IMAGING-PAM M-Series

The IMAGING-PAM family comprises the versions MAXI, MINI and MICROSCOPY. The different versions employ the same Multi Control Unit IMAG-CG. Also, the same camera can be used for different versions. This modularity permits easy and most cost-effective switching between various applications and magnifications provided by the IMAGING-PAM M-Series.

The MAXI and MINI versions can be combined with the Gas-Exchange System GFS-3000 and, thus, integrates detailed spatial information on photosynthesis with exact analysis of photosynthetic carbon fixation.


IMAG-3D PAM and projector setup

Most recently, an advanced method to create three-dimensional plant images has been added to the MAXI version. This new configuration opens the way to project data of fluorescence analysis on the three-dimensional plant image so that photosynthesis can be analyzed in the context of whole plant architecture.

 

Elevating Chlorophyll Fluorescence to the 3D Level

The brand-new integration of a precise 3D scanner into the proven MAXI version of the IMAGING-PAM fluorometer combines saturation pulse analysis with knowledge on size, number, position and angle of leaves. As a result, the effective light intensity per unit of leaf area can be considered when light effects on photosynthesis or photoinhibition are evaluated.

The 3D configuration provides a multitude of additional morphological data for structural phenotyping of plants. Together, morphological parameters and fluorometrically characterized, functional parameters form a new realm in plant phenotyping.


Threedimensional RGB color image of Arabidopsis thaliana

 

Chlorophyll Fluorescence and
PAM Fluorometry

Chlorophyll fluorescence is a very sensitive indicator of photosynthesis. Quantitative information on the quantum yield of photosynthetic energy conversion is obtained by PAM fluorometry and the saturation pulse method (Schreiber U (2004) Pulse-Amplitude-Modulation (PAM) Fluorometry and Saturation Pulse Method: An Overview, pp. 279-319. Kluwer Academic Publishers, Dordrecht, The Netherlands).

A wide range of photosynthetic parameters can be derived from fluorescence measurements, giving insight into the physiological state of all photosynthetically active organisms, including higher plants, mosses and ferns as well as various types of algae, phytoplankton and biofilms.

Chlorophyll Fluorescence Imaging

With the advance of highly sensitive CCD cameras and extremely strong light emitting diodes (LED) development of IMAGING-PAM fluorometers has become possible that not only measure images of chlorophyll fluorescence but are also fully competent in providing all relevant chlorophyll fluorescence parameters using the saturation pulse method. In this way, images of photosynthetic activity and its spatio-temporal variations can be obtained.

All IMAGING-PAM fluorometers provide images for 17 different parameters. The fluorescence parameter Ft is continuously monitored. Fo and Fm are assessed after dark adaptation, serving as reference for fluorescence quenching analysis by the saturation pulse method.

Besides Fv/Fm, the PS II quantum yield after dark acclimation, also the PS II quantum yield during illumination, Y(II), and the quantum yields of regulated and non-regulated energy dissipation, Y(NPQ), Y(NO) as well as the apparent electron transport rate (ETR and PS) can be imaged.

A routine for measuring a PAR-absorptivity image is provided for the MAXI and the MINI version of the IMAGING-PAM (Abs.-image based on images of NIR and red light remission). From this parameter also the apparent rate of photosynthesis is calculated and does not need to use the commonly known PAR-absorptivity mean value of 0.84 (ETR) for all areas anymore.

The parameter PS/50 is displayed in order to depict the apparent photosynthesis with the help of the false color code that is also used for the other photosynthesis parameters.

 

The new 3D version of IMAGING-PAM is particularly suited for precise measurements of whole above-ground parts of sample plants. It measures exactly leaf heights and provides a clear numerical description of single leaves (size, shape, angle, balance point) and entire plants (rosette radius, surface area and number of leaves).

For different applications the MAXI, MINI and MICROSCOPY versions are available in various configurations differing in optical geometries and excitation wavelengths. While blue excitation light is normally used for fluorescence imaging of higher plants and algae, red-orange excitation is required for cyanobacteria.

Configurations of the 3D, MAXI, MINI and MICROSCOPY Versions

 

Measuring heads can also be equipped with special LEDs and filter sets for imaging fluorescence from reporter molecules like GFP. The use of power LEDs allows actinic intensities of up to 5000 μmol m-2 s-1 (depending on the M-Series member and configuration).

For the measurement of suspensions additional filter plates are available enhancing the image quality even with reflective surfaces. For PSII measurements under ambient light conditions also the communication with our Universal Light Meter ULM-500 is possible, so that real time light values directly can be taken over to the IMAGING-PAM report file.

For extended analysis options system combination adapters are available facilitating imaging under climate control and combined with gas exchange analysis.

 

The basis of the IMAG-3D is the successful MAXI IMAGING head expanded by a 3D scanner. Using algorithms developed by the Fraunhofer Institute for Integrated Circuits IIS, the system creates a 3D mesh from images of structured light casted on the plant by a projector. The projector is mounted on the backside of the instrument whereas the camera is positioned above the plant. For 3D imaging, this camera is used in the high resolution mode (1280 x 960 pixels) to precisely digitize the sample surface. For fluorescence measurements, highest sensitivity is achieved by switching the camera to 4 pixel binning which results in larger pixel size.


IMAG-3D during saturating flash (up to 5000 μmol m-2 s-1)
 

A dedicated tabletop housing offers ideal darkening for 3D and PSII analysis. The housing is optimized for high precision and cost efficiency. A manual turntable enables researchers to measure single potted plants with a rosette of 10 cm maximum diameter. The 3D version has a slim footprint so that it fits on almost every laboratory desk. For 3D recalibration purposes, the instrument is shipped together with two calibration patterns and a holder so that a quick calibration of the whole system is possible at any time.


Configuration of the 3D Version


 

Multi Control Unit

  IMAG-CG Multi Control Unit to connect Measuring Head and GigE –Vision® CCD camera as M-Series IMAGING-PAM MAXI, MINI and MICROSCOPY version including ImagingWin GigE Software.

 

Measuring Head

  IMAG-MAX/L (blue measuring light version, 450 nm, standard applications)

 

Camera and Lens

  IMAG-K6/MOD3D with increased NIR sensitivity (2/3" chip, primary resolution 1280 x 960 pixels ) with prime lens objective (F1.4/f=16 mm, c-mount). A filter slider with ND filter for 3D

Mounting

  IMAG-3D/GS Stand. Darkening and rack for IMAGING-PAM 3D version

 

Computer

  IMAG-3D/PC a brand-name notebook PC (with Intel Core i5-5xx or higher CPU, NVDIA® graphics board (2 GB or more dedicated memory), SSD, 8GB RAM, internal Gigabit Ethernet (GigE), HDMI interface, Windows 10 OS)

 

System Combination

  Since the instrument is designed as a compact stand-alone unit there are currently no combinations with other Walz Instruments possible.

 

The MAXI version of the IMAGING-PAM M-Seriesemploys a very compact and powerful 300 W LED array for homogeneous illumination of up to 10 x 13 cm areas (microtiter plates) with pulse-modulated excitation, actinic light and saturation pulses.

Two different CCD-cameras are available. For high sensitivity applications the IMAG-K6 (2/3" chip, 1392 x 1040 pixels with 4-pixel-binning) is recommended. For standard applications the IMAG-K7 (1/2" chip, 640 x 480 pixel) is available, which can be used in conjunction with the powerful K7-MAX/Z zoom objective (F1.0/f=8-48mm). So the system features an imaged area from 10 x 13 cm stageless down to 13 x 22 mm at constant resolution.


IMAGING-PAM M-Series in the MAXI Version

A special Mounting Stand with Eye Protection is provided (IMAG-MAX/GS), which features a red perspex glass hood, through which the red chlorophyll fluorescence can be observed. On the bottom of the Mounting Stand IMAG-MAX/GS an x-y stage for variable sample positioning or a multiwell plate can be placed at defined working distance of 18.5 cm.


System combination IMAGING‐PAM MAXI Version with Adapter IMAG‐MAX/GWK1
on Gas‐Exchange Chamber 3010‐GWK1

The bottom plate can be removed so that the whole stand can be jacked up (legs provided) for imaging of plants growing on trays or in pots – additionally the combination with the large gas exchange cuvette 3010-GWK1 for the GFS-3000 is possible using optional adapter sets.

Configurations of the MAXI Version

Multi Control Unit

  IMAG-CG Multi Control Unit to connect Measuring Head and GigE –Vision® CCD camera as M-Series IMAGING-PAM MAXI, MINI and MICROSCOPY version including ImagingWin GigE Software.

 

Measuring Head

  IMAG-MAX/L (blue measuring light version, 450 nm, standard applications) extendable with optional filter plate IMAG-MAX/F for samples with mirror-surface properties
  IMAG-MAX/LR (red measuring light version, 650 nm, e.g. for cyanobacteria) no additional filter plate needed by measurements with mirror-surface properties

 

CCD Camera and Lenses

  IMAG-K7 (1/2" chip, 640 x 480 pixel) with K7-MAX/Z zoom objective (F1.0/f=8-48mm) or K7-MAX/S prime lens objective (F1.2/f=12 mm)
  IMAG-K6 increased sensitivity (2/3" chip, primary resolution 1392 x 1040 pixels) with K6-MAX/S prime lens objective (F1.4/f=12.5 mm)

 

Camera Mounting Set

  all cameras need a mounting set K7-MAX/M or K6-MAX/M to be mountable on the illumination unit.

 

Mounting

  IMAG-MAX/GS Mounting Stand with Eye Protection, protects the eyes and allows direct observation of the red chlorophyll fluorescence
  IMAG-MAX/B Leaf Distance Holder for experiments under ambient light and fixed working distance of 18.5 cm
  St-101 Laboratory stand with wooden baseplate
  IMAG-MAX/HF the fruit adapter helps to keep fruit like apple or capsicum in place for the PAM experiment

 

Computer

  IMAG-PC a brand-name notebook PC

 

Optional

  IMAG-MAX/F filter plate in conjunction with IMAG-MAX/L for samples with mirror-surface properties. A filter plate is factory provided in conjunction with IMAG-MAX/LR.

 

System Combination

  IMAG-MAX/GWK1 Adapter for Gas-Exchange Chamber 3010-GWK1 with Eye Protection

 

Due to its compact design, the MINI version is well suited for field applications. As the imaged area is much smaller than that of the MAXI version (17 x magnification of the area at the same resolution), maximal light intensities are higher, whereas power consumption is lower. For this reason the MINI version can easily be used as handy version independently from mains power.


M-Series MINI version with IMAG-MIN/BK Leaf Holder

The measuring head can also be mounted on the Standard Measuring Head 3010-S of the Portable Gas Exchange Fluorescence System GFS-3000 so that ImagingWin can directly communicate with GFS-Win for control and the exchange of data.


M-Series MINI version with IMAG-MIN/BK Leaf Holder mounted on tripod


M-Series MINI version mounted on a Standard Measuring Head 3010-S of the
GFS-3000 Gas Exchange & Fluorescence System


M-Series MINI version with IMAG‐MIN/BK Leaf Holder mounted on a tripod

 

Configurations of the MINI Version

Multi Control Unit

  IMAG-CG Multi Control Unit to connect Measuring Head and GigE –Vision® CCD camera as M-Series IMAGING-PAM MAXI, MINI and MICROSCOPY version including ImagingWin GigE Software.

 

Measuring Head

  IMAG-MIN/B (blue measuring light version, 460 nm, standard applications)
  IMAG-MIN/R (red measuring light version, 620 nm, e.g. for cyanobacteria)
  IMAG-MIN/GFP (blue measuring light version, 470 nm, GFP imaging and PSII fluorescence) using a high sensitivity camera IMAG-K6, K6-MIN prime lens and Detector Filter Slide K6-MIN/FS

 

CCD Camera and Lenses

  IMAG-K7 (1/2" chip, 640 x 480 pixel) with K7-MIN prime lens (F1.4/f=16 mm)
  IMAG-K6 increased sensitivity (2/3" chip, primary resolution 1392 x 1040 pixels) with K6-MIN prime lens objective (F1.4/f=12.5 mm)

 

Camera Mounting Set

  all cameras need a mounting set K7-MIN/M or K6-MIN/M for robust coupling to the MINI-Head.


MINI version with IMAG‐MIN/GFP; imaging of GFP and PSII fluorescence

Mounting

  IMAG-MIN/BK Leaf holder fixes a leaf to the MINI-Head sample frame
  ST-1010 compact tripod with UNC1/4-20 screw threads tripod head
  IMAG-MIN/ST fine drive with 120 mm traverse path for adjustment of the working distance coupled with a tripod adapter for mounting the MINI-Head onto a UNC1/4-20 screw threads tripod head
  IMAG-S Fine Drive Laboratory Stand with 50 mm traverse path high performance rack-and-pinion drive and 25 x 17 cm platform base.

 

Computer

  IMAG-PC a brand-name notebook PC

 

System Combination

  IMAG-MIN/GFS Adapter for mounting MINI-Heads on Standard Measuring Head 3010-S of GFS-3000


Sample image of beginning deletion in a lime tree leaf

The MICROSCOPY version of the IMAGING-PAM M-Series features imaging analysis on single cell level. Providing highes sensitivity in conjunction with a modified Axio Scope.A1 (Zeiss, Göttingen) epifluorescence microscopes and the IMAG-K6 CCD Camera (2/3" chip with 1392 x 1040 pixels primary resolution and 4-pixel-binning).

The ImagingWin software takes account of the particular needs of microscopy applications. A life video image of the object can be obtained using the standard through-light condenser-illuminator of the microscope. A special saturation pulse routine is provided for optimal assessment of Fo, Fm and Fv/Fm at low levels of excitation intensity.


M-Series MICROSCOPY version with CCD‐Camera IMAG‐K6 on a Zeiss Axio ScopeA.1 microscope


M-Series MICROSCOPY version with Red‐Green‐Blue Microscopy LED Lamp IMAG‐RBG
on a Zeiss Axio ScopeA.1 microscope

For standard applications a single high power Luxeon LED (470 nm or 625 nm) is provided for excitation, actinic illumination and saturation pulses . Alternatively a sophisticated Red-Green-Blue-Microscopy LED Lamp is available allowing differentiating between various groups of algae (including cyanobacteria) in biofilms, in analogy to the PHYTO-PAM.

With our Universal Light Meter ULM-500 internal light list calibration becomes easy. Just plug in the ULM, place the light sensor MC-MQS-OVV in the focal plane and start the calibration procedure.


Filamentous algae sample with epiphytic population (Ft image)


Filamentous algae sample with epiphytic population (Fm image)

Configurations of the MICROSCOPY Version

Multi Control Unit

  IMAG-CG Multi Control Unit to connect Measuring Head and GigE–Vision® CCD camera as M-Series IMAGING-PAM MAXI, MINI and MICROSCOPY version including ImagingWin GigE Software.

 

Measuring Head

  IMAG-L470M Microscope LED Lamp Module 470 nm (blue) for fluorescence excitation of chlorophyll fluorescence
  IMAG-L625M Microscope LED Lamp Module 625 nm (red-orange) for fluorescence excitation of chlorophyll fluorescence of most algae groups including cyanobacteria
  IMAG-RGB Red-Green-Blue Microscopy LED Lamp Module allowing computer-assisted deconvolution of major algae groups. Fluorescence excitation and actinic illumination using red (620 nm), green (520 nm), blue (460 nm) or white light (mixed 620, 520 and 460 nm).

 

CCD Camera

  IMAG-K6 increased sensitivity (2/3" chip, primary resolution 1392 x 1040 pixels)


Closeup on epiphytes growing on filamentous algae sample (Ft image)

Microscope

  IMAG-AXIOSCOPE Axio Scope. A1 from Zeiss adapted for IMAGING-PAM applications. Detector filter RG665, dichroic mirror 420-640 nm, video adapter 60N-C 2/3" 0,5x and standard lens Zeiss Fluar 20x.

 

Computer

  IMAG-PC a brand-name notebook PC

 

Optional

  IMAG-AX-REF Reflector Module with Filter Set consisting of a beam splitter filter (420-640 nm) and a detector filter (665 nm), mounted in a Zeiss reflector module frame for convenient measurements using more than one LED Lamp Module.


Epidermal strip with stomates (Ft image)

General Features and Graphical User Interface

The IMAGING-PAM M-Series GigE instruments are fully controlled by the ImagingWinGigE software. When started, the ImagingWinGigE software opens with the image window that occupies most of the user surface showing the Ft value as starting parameter.
ImagingWin software: Ft-values of Arabidopsis seedlings shown in false color

Values are represented in a false color scale ranging from black (0.0) to white (1.0) with red, orange yellow, blue and violet to purple in between. At first a standard AOI (area of interest) is already present after the start of the software. Different shapes and up to 100 AOIs can be defined. AOIs` positions can be moved by the new Edit function.


Kinetics window with evaluation of an automated illumination phase, called induction curve plus recovery time

The customer can chose between 18 different parameters (Ft, Fo, Fm, F, Fm’, Fv/Fm, Y(II), Y(NPQ), Y(NO), PS/50, Abs, Red, NIR, NPQ/4, qN, qP, qL, Inh.) that could be displayed in the image window in different color modes. In this tab the alteration of the parameters can be observed in real-time during the experiment.

The kinetics window shows various parameter values for some or all AOIs of the currently chosen experiment plotted versus time. It serves for the evaluation of dynamic dark / light phenomena (Kautsky curve or Induction curve).

Some of the possible experiments are already preset in this and the following light curve window so that also the beginner finds an easy starting point for his first experiments. For advanced users it is also possible to program script files with more complex structure.


RGB‐FIT window MICROSCOPY version; green filamentous algae with diatom epiphytes

 

Easy light calibration using the ULM-500 Light Meter & Logger. ImagingWinGigE in communication with the ULM-500 provides an automated light calibration routine to generate a calibrated internal light list and furthermore offers to follow an external illumination.

Some new features can be provided solely for the ImagingWinGigE software, not for the ImagingWin software suitable for FireWire camera versions of the IMAGING-PAM M-Series.


MINI version with IMAG‐MIN/GFP; imaging of GFP and PSII fluorescence