Our Technologies and Equipment
Our research goals rely on the visualization of physiological processes and structures of the living plant (in vivo imaging). For instance, central metabolic pathways and the accumulation/degradation of storage products (lipids, starch, proteins) need to be analyzed space-resolved within the relevant biological structure (seed, stem, etc.). Thus, our range of methods comprises especially procedures/tools which allow a spatially resolved analysis.
If you have any specific requests/questions, please contact: Ljudmilla Borisjuk (NMR/MRI) or Hardy Rolletschek (biochemical analytics and sensors).
- Magnetic Resonance Imaging (MRI)
- Orbitrap technology for metabolite profiling
- Spectroscopy (NIRS, FTIRS, UV/Vis) and chemical imaging
- Sensor technologies (sensor spots, microsensors, sensor foils)
- Time-Domain-Nuclear Magnetic Resonance (TD-NMR) with sample robot
- Analytics for photosynthesis
- Molecular analytics
- Laser microdissection
- Histological tools
Magnetic Resonance Imaging (MRI)
The Bruker Avance III HD 400 NMR-Spectrometer and cryogenically-cooled double-resonant 1H-13C-probehead is established in our group for advanced imaging applications on intact seeds and other plant organs. Various plant species and transgenic models can be safely investigated owing to the close vicinity of NMR-laboratory to the our analytic and growth facilities (climate chambers, greenhouses). Together with Penn State University (USA) and University of Würzburg (Germany) we are providing a powerful technical platform for high resolution functional imaging of plants.
Different methods for the acquisition of NMR images are established. For example, we apply MRI to obtain three-dimensional models of plant organs, internal structures and characterize metabolite distribution with high spatial resolution (Borisjuk et al., Plant Journal 2012; Munz et al., Biochimie 2016). Diligent adaption of NMR pulse sequences provides the means to track the flow of sugars within a living seed (Melkus et al., Plant Biotechnology 2011), to quantitatively map the distribution of storage lipids in seeds (Borisjuk et al., Progress in Lipid Research 2013) and to monitor the germination of seeds (Munz et al., New Phytologist 2017).
Orbitrap technology for metabolite profiling
Targeted and untargeted metabolite profiling technologies are used to detect and quantify metabolic intermediates of central metabolism. In the past, we primarily relied on triple quadrupol mass spectrometers which generally provide highest sensitivity and easy metabolite identification by recording specific fragmentations and ion traces. The pattern of ~ 100 intermediates (sugars, amino acids, nucleotides, cofactors) can be measured when combining MS instruments with both ion chromatography and HILIC mode of analyte separation. Based on external calibration routines we can determine real metabolite concentrations, necessary for quantitative biology approaches (Schwender et al., Plant Physiology 2015). When coupling capillary electrophoresis with mass spectrometry we can further improve sensitivity as needed for microsampling approaches.
Most recently, we established on Orbitrap mass spectrometer (Q Exactive), which enables sensitive and specific measurement of known and unanticipated metabolites in parallel. The currently established methods capitalize on the mass-resolving power of the Orbitrap technology. In combination with newest ion chromatography, we focus on water-soluble, polar species, which represent the majority of intermediates in central metabolism (glycolysis, TCA cycle, amino acid and sugar metabolism, cofactors, etc.). The number of detectable, chemically identified metabolites could already be tripled, and further improvements can be expected.
Time-Domain-Nuclear Magnetic Resonance (TD-NMR) with sample robot
The Bruker mq60 device (Bruker GmbH) is an instrument, which can be used for low-field TD-NMR experiments. The instrument has a magnetic field strength of 1.5 tesla and has traditionally allowed determining the lipid content of samples. We have achieved significant improvements in instrument performance by developing a novel, rapid, accurate procedure, designed to simultaneously quantify a number of basic seed traits without any seed destruction. Using this instrument, the procedure gives a high accuracy measurement of oil content (R2=0.98), carbohydrate content (R2=0.99), water content (R2=0.98), and both fresh and dry weight of seeds/grains (R2=0.99). The non-invasive method requires a minimum of ~20mg biomass per sample, and thus enables to screen individual, intact seeds. When combined with an automated sample delivery system (sample robot), a throughput of ~1,000 samples per day is achievable. For details, see Rolletschek et al., Plant Biotech J 13, 2015). We currently develop further routines/procedures to widen the analytical spectrum and applicability of TD-NMR for plant seeds of various species.
Needle-type microsensors are an elegant tool for measuring in small volumes of sample or for measuring oxygen concentration profiles across plant tissues. We routinely apply sensor probes to map oxygen distribution in seeds and other plant tissues. For overview on the use of oxygen microsensors in seed research, see Rolletschek et al. (Sensors 2009), and Borisjuk & Rolletschek (New Phytologist 2009). We further employ oxygen-sensitive planar sensor foils – a unique tool for the quantitative mapping of local respiratory activities (Tschiersch et al., New Phytologist 2012). In addition, we recently developed tools (VisiSens TD with sensor spots) to trace respiration at high sample throughput for various applications in seed biology (Keil et al., 2017).
Spectroscopy (NIRS, FTIRS, UV/Vis-spectrometer) and chemical imaging
The MultiPurposeAnalyzer (MPA Bruker) is an instrument allowing Fourier-transform near infrared (FT-NIR) spectroscopy of both liquid and solid samples. In our hands, we apply the MPA for routine, high-throughput analysis of sugars, starch, lipids and proteins in leaf, stem and seed tissues of plants. The HYPERION 3000 is a fully automated Fourier-transform infrared (FTIR) imaging microscope with modern focal plane array (FPA) detector technology. With the FPA detector up to 16,384 spectra can be measured simultaneously covering sample areas of up to 340 x 340 µm with a pixel resolution of 2.7 µm. Using HYPERION we analyze the distribution pattern of different compounds like lipid, protein and sugars in heterogeneous plant tissues. We further apply standardized assays using spectrophotometers (UVIKON) to profile enzymatic activities as well as various plant compounds.
Analytics for photosynthesis
The imaging-PAM fluorometer is a specialized device for the study of two-dimensional heterogeneities of photosynthetic activity in plant tissue. Using the Saturation Pulse method for the estimation of key chlorophyll fluorescence parameters it provides a (non-destructive) means of analyzing the photosynthetic performance of plants in a spatially resolved manner. It allows measuring the quantum yield of energy conversion at PSII reaction centers and thus the calculation of photosynthetic electron transport rates. The mapping of photosynthetic activity across developing seeds is possible with a spatial resolution of approx. 20 µm. For details, see Tschiersch et al., Biosystems 103 (2011). We further apply standard LICOR equipment (LI-6400XT) for analysis of gas exchange and fluorescence of (source) leaf.
Workstation for 3D-modelling
By using a high-end computer work station in combination with the AMIRA software package, we perform three dimensional (3D) modelling of seeds and other plant organs. Based on 3D-datasets, derived from NMR/MRI or x-ray computed tomography, we generate 3D-models, allowing to visualize structures or calculate volumes of (sub-)organs and their dynamic changes during growth and development.
Microscopy/ Histological tools
Our ZEISS Axioplan-2 Imaging fluorescence microscope allows the investigation of tissues in darkfield, phase contrast, and differential interference contrast. Furthermore, it is equipped with ApoTome, which allows creating optical sections of the specimen increasing axial resolution of fluorescence microscopy by structured illumination.
We use several histological techniques to visualize gene activity: GUS staining in order to localize and analyze β-glucuronidase reporter gene activity within tissues; in situ hybridization of DIG-labeled riboprobes to localize specific gene transcript within individual cells in semi-thin tissue sections; and finally immunohistochemistry in order to localize gene products in cells and tissues by specific antibodies detected either by: chromogenic reaction or fluorescence. Additionally, our group performs in situ Terminal deoxynucleotidyl transferase dUTP Nick End Labeling (TUNEL assay) for the identification and visualization of plant cells undergoing programmed cell death.
Our group is further equipped with Leica EG1150 embedding station incorporating two separate components, the Leica EG1150 C cold plate and the Leica EG1150 H heated paraffin dispensing module, to facilitate and accelerate the embedding process of the tissue.