Walz MINI-PAM User manual

PHOTOSYNTHESIS YIELD
ANALYZER MINI-PAM
Portable Chlorophyll Fluorometer
Handbook of Operation
2.115 / 04.96
2. Edition: August 1999
minip_1ea.doc
Heinz Walz GmbH, 1999
Heinz Walz GmbH •Eichenring 6 •91090 Effeltrich •Germany
Phone [phone] •Telefax +49-(0)9133/5395
E-mail [email protected] •Internet www.walz.com
Printed in Germany

CONTENTS
1Safety instructions ........................................................................ 1
1.1 General safety instructions........................................................1
1.2 Special safety instructions.........................................................1
2General Information .................................................................... 2
3Basic Operation of the MINI-PAM............................................. 4
4Description of the eight Keyboard Functions ............................ 7
4.1 Single key operations ................................................................ 7
4.2 Double key operations............................................................... 8
5Important Points for Correct YIELD-Measurements ............ 10
6Description of the Memory-Function ....................................... 12
7The Mode-Menu ......................................................................... 14
7.1 List of Menu points .................................................................15
7.2 Description of the Mode-menu points.....................................17
8Components of the MINI-PAM................................................. 29
8.1 Main Control Unit...................................................................29
8.1.1 Fluorescence excitation and detection ..............................30
8.1.2 Special information on MINI-PAM/B ..............................31
8.1.3 Internal halogen lamp as actinic light source.................... 33
8.1.4 Rechargeable battery.........................................................34
8.1.5 LC-display.........................................................................35
8.1.6 Electronic components...................................................... 36
8.1.7 Description of the connectors ...........................................37
8.2 Fiberoptics MINI-PAM/F and Miniature Fiberoptics
MINI-PAM/F1 ........................................................................38
8.3 Leaf-Clip Holder 2030-B ........................................................41
8.4 Micro Quantum/Temp.-Sensor 2060-M..................................45
8.5 External Halogen Lamp 2050-HB........................................... 45
8.6 Dark Leaf Clip DLC-8 ............................................................47
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CONTENTS
9Data Transfer.............................................................................. 48
10 Operation of the MINI-PAM via a PC-Terminal and the
RS 232 Interface ......................................................................... 51
11 Maintenance................................................................................ 53
11.1 Internal battery and its replacement ........................................ 53
11.2 Halogen lamp and its replacement .......................................... 54
11.3 Fuse replacement.....................................................................55
11.4 EPROM and its replacement...................................................55
12 Chlorophyll Fluorescence Measurements with the
MINI-PAM.................................................................................. 57
12.1 Chlorophyl fluorescence as an indicator of photosynthesis .... 58
12.2 The PAM measuring principle ................................................64
12.3 Assessment of photosynthesis with the MINI-PAM: Outline
of the most important functions in practical applications. ......67
12.3.1 Maximal photochemical yield Fv/Fm ............................... 67
12.3.2 ML-BURST (menu point 5).............................................. 68
12.3.3 AUTO-ZERO (menu point 2) ........................................... 69
12.3.4 Fo, Fm (menu point 25) ....................................................70
12.3.5 INT.TEMP (menu point 35).............................................. 71
12.3.6 qP, qN and NPQ (menu points 26 and 27)........................ 73
12.3.7 YIELD-measurements of illuminated samples ................. 75
12.3.8 ACT-LIGHT and ACT+YIELD (menu points 12 and 13)76
12.3.9 LIGHT CURVE (menu point 17) and LIGHT-
CURVE+REC (menu point 18) ........................................79
12.3.10YIELD- and ETR-averaging (menu point 11) ..................82
12.3.11INDUCTION CURVE (menu point 21) and
INDUCTION CURVE+RECOVERY (menu point 22).... 83
12.3.12Repetition Clock (menu point 28: REP-CLOCK and
double key function ON+MEM)....................................... 85
II

CONTENTS
13 Appendix ..................................................................................... 87
13.1 Technical specifications ..........................................................87
13.2 List of warnings and errors......................................................91
13.3 PIN-assignments......................................................................93
13.4 List of commands for operation of MINI-PAM via
PC-terminal by user-written software ..................................... 94
13.5 Selected reviews on chlorophyll fluorescence and related
topics .......................................................................................98
14 Rechargeable battery ............................................................... 104
15 Warranty conditions ................................................................ 105
III

CHAPTER 1 SAFETY INSTRUCTIONS
1 Safety instructions
1.1 General safety instructions
1. Read the safety instructions and the operating instructions first.
2. Pay attention to all the safety warnings.
3. Keep the device away from water or high moisture areas.
4. Keep the device away from dust, sand and dirt.
5. Always ensure there is sufficient ventilation.
6. Do not put the device anywhere near sources of heat.
7. Connect the device only to the power source indicated in the
operating instructions or on the device.
8. Clean the device only according to the manufacturer’s
recommendations.
9. Ensure that no liquids or other foreign bodies can find their way
inside the device.
10. The device should only be repaired by qualified personnel.
1.2 Special safety instructions
1. The MINI-PAM Photosynthesis Yield Analyzer is a highly
sensitive research instrument which should be used only for
research purposes, as specified in this manual. Please follow the
instructions of this manual in order to avoid potential harm to the
user and damage to the instrument.
2. The MINI-PAM employs high intensity light sources which may
cause damage to the eye. Avoid looking directly into these light
sources during continuous illumination or saturation pulses.
1

CHAPTER 2 GENERAL INFORMATION
2 General Information
The Photosynthesis Yield Analyzer MINI-PAM has been
developed with special attention to the quick and reliable assessment
of the effective quantum yield of photochemical energy conversion
in photosynthesis. The most relevant information is obtained by a
single key operation within a second and up to 4000 data sets can be
stored for later analysis. Due to a novel opto-electronic design and
modern microprocessor technology, the MINI-PAM is extremely
compact and at the same time highly sensitive and selective. It is
ideally suited for rapid screening of photosynthetic activity in the
field, green house and laboratory and due to its robust, waterproof
housing it can be used even in extreme environments.
The MINI-PAM, like all PAM Fluorometers, applies pulse-
modulated measuring light for selective detection of chlorophyll
fluorescence yield. The actual measurement of the photosynthetic
yield is carried out by application of just one saturating light pulse
which briefly suppresses photochemical yield to zero and induces
maximal fluorescence yield. The given photochemical yield then
immediately is calculated, displayed and stored. Numerous studies
with the previously introduced PAM Fluorometers have proven a
close correlation between the thus determined YIELD-parameter
(∆F/Fm') and the effective quantum yield of photosynthesis in leaves,
algae and isolated chloroplasts. With the help of the optional Leaf-
Clip Holder 2030-B also the photosynthetic active radiation (PAR)
can be determined at the site of fluorescence measurement, such that
an apparent electron transport rate (ETR) is calculated. In addition to
this central information, the MINI-PAM also provides the possibility
of measuring fluorescence quenching coefficients (qP, qN, NPQ),
applying continuous actinic light for measurement of induction
curves (Kautsky-effect) and automatic recordings of light-saturation
2

CHAPTER 2 GENERAL INFORMATION
curves with quenching analysis. For these purposes, an extensive
MODE-menu is provided.
While the MINI-PAM was conceived as a typical stand-alone
instrument for field experiments, it can also be operated under
laboratory conditions in conjunction with a PC and the special
Windows-software WinControl. When the MINI-PAM is connected
to a PC, the information on instrument settings and data registration
is continuously exchanged, such that both ways of operation are
equivalent.
The WinControl software provides so-called "tooltips" with short
explanations of the numerous functions of the MINI-PAM. Hence,
use of WinControl is recommended particularly to the beginner for
becoming acquainted with the principles of operation and of
chlorophyll fluorescence information. It should be emphasized that
there is no risk of serious mistakes causing damage. Hence,
beginners may feel free to "play" with the system, trying out all
functions. For this purpose, the Chart-window is particularly useful,
as it records all fluorescence changes like a chart recorder.
This manual deals mainly with the MINI-PAM as such, operated
as a stand alone unit. A separate manual will be provided for the
WinControl software.
3

CHAPTER 3 BASIC OPERATION OF THE MINI-PAM
3 Basic Operation of the MINI-PAM
The MINI-PAM is very easy to operate. It has a two-line LC-
display and a small tactile keyboard with eight function keys (ON,
OFF, MODE, MEM, ∧, ∨, START, SET). In order to get started, only
the fiberoptics have to be connected and the ON-key is pressed. Now
the system is ready for recording fluorescence yield of any sample
which is close (5-20 mm) to the free end of the fiberoptics. The
actual measurement of the most relevant YIELD-parameter (quantum
yield of photochemical energy conversion) just involves pressing the
START-key. Then on the display, for example, the following
information is shown:
1: 445F 1739M ..C
F: 448 745Y ..E ..L
The meaning of the various displayed parameters is as follows:
1: Number denoting the standard MODE-menu position 1
which is automatically installed whenever the MINI-PAM
is switched on or a YIELD-determination is carried out
via START.
445F Fluorescence yield (F) measured briefly before the last
saturating light pulse triggered by START.
1739M Maximal fluorescence yield (M = Fm or Fm') measured
during the last saturating light pulse triggered by START.
..C Temperature in degree Celsius, display of which requires
optional Leaf-Clip Holder 2030-B.
F: 448 Momentary fluorescence yield displaying small
fluctuations.
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CHAPTER 3 BASIC OPERATION OF THE MINI-PAM
745Y The most relevant YIELD-parameter determined by the
last saturating light pulse triggered by START, calculated
as follows:
YIELD = Y/1000 = (M-F)/M = ∆F/M = ∆F/Fm'
(Genty-parameter)
With a dark-adapted sample ∆F/Fm = Fv/Fm,
corresponding to the maximal yield of photochemical
energy conversion.
..E Relative rate of electron transport (ETR), display of
which requires optional Leaf-Clip Holder 2030-B. It is
calculated by the formula:
ETR = E = YIELD x PAR x 0.5 x ETR-factor
..L Light intensity in terms of PAR (quantum flux density of
photosynthetically active radiation), display of which
requires Leaf-Clip Holder 2030-B.
After every operation of START the obtained data set with the
corresponding time and date is entered into a RAM-memory, with a
storage capacity of 4000 data sets. The stored data can be called on
the display via the MEM-key. Previously recorded data can be
recalled by using the arrow-keys (∧or ∨). Stored data can be printed
out via an RS 232 interface or transferred on a PC for further
analysis.
The MINI-PAM has been pre-programmed at the factory with
standard settings (see list in 7.1) for all relevant measuring
parameters (for example Measuring Light Intensity, Gain, Damping,
Saturation Pulse Intensity, Saturation Pulse Width etc.). These
standard settings are optimized for measurements with standard leaf
samples at approx. 12 mm distance between fiberoptics and leaf
surface. For special applications, there is great flexibility for
appropriate adjustment of all measuring parameters with the help of
the extensive MODE-menu, using the arrow-keys (∧and ∨) in
5

CHAPTER 3 BASIC OPERATION OF THE MINI-PAM
combination with the SET-key. Details are given in the MODE-menu
list (see 7.2).
6

CHAPTER 4 DESCRIPTION OF KEYBOARD FUNCTIONS
4 Description of the eight Keyboard Functions
Fig. 1: Photosynthesis Yield Analyzer MINI-PAM
4.1 Single key operations
ON To switch MINI-PAM on (short pressing of the key).
To activate the backlighting of the display (switches
automatically off when no key operation for 50 s; power
saving for field use); requires 3 s pressing of the key.
OFF To switch MINI-PAM off; will occur automatically, if no
key operation for 4 min (power saving for field use), unless
disabled via menu point 10.
MODE To return to MODE-menu after using the MEM- or SET-
keys.
7

CHAPTER 4 DESCRIPTION OF KEYBOARD FUNCTIONS
MEM To enter the MEMORY-level of stored data with the last
stored data set being displayed.
∧, ∨To select one of 51 points of the MODE-menu or one of
4000 data sets when MEMORY is activated:
To change a particular parameter setting in the MODE-
menu after operating the SET-key.
For advancement by several steps these keys can be kept
pressed.
START To trigger a saturating light pulse for assessment of YIELD
and related fluorescence parameters.
SET To start and stop selected function.
4.2 Double key operations
Besides the single key operations, there is a number of double
key operations which can serve as short-cuts for selecting/carrying
out certain items/commands in the MODE-menu. For this purpose,
the first key must be kept firmly pressed before briefly pressing the
second key.
MODE+START To return to standard display (menu position 1).
MODE+SET To move from one functional block in the MODE-
menu to the next (see list in 7.1).
MODE+∧To move to MODE-menu point 17: LIGHT CURVE
(carried out via SET).
MODE+∨To move to MODE-menu point 21: IND.CURVE.
MODE+ON To switch measuring light on/off.
MODE+MEM To move to MODE-menu point 28: REP-CLOCK.
8

CHAPTER 4 DESCRIPTION OF KEYBOARD FUNCTIONS
ON+SET To switch actinic light on/off.
ON+START To start/stop actinic illumination with yield-
measurement (see menu point 13).
ON+MEM To start/stop the clock for repetitive triggering of
selected function (e.g. saturation pulses when 29:
CLOCK-ITEM in position SAT).
ON+∧To start/stop a LIGHT CURVE (equivalent to menu
point 17).
ON+∨To start/stop an INDUCTION CURVE (equivalent to
menu point 21).
SET+OFF To reset program, if MINI-PAM for some reason
does not respond to key-operations.
If the MINI-PAM is switched on by RS 232-access the key-
controller may not respond. In this case push the ON-key once.
Note:Whenever a command is given which involves the switching
on and off of the actinic halogen light source, a short beep-
sound confirms that the command is carried out. In addition,
there is a more extended beep for the duration of a saturating
light pulse.
9

CHAPTER 5 IMPORTANT POINTS
5 Important Points for Correct YIELD-
Measurements
The main purpose of the MINI-PAM is the reliable determination
of the YIELD-parameter ∆F/Fm (Genty-parameter). This task is
carried out by the MINI-PAM with exceptional sensitivity and
reproducibility. Because of the central importance of this particular
type of measurement, a special section is devoted to it in this
handbook (see section 12.3). Here just the most important practical
aspects are outlined which are essential for correct YIELD-
measurements:
1) The distance between sample and fiberoptics should be approx.
10-15 mm, such that a normal leaf at standard settings gives a
signal of 200-500 units.
2) The AUTO-ZERO function (MODE-menu point 2) should be
applied (while sample is removed), in order to suppress any
unavoidable background signal which otherwise would cause
some lowering of the YIELD-reading (see 12.3.3).
3) In practice, YIELD-measurements make sense only, if the light
conditions of the sample are well controlled. For example, a leaf
may be severely damaged in Calvin cycle activity and still show a
high YIELD-value when dark-adapted or in weak light. The
overall photosynthetic performance should be assessed during
steady state illumination at a photon flux density which is
somewhat below saturation in a control sample. For highest
accurracy it is essential that the PAR is measured close to the spot
of the sample where also fluorescence is detected. For this
purpose the optional Leaf-Clip Holder 2030-B is available (see
8.3). On the basis of the measured YIELD- and PAR-data an
apparent electron transport rate (ETR) is calculated and displayed
10

CHAPTER 5 IMPORTANT POINTS
(...E). The plot of ETR vs. PAR corresponds to a light-response
curve of photosynthesis (see 12.3.9).
4) When YIELD is measured under field conditions, it is essential
that the leaf position and effective PAR are not inadvertently
changed. During the actual measurement, the fiberoptics must be
stably fixed with respect to the leaf surface for ca. 2 s, e.g. with
the help of the Leaf-Clip Holder 2030-B.
5) Dark YIELD-measurements require special conditions (see also
12.3.1). As already pointed out in 3), such measurements cannot
give information on the overall photosynthetic performance. They
are useful to specifically assess the state of PS II, for example
following light stress treatment. In this case, it is essential, that
the measuring light does not induce any significant increase of
fluorescence yield. For this purpose, the MODE-menu point 5
provides the possibility of applying the measuring light in short
pulse bursts, thus cutting its integrated intensity to 1/5 (see
12.3.2).
11

CHAPTER 6 DESCRIPTION OF THE MEMORY-FUNCTION
6 Description of the Memory-Function
All data recorded via START are automatically stored in RAM-
memory with a capacity of 4000 data sets. They can be recalled on
display via the MEM-key. Then, for example, the following
information is shown:
MEM 382: 12:27 27/MAY/95
A: 322Y 21.1E 157L
In the top line it can be seen that the data set Nr. 382 of the
current MEMORY was recorded at 12:27 o'clock on May 27th 1995.
The bottom line shows that a sample of type A was used (see
MODE-menu point 51), which displayed a YIELD-value (Y) of
0.322 and an apparent ETR-value (E) of 21.1 at an incident light
intensity (L) of 157 µmol quanta m-2 s
-1 of the photosynthetically
active radiation (PAR).
More information relating to this particular data set can be
displayed in the top line by SET-operation:
MEM 382:390F 576M 19.9C
A: 322Y 21.1E 157L
After the first SET, the top line shows that the fluorescence yield
(F) measured briefly before the saturating light pulse was 390, that
the maximal fluorescence (M) amounted to 576 and that temperature
was 19.9 °C.
MEM 382:645P 759N 1.557Q
A: 322Y 21.1E 157L
After the second SET, the top line shows the quenching
coefficients qP=0.654, qN=0.759 and NPQ=1.557, which will be
meaningful only if for this particular sample a Fo-Fm determination
12

CHAPTER 6 DESCRIPTION OF THE MEMORY-FUNCTION
(MODE-menu point 25) had been carried out beforehand (see
12.3.4).
Further operation of SET (2x) leads back to the original display
with time and date.
Using the arrow keys ∧and ∨one can move within the memory
and display any previously recorded data sets.
All data stored in MEMORY can be cleared by the CLEAR
MEMORY function (MODE-menu point 39). For safety's sake, this
command does not only require execution by SET, but in addition
confirmation by the ∧-key. The memory is organized in form of a
ring storage and its clearance normally is not required, as old data
will be automatically overwritten.
The MEMORY-front normally corresponds to the MEM-No.
under which the last set of data was stored. It can be moved to any
number between 1 and 4000 with the help of MODE-menu point 38.
After any change in instrumental settings, the complete set of
settings will be stored upon the next YIELD-measurement in the
Report-file of the WinControl program (see separate manual). This is
indicated by "Saved Settings" in the MEMORY-display.
Data stored in MEMORY can be readily transferred to a PC via
the RS 232 cable (see section 9).
13

CHAPTER 7 THE MODE-MENU
7 The Mode-Menu
The MODE-menu contains 51 items corresponding to a variety
of measured values, instrumental settings or special commands. The
positions of the various menu points were arranged for optimal
practicability, with the most frequently used functions being closest
to the standard position 1.
Increasing or decreasing position numbers are selected by the ∧-
or ∨-arrow keys, respectively. Changes are terminated via SET or
MODE. Starting from position 1, at increasing numbers there are
mostly MODE-points involving commands (for example, 2: AUTO-
ZERO), while at decreasing numbers the MODE-points for
instrumental settings prevail (for example, 50: MEASURING
LIGHT INTENSITY). Some of the MODE-menu positions can be
directly reached via double key operations (see list in section 4.2
above).
Irrespective of the selected menu position, a YIELD-
measurement can be initiated at any time by pressing the START-key.
Normally, the system then automatically returns to the menu position
1 where the measured data set is displayed. The only exceptions are
menu-positions 11, 25-27 and 34, where the displayed values are of
primary interest.
The operations related to the various points of the MODE-menu
are either directly carried out via SET (e.g. 2: AUTO-ZERO: 50) or
initiated/terminated (e.g. 50: MEAS-INT: 8) by pressing SET.
Settings are changed by arrow key operations (∧, ∨) and become
immediately effective. The numbers following the double points
show the present settings.
14