FKM multispectral fluorescence dynamic microscopy imaging system

The FKM (Fluorescence Kinetic Microscope) multispectral fluorescence dynamic microscopy imaging system is currently the most powerful and comprehensive instrument for studying plant microfluorescence. It is a customized microscopy imaging system based on FluorCam chlorophyll fluorescence imaging technology. It consists of an enhanced microscope with expandable components, a high-resolution CCD camera, an excitation light source group, a spectrometer, a temperature control module, corresponding control units, and dedicated workstations and analysis software. It can not only perform Fv/Fm, Kautsky induction effect, fluorescence quenching, OJIP rapid fluorescence response curve, QA re oxidation and other chlorophyll fluorescence and MCF multispectral fluorescence imaging analysis on microalgae, single cells, single chloroplasts and even granule matrix thylakoid fragments; It is also possible to measure any fluorescence excitation and fluorescence release band by exciting the light source group, thereby performing imaging analysis of fluorescent proteins such as GFP, DAPI, DiBAC4, SYTOX, CTC, fluorescent dyes, as well as algal specific fluorescent pigments such as phycocyanin, phycoerythrin, and phycobilin; It is also possible to use a spectrometer for spectral analysis of various fluorescent substances, distinguish between different chromophores (such as PSI and PSII, and various light harvesting pigment complexes), and conduct in-depth analysis.

The FKM multispectral fluorescence dynamic microscopy imaging system has truly made fluorescence imaging technology a probe for studying the mechanism of photosynthesis, enabling researchers to deeply understand the process of photosynthesis and the various changes that occur during it at the cellular and subcellular levels in algae and higher plants. It provides the most powerful tool for directly studying the working mechanism of the photosynthetic system in chloroplasts. FKM, as a dual tool for the microscopic study of algal/plant phenotypes and genotypes, has been widely recognized by the academic community and has achieved a large number of scientific research results.

Functional Features

Built in all programs for current chlorophyll fluorescence research, such as Fv/Fm, Kautsky induction effect, fluorescence quenching, OJIP rapid fluorescence response curve, QA re oxidation, etc., can obtain more than 70 parameters.

Equipped with 10x, 20x, 40x, 63x, and 100x specialized bioluminescence objectives, chloroplasts and their emitted fluorescence can be clearly observed.

The excitation light source group includes infrared light, red light, blue light, green light, white light, ultraviolet light, and far red light. Through the three colors of red, blue, and green, any color in the visible spectrum can be extracted, and any pigment molecule or chromophore in plants/algae can be studied.

• Can perform imaging analysis of fluorescent proteins and dyes such as GFP, DAPI, DiBAC4, SYTOX, CTC, etc

High resolution spectrometers can deeply analyze various fluorescent spectra.

The temperature control system can ensure that the experimental samples are measured under the same temperature conditions, improve experimental accuracy, and can also be used for high/low temperature stress research.

application area

Microstructure and plant photosynthetic physiology research of single cells, single chloroplasts, and granule matrix thylakoid fragments of microalgae, macroalgae/higher plants

• Algae/Plant Stress Research

Research on biotic and abiotic stress

Research on the stress resistance and susceptibility of algae/plants

Mutant screening and photosynthetic mechanism research

• Assessment of Algae Growth and Yield

The relationship between algal specific pigments and photosynthesis

• Algae/Plant Microbial Interaction Study

Research on the Interaction between Algae/Plants and Protozoa

Genetic Engineering and Molecular Biology Research

Measure the sample

• Plant live slices

• Plant epidermis

• Plant cells

Various single-cell and multicellular microalgae such as green algae and blue-green algae

Chloroplast extract

• thylakoid extract

Protozoa containing chloroplasts

Working principle

During the FKM analysis process, dynamic fluorescence of various chromophores in plant samples is excited by a series of filters and spectroscopes embedded in a 6-position filtering wheel and an excitation light source group connected to the microscope. The fluorescence excited by the sample is magnified under a microscope for fluorescence spectroscopy analysis and fluorescence kinetic imaging analysis. The SM 9000 spectrometer is connected to a microscope through optical fibers for excitation fluorescence spectroscopy analysis. The high-resolution CCD camera installed on top of the microscope is used for fluorescence dynamics imaging analysis. The entire work process is automatically carried out through workstations and control units according to pre-set programs. During the measurement process, the temperature of experimental samples such as algae and plant cells can be regulated through a temperature control module. Peristaltic pumps can achieve continuous measurement of algae cultivation.

Instrument composition

1. Enhanced microscope

2. High resolution CCD camera

3. Excitation light source group

4. SM 9000 spectrometer

5. Main control unit

6. Workstation and software

7. Control unit of temperature control module

8. 6-digit filtering wheel

Technical Parameter

• Measurement parameters

Fo, Fo’, Fs, Fm, Fm’, Fp, FtDn, FtLn, Fv, Fv'/ Fm', Fv/ Fm ,Fv',Ft,ΦPSII, NPQ_Dn, NPQ_Ln, Qp_Dn, Qp_Ln, qN, qP，QY, QY_Ln, Rfd, ETR Waiting for over 50 chlorophyll fluorescence parameters, each parameter can display a 2D fluorescence color image

OJIP rapid fluorescence curve: Determination and analysis of OJIP curve and more than twenty related parameters, including: Fo, Fj, Fi, P or Fm, Vj, Vi, Mo, Area, Fix Area, Sm, Ss, N (QA reduction turnover quantity), Phi ¬¬¬ P_o, Psiuo, Phi-Eo, Phi-Do, Phiepav, ABS/RC (absorbed light quantum flux per unit reaction center), TRo/RC (initial captured light quantum flux per unit reaction center), ETO/RC (initial electron transfer light quantum flux per unit reaction center), DIo/RC (energy per unit reaction center) Loss, ABS/CS (absorption photon flux per unit sample cross-section), TRo/CSo, RC/CSx (reaction center density), PIABS (performance index or survival index based on absorption photon flux), PIcs (performance index or survival index based on cross-section), etc. (optional)

Imaging analysis of fluorescent proteins and dyes such as GFP, DAPI, DiBAC4, SYTOX, CTC, etc. (optional)

QA re oxidation kinetics curve (optional)

 Spectrum fluorescence spectrum (optional)

• Equipped with a complete automatic measurement protocol, allowing for free editing of the automatic measurement program

Fv/Fm: Measurement parameters include Fo, Fm, Fv, QY, etc

Kautsky induced effect: Fluorescence parameters such as Fo, Fp, Fv, Ft_Lss, QY, Rfd, etc

Fluorescence quenching analysis: Fo, Fm, Fp, Fs, Fv, QY, Φ II, NPQ, Qp, Rfd, qL and more than 50 parameters, 2 sets of standard programs

Light response curve LC: Fluorescence parameters such as Fo, Fm, QY, QYLn, ETR, etc

Dyes&FPs steady-state fluorescence imaging measurement

OJIP rapid fluorescence kinetics analysis: 26 parameters including Mo (initial slope of OJIP curve), OJIP fixed area, Sm (measurement of energy required to close all photoreaction centers), QY, PI, etc. (optional)

QA re oxidation kinetics (optional)

Spectrum fluorescence spectroscopy analysis (optional)

Fluorescent excitation light source: infrared light, red light, orange light, blue light, green light, white light, ultraviolet light, etc. can be selected, and customized light source groups can be made according to customer requirements

• Transmitting light source (optional): white light, far red light

High resolution TOMI-2 CCD sensor:

Scan CCD line by line

Maximum image resolution: 1360 × 1024 pixels

Time resolution: up to 20 frames per second at the highest image resolution

A/D conversion resolution: 16 bits (65536 grayscale levels)

Pixel size: 6.45 µ m × 6.45 µ m

Operation mode: 1) Dynamic video mode, used for measuring chlorophyll fluorescence parameters; 2) Snapshot mode, used for measuring fluorescent proteins such as GFP and fluorescent dyes

Communication mode: Gigabit Ethernet

• Microscope: Axio Imager M2， Optional Axio Scope A1 Lite or Axio Imager Z2 Premium

Objective lens turntable: Research grade 7-hole automatic objective lens turntable

Transmitting light shutter

Concentrator Achr Apl 0.9 H

6-position reflector dial

Binocular tube (100:0/30:70/0:100)

Mechanical stage: 75 × 50mm, hard anodized surface

Sample rack: 76 × 26mm

Objective lenses: 10x, 20x, 40x, 63x, and 100x specialized bioluminescence objectives (optional)

6-digit filtering wheel: chlorophyll fluorescence, GFP/SYTOX, DAPI/CTC, etc

SM9000 spectrometer

Incident slit: 70 µ m × 1400 µ m

 Grating: flat field correction

Spectral range: 200-980nm

Absolute precision of wavelength:< 0.5nm

Reproducibility:< 0.1nm

Temperature drift:< 0.01nm/K

Temperature regulation module: Temperature regulation range 5 ℃ -70 ℃, accuracy 0.1 ℃

Peristaltic pump (optional): flow rate of 10-5600 µ l/min, used for continuous cultivation measurement of algae

FluorCam chlorophyll fluorescence imaging analysis software features: with menus for Live (live testing), Protocols (experimental program selection customization), Pre processing (imaging preprocessing), Result (imaging analysis results), and other functions

• Customized experimental protocol for customers: Time (such as measurement light duration, photochemical light duration, measurement time, etc.), light intensity (such as photochemical light intensity of different qualities, saturation flash intensity, modulated measurement light, etc.) can be set, with dedicated experimental programming language and scripts. Users can also freely create new experimental programs using the wizard program template in the Protocol menu

• Automatic measurement and analysis function: An experimental program (Protocol) can be set to automatically perform unmanned cyclic imaging measurements, with customizable repetition times and interval times. Imaging measurement data is automatically stored in the computer according to the time and date (with a timestamp)

• Snapshot mode: Through snapshot imaging mode, the light intensity, shutter time, and sensitivity can be freely adjusted to obtain clear and prominent steady-state and instantaneous fluorescence images of plant samples

• Imaging preprocessing: The program software can automatically identify multiple plant samples or regions, or manually select regions of interest (ROI). The shape of manual selection can be square, circular, any polygon, or sector. The software can automatically measure and analyze the fluorescence kinetics curves and corresponding parameters of each sample and selected area, with no limit on the number of samples or areas (>1000)

• Data analysis mode: It has "signal calculation and re averaging" mode (arithmetic mean) and "signal average and re calculation" mode. In high signal-to-noise ratio situations, the "signal calculation and re averaging" mode is selected, and in low signal-to-noise ratio situations, the "signal average and re calculation" mode is selected to filter out errors caused by noise

• Output results: High time resolution fluorescence dynamic images, fluorescence dynamic change videos, fluorescence parameter Excel files, histograms, imaging images with different parameters, fluorescence parameter lists for different ROIs, etc

Chlorophyll fluorescence and spectral analysis results

Typical applications:

Origin: Czech Republic

reference:

1.Küpper H, et al. 2019. Analysis of OJIP Chlorophyll Fluorescence Kinetics and QA Reoxidation Kinetics by Direct Fast Imaging. Plant Physiology 179: 369-381

2.Konert G, et al. 2019. Protein arrangement factor: a new photosynthetic parameter characterizing the organization of thylakoid membrane proteins. Physiologia Plantarum 166: 264-277.

3.Exposito-Rodriguez M, et al. 2017. Photosynthesis-dependent H2O2 transfer from chloroplasts to nuclei provides a high-light signalling mechanism. Nature Communications, 8: 49

4.Higo S, et al. 2017. Application of a pulse-amplitude-modulation (PAM) fluorometer reveals its usefulness and robustness in the prediction of Karenia mikimotoi blooms: A case study in Sasebo Bay, Nagasaki, Japan. Harmful Algae, 61:63-70

5.Jacobs M, et al. 2016. Photonic multilayer structure of Begonia chloroplasts enhances photosynthetic efficiency. Nature Plants, doi:10.1038/nplants.2016.162

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8.Fujise L, et al. 2014. Moderate Thermal Stress Causes Active and Immediate Expulsion of Photosynthetically Damaged Zooxanthellae (Symbiodinium) from Corals. PLOS ONE, DOI:10.1371/journal.pone.0114321

9.Gorecka M, et al. 2014. Abscisic acid signalling determines susceptibility of bundle sheath cells to photoinhibition in high light-exposed Arabidopsis leaves. Philosophical Transactions of the Royal Society B, 369(1640), DOI: 10.1098/rstb.2013.0234

10.Mishra S, et al. 2014. A different sequence of events than previously reported leads to arsenic-induced damage in Ceratophyllum demersum L. Metallomics, 6: 444-454

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