Fluorescence Kinetic Microscope FC 2000-Z
Fluorescence Kinetic Microscope (FKM) is designed to be the most versatile tool for lab-based research. In addition to the capabilities of the standard Micro-FluorCam, the FKM allows 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 system consists of an excitation module, a detection module and the microscope stand. All parts and functions can be controlled by the FluorCam software depending on individual configuration.
Fluorescence Kinetic Microscope: Scheme of the Device
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 spectrally resolved measurements are done via the Spectrometer SM 9000 synchronised 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.
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.
The FKM can perform the following tasks:
- 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.
References:
- Andresen E. et al. (2009): New Phytologist.
- Mijovilovich A. et al. (2009): Plant Physiol. DOI: 10.1104/pp.109.144675.
- Kuepper H. et al. (2009): Plant Physiol. DOI:10.1104/pp.109.139717.
- Kuepper H. et al. (2009): BBA 1787: 155-167.
- Rocchetta I. and Kuepper H. (2009): New Phytologist 182: 405-420.
- Kuepper H. et al. (2008): New Phytologist 179: 784-798.
- Kuepper H. et al. (2007): New Phytologist 175: 655-674.
- Gachon C.M.M. et al. (2006): Eur. J. Phycol. 41: 395-403.
- Setlikova E. et al. (2005): Photosynth. Res. 84: 113-120.
- Kuepper H. et al. (2004): Plant Physiol. 135: 2120-2133.
- Ferimazova et al. (2002): Photochem. Photobiol. 76: 501-508.
- Berman-Frank I. et al. (2001): Science 294: 1534-1537.
- Kuepper H. et al. (2000): Photosynthetica 38: 553/570.
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Optional Features and Accessories:
QA-Reoxidation Option
High-end option that allows to determine the kinetics of QA-reoxidation.
