,

·and pulsed fluorescence signal induced by the modulating beam (B, Figure 1-2).

Addition of ambient light changes the physiology of the sample, altering the proportion of energy

that is re-emitted as fluorescence. Thus the magnitude of the peaks of the pulsed fluorescence

signal that are induced by the modulating beam will reflect changes sample physiology. It is these

signals that the rapid pulse peak tracking electronics amplify and measure; ambient and non-

pulsed fluorescence signal are discarded (C, Figure 1-2).2.

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