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Fluorescence Kinetic Microscope

Fluorescence Kinetic Microscope (FKM) is designed to be the most versatile tool for lab-based research. It extends the complete capacity of kinetic chlorophyll or multicolor fluorescence imaging to the realm of individual cells and sub-cellular structures. All conventional fluorescence parameters can be mapped with micro-meter resolution so that individual chloroplasts or even grana-stroma thylakoid segments can be investigated. The modular setup of the FKM is designed for kinetic fluorescence measurements with various user-selectable excitation and emission wavelengths as well as the combination of imaging measurements with spectrally resolved or ultra fast (µs) spot measurements of fluorescence and absorbance kinetics.

The FKM allows imaging measurements of two-dimensional resolved multicolor fluorescence transients induced by complex irradiance protocols. The fluorescence emission is induced by an appropriate LED light source.

In the standard configuration, the wavelength selection is the same as in other research-grade fluorescence microscopes by the use of a white excitation light source combined with a set of excitation filters, dichroic mirrors, and emitter filters. As an additional option, a light source with tuneable spectra for both measuring and actinic/saturating light is available. It increases the range of measurable chromophores and improves signal/noise ratio. The system is designed to allow automatic switching between different excitation wavelengths even during same measurement. In contrast to conventional fluorescence microscopes, the ultrahigh sensitivity of the measuring camera combined with modulated light measurement makes it possible to perform imaging at extremely low light levels that do not disturb the metabolism of the cell.

The Fluorescence Kinetic Microscope can be combined with spectrally resolved measurements which are done via the Spectrometer SM 9000 synchronized to the measuring camera .The ultrafast kinetic measurements are performed by a microscope-adapted version of the Double Modulation Fluorometer FL 3500, which again can be synchronised to the measuring camera. In this way, the spectrally resolved or ultrafast kinetic spot measurements can be done simultaneously on the same object and controlled by the same measuring protocol as the imaging kinetic record.

All parts and functions can be controlled by the FluorCam software depending on individual configuration.

Fluorescence Kinetic Microscope
Device Description: 1: Measuring camera; 2: 3-port motorised video adapter; 3: 2-port manual switching adapter; 4: Fibreoptics adapter for spectrometer; 5: Spectrometer; 6: C-mount adapter for photo camera; 7: Motorised wheel for filter cubes (8 cubes); 8: filter wheel for switching excitation while maintaining emission filters; 9: FKM control unit; 10: Peristaltic pump for liquid media; 11: Air pump; 12: Temperature control unit; 13: Flow-through thermostat.

Scheme of the Device
Fluorescence Kinetic Microscope: Scheme of the Device

  • Pulse-amplitude modulated measurement of in vivo chlorophyll fluorescence kinetics and its imaging.
  • Various parts of the light harvesting antenna (e.g. different phycobiliproteins vs. Chl-protein complexes) can be excited by selecting the optimal excitation wavelength. Different parts of the antenna can be excited in the same measurement by automatic switching between different excitation wavelengths.
  • The complete Kautsky kinetics, including the analysis of photochemical and non-photochemical quenching, can be analysed with spectral resolution. In this way it is possible to decide, for example, which of the pigment-protein complexes of a photosynthetic system contributes most to photochemical or non-photochemical quenching. This can be decisive in analysing the mechanisms of changes in photosynthetic performance.
  • Analysis of any non-chlorophyll fluorescence kinetics, so that other physiological processes can be monitored in vivo (via their autofluorescence or via specific fluorescent dyes) and compared to the performance of photosynthesis of the same cells. In contrast to conventional fluorescence microscopes, the ultrahigh sensitivity of the measuring camera combined with modulated light measurement allows for imaging at extremely low light levels that do not disturb the metabolism of the cell.
  • Investigating fast processes that cannot be captured by the speed of currently available cameras, e.g. direct (not pump-and-probe) measurements of Qa reoxidation, connectivity, or antenna size.
  • Corn Leaf Image
    Corn Leaf Image
  • 10 µs to 20 ms exposure time per frame
  • Ten or three light excitation module generating measuring and actinic light (tunable light spectra)
  • Multiple fluorophore imaging
  • 70 % peak quantum yield with about 4 electrons readout noise
  • Automated software-based system operation
  • Spectrally resolved or ultra fast (µs) spot measurements of fluorescence and absorbance kinetics (optional)
  • Fv/Fm
  • Kautsky induction
  • Quenching analysis
  • Light curve
  • Steady state fluorescence, e.g., ChlF, GFP and other FPs
  • Multicolor fluorescence, e.g., ChlF, plant blue-green fluorescence
  • QA re-oxidation (needs optional electronic module)
  • Fast fluorescence induction (OJIP) with 1µs resolution (needs optional electronic module)
  • Spectrally resolved absorbance kinetic (needs optional Spectrometer SM 9000)
  • Chlamydomonas Image
    Chlamydomonas Image
  • Fully automated control of the whole FluorCam system
  • Image acquisition via automated experimental protocols:
    - numerous predefined protocols
    - possibility to create user defined protocols via a program Wizard
    - multiple (automatically repeated) experiments
  • Image processing tools:
    - automatic or manual image segmentation – e.g., labeling of individual plants
    - analysis of kinetic data from all samples within the field of view
    - numerous image manipulation tools
    - export to text file, avi, bmp or raw data formats
  • Windows 2000, XP, Vista, W7 compatible
* Windows is a registered trademark of Microsoft Corporation
  • Axio Imager M2
    Included components:
    - Axio Imager M2 tripod Mot
    - Objective holder, 7-mounted, encoded
    - Transmitted light shutter
    - Optics for FL
    - Condenser Achr Apl 0.9 H
    - Reflector revolver, 6x mot
    - Binocular photo tube (100:0/30:70/0:100)
    - Mechanical stage 75x50 R basic, hardcoat anodized surface
    - Sample holder 76 x 26 mm
    - Adapter 60N C 2/3" 0.63x
    - Adapter 60 C 1/3" 0,4x

    * The Microscope body allowes motorized stage movement in Z axis (up and down)
    * Output for Spectrometer connection is not included (special optical switch may be added as an option)
  • Axio Scope A1
    Included components:
    - Microscope stand Axio Scope.A1 6x HD DIC
    - Stage holder D/A, vertically adjustable
    - Condenser carrier with vertical adjustment on both sides
    - Condenser - achromatic-aplanatic 0.9 H
    - Reflector turret 6x man., changeable, for P&C modules (filter cubes)
    - Mechanical stage 75x50 R ergonomic drive, fixed position
    - Sample holder 76 x 26 mm
    - Binocular photototubus 30°/23 (100:0/0:100)
    - Adapter 60N C 2/3" 0.5x

    * The Microscope body allowes manual movement in X, Y, Z axis
  • Microscope Body
  • 10-LED Light Module
    Included components:
    - Control unit for 5 flash lights and 5 continual trans-illumination lights
    - Epi-illumination light source: FCMLS 5LEDC, FCMLS 5LEDF, OFUSER (LED colors: UV, blue, green, white, red)
    - Trans-illumination light source TLS (white and infra LED)
    - Power supply for Microscope control unit
  • 3-LED Light Module
    Included components:
    - FCM3 Control unit
    - Epi - illumination light source FCMLS  (LED colors: FL – white, Cont – white, UV)
    - Trans-illumination light source TLS (white and infra LED)
    - Power supply for Microscope control unit
  • Microscope Body
  • High resolution camera
  • High sensitivity camera
  • FKM
  • Chlorophyll fluorescence filter cube
  • GFP filter cube
  • FKM
  • Spectrometer SM 9000
  • Optical switch – double adapter for P&C; T2-2x60N
  • PSI thermoregulated module
  • Objectives (according to customer's needs and requirements)
  • Spectrometer SM 9000
    Spectrometer SM 9000
  • FERIMAZOVA N., FELCMANOVA K., ŠETLIKOVA E. et al. (2013): Regulation of photosynthesis during heterocyst differentiation in Anabaena sp. strain PCC 7120 investigated in vivo at single-cell level by chlorophyll fluorescence kinetic microscopy. Photosynth Research. Volume 116, Pages 79-91. DOI: 10.1007/s11120-013-9897
  • ANDRESEN E., LOHSCHEIDER J., ŠETLÍKOVÁ E. et al. (2010): Acclimation of Trichodesmium erythraeum ISM101 to high and low irradiance analysed on the hysiological, biophysical and biochemical level. New Phytologist. Volume 185, Pages 173–188. DOI: 10.1111/j.1469-8137.2009.03068.x
  • KOMÁREK O., FELCMANOVÁ K., ŠETLÍKOVÁ E. et al. (2010): Microscopic Measurements of the Chlorophyll A Fluorescence Kinetics. Developmnet in Applied Phycology. Volume 4, Pages 91-101. DOI: 10.1007/978-90-481-9268-7_5
  • MIJOVILOVICH A., LEITENMAIER B., MEYER-KLAUCKE W. et al. (2009): Complexation and Toxicity of Copper in Higher Plants. II. Different Mechanisms for Copper versus Cadmium Detoxification in the Copper-Sensitive Cadmium/Zinc Hyperaccumulator Thlaspi caerulescens. Plant Physiology. Volume 151, Pages 715-731. DOI: 10.1104/pp.109.144675
  • KÜPPER H., GÖTZ B., MIJOVILOVICH A. et al. (2009): Complexation and toxicity of copper in higher plants. I. Characterization of copper accumulation, speciation, and toxicity in Crassula helmsii as a new copper accumulator. Plant Physiology. Volume 151, Pages 702-14. DOI: 10.1104/pp.109.139717
  • KÜPPER H., ANDRESEN E., WIEGERT S. et al. (2009): Reversible coupling of individual phycobiliprotein isoforms during state transitions in the cyanobacterium Trichodesmium analysed by single-cell fluorescence kinetic measurements. Biochim Biophys Acta. Volume 1787, Pages 155-67. DOI: 10.1016/j.bbabio.2009.01.001
  • ROCCHETTA I. AND KÜPPER H. (2009): Chromium- and copper-induced inhibition of photosynthesis in Euglena gracilis analysed on the single-cell level by fluorescence kinetic microscopy. New Phytologist. Volume 182, Pages 405-420. DOI: 10.1111/j.1469-8137.2009.02768.x
  • KÜPPER H. ŠETLÍK I., SEIBERT S. et al. (2008): Iron limitation in the marine cyanobacterium Trichodesmium reveals new insights into regulation of photosynthesis and nitrogen fixation. New Phytologist. Volume 179, Pages 784-798. DOI: 10.1111/j.1469-8137.2008.02497.x
  • KÜPPER H., PARAMESWARAN A., LEITENMAIER B. et al. (2007): Cadmium-induced inhibition of photosynthesis and long-term acclimation to cadmium stress in the hyperaccumulator Thlaspi caerulescens. New Phytologist. Volume 175, Pages 655-674. DOI: 10.1111/j.1469-8137.2007.02139.x
  • VÁCHA F., SARAFIS V., BENEDIKTYOVÁ Z. et al. (2007): Identification of Photosystem I and Photosystem II enriched regions of thylakoid membrane by optical microimaging of cryo-fluorescence emission spectra and of variable fluorescence. Micron. Volume 38, Pages 170-5. DOI:10.1016/j.micron.2006.07.013
  • GACHON C. M. M., KÜPPER H., KÜPPER F. C. et al. (2006): Single-cell chlorophyll fluorescence kinetic microscopy of Pylaiella littoralis (Phaeophyceae) infected by Chytridium polysiphoniae (Chytridiomycota). European Journal of Phycology. Volume 41, Pages 395-403. DOI: 10.1080/09670260600960918
  • ŠETLIKOVA E., ŠETLÍK I., KÜPPER H. et al. (2005): Photosynth. Res. 84: 113-120. The photosynthesis of individual algal cells during the cell cycle of Scenedesmus quadricauda studied by chlorophyll fluorescence kinetic microscopy. Photosynthesis Research. Volume 84, Pages 113-20. DOI: 10.1007/s11120-005-0479-6
  • KÜPPER H., FERIMAZOVA N., ŠETLÍK I. et al. (2004): Traffic Lights in Trichodesmium. Regulation of Photosynthesis for Nitrogen Fixation Studied by Chlorophyll Fluorescence Kinetic Microscopy. Plant Physiology. Volume 135, Pages 2120-2133. DOI:10.1104/pp.104.045963
  • FERIMAZOVA N., KÜPPER H., NEDBAL L. et al. (2002): New Insights into Photosynthetic Oscillations Revealed by Two-dimensional Microscopic Measurements of Chlorophyll Fluorescence Kinetics in Intact Leaves and Isolated Protoplasts. Photochem. Photobiol. Volume 76, Pages 501-508. DOI: 10.1562/0031-8655(2002)0760501NIIPOR2.0.CO2
  • BERMAN-FRANK I., LUNDGREN P., BU CHEN Y., ET AL. (2001): Segregation of Nitrogen Fixation and Oxygenic Photosynthesis in the Marine Cyanobacterium Trichodesmium. Science. Volume 294, Pages 1534-1537. DOI: 10.1126/science.1064082
  • KÜPPER H., ŠETLÍK I., TRTÍLEK M. et al. (2000): A microscope for two-dimensional measurements of in vivo chlorophyll fluorescence kinetics using pulsed measuring radiation, continuous actinic radiation, and saturating flashes. Photosynthetica. Volume 38, Pages 553-570. DOI: 10.1023/A:1012461407557


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  • Fluorescence Kinetic Microscope FC 2000-Z
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Optional Features and Accessories

QA-Reoxidation Option
High-end option that allows to determine the kinetics of QA-reoxidation.



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