ProVIS - Centre for chemical microscopy

The aim of ProVIS is to visualise and quantify microbially mediated biochemical processes in natural and polluted environments using innovative high-resolution chemical and surface imaging techniques employing optical, electron and ion probes. Information on elemental and isotopic composition as well as molecular structural units of small objects can be obtained using the following techniques: nano-scale secondary ion mass spectrometry (nanoSIMS), time of flight secondary ion mass spectrometry (ToF-SIMS), helium ion microscopy (HIM), MALDI imaging mass spectrometry, scanning electron microscopy (SEM), atomic force microscopy (AFM), confocal RAMAN micro-spectroscopy, laser ablation ICP-MS with isotope resolution  as well as laser micro-dissection (LMD) combined with fluorescence microscopy. The chemical microscopic approach is complemented by a high resolution mass spectrometry laboratory for metabolomics and proteomics comprising Fourier transform ion cyclotron resonance mass spectrometer (FT-ICR-MS) and an Orbitrap mass spectrometer.

Using these techniques in combination with stable isotope tracer experiments and fluorescence in situ hybridisation (FISH) a direct correlation of the phylogenetic/morphological identity of micro-organisms with their metabolic activity can be obtained. The final aim is to determine the contribution of specific populations to a certain biochemical process, e.g. metal and element cycling, and therefore the micro-organism's role in ecosystem functioning.

Current topics:

• Microbe-metal/mineral interactions e.g. bio-films on metal surfaces, intracellular and extracellular sequestration of metals on terrestrial pristine and polluted environments;
• Carbon, nitrogen and sulphur fluxes in microbial communities;
• Microbial ecology of contaminant degradation / transformation of organic chemicals in microbial communities;
• Tracer experiments in oxic/anoxic conditions, aerobic/anaerobic cultivation methods;

Development of novel methods using isotope-containing fluorescent nano-markers for cell identification, imaging of molecules (proteins, mRNA) and activity tracing.

We have established a work-flow from sample preparation to chemical microscopy and high-resolution mass spectrometric analysis. Our work-flow allows for a flexible sample preparation using a combination of various techniques, depending on the particular target. Cryo-preparation is possible for a number of applications. A broad variety of techniques for molecular work, biological sample preparation and surface treatment has been established.  The preparation of samples for high-vacuum environment, as needed for electron or ion microscopy, will involve high-pressure freezing, freeze substitution, cryo-ultramicrotomy, cryo-sublimation drying, cryo-fracturing, cryo-etching, cryo-coating, and critical-point drying. Magnetron sputter coating and electron beam evaporation will be used for conductive layer deposition needed for electron microscopy on insulating samples.

Methods and instruments:

Sample preparation:

• high pressure freezing
• freeze substitution
• cryo-sublimation/etching
• cryo-fracturing
• ultramicrotomy in cryogenic environment
• critical point drying
• thin layer deposition by sputter coating and electron beam evaporation
• A well-equipped molecular biological laboratory allows application of molecular biology techniques including PCR, clone libraries, DNA sequencing, and DNA/RNA-stable isotope probing for sample preparation and analysis.

Microscopy:

• Epifluorescence-microscope, stereo-microscope with epifluorescence option for visual inspection and sample preparation.
• Optical profilometer (Sensofar) with objective AFM option (NanoSurf) enables high resolution monitoring to the surface topography of objects.
• Laser micro-dissection system (LMD, Palm IV system, Zeiss) combined with optical tweezers and epifluorescence microscope. The LMD in combination with laser tweezers allows for cell marking, cell separation, isolation and sample preparation in combination with epifluorescence microscopy for example for FISH.
• Confocal Raman microscope combined with atomic force microscopy (AFM) option. The combination of confocal Raman with Atomic Force Microscopy (ALPHA 300 RA+ instrument, WITec) will enable chemical imaging along with exploration of sample surface topography and physical properties. 
• Scanning electron microscope (SEM, Merlin VP Compact, Zeiss) equipped with cryo-sample transfer system and cryo-sample holder allows element mapping with energy dispersive X-ray analysis (EDX), transmission electron detection for organic thin section, material-contrast imaging with secondary electron detection as well as with energy-filtered back-scattered electron detection.

High resolution surface imaging and treatment by:

• Helium ion microscope (HIM, ORION NanoFab, Carl Zeiss) will facilitate the high-resolution imaging of virus, microbial cells or nano objects. Its Atomic Level Ion Source (ALIS) delivers a highly focussed He⁺-beam or Ne⁺-beam with resolutions of 0.35 nm and 1.9 nm, respectively. The He⁺-ion-beam is used for imaging with high surface sensitivity whereas the Ne⁺-ion beam shows an enhanced sputtering rate as needed for lithography, sample preparation and HIM-SIMS technique for elemental analysis.

Visualisation of elemental/isotopic ratio distribution or molecular ion mapping by secondary ion microscopy comprise:

• Nano-scale secondary ion mass spectrometry (NanoSIMS 50L, Cameca) allows for mapping of elements and isotopes at a lateral resolution down to 50nm.
• Detailed information on molecular composition of organic substances will be gained with a  time of flight secondary ion mass spectrometer (ToF-SIMS-5, IonTOF) comprising cluster ion sources (Bi, Ar) and focussed Ga ion beam source (FIB) for high-resolution depth profiling and molecular ion mass spectrometry.
• Maldi imaging of biological samples using  Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS, 12 tesla-Solarix 120 Hybrid,  Bruker)  or  TOF Mass spectrometers (Autoflex Speed LRF, Bruker).
• Laser Ablation System (LA, Analyte G2, S-Prep) equipped with 193 nm ArF-Excimerlaser connected to an ICP-MS System (Spectro MS, Ametek) allows imaging and element mapping. When the LA system is connecting to a multi-collector ICP-MS (Neptune, ThermoScientific) isotope analysis of elements with high mass resolution is possible.

Chemical methods for proteomics, metabolomics and isotope analysis

We have established a unit for high-end proteomics, metabolomics and isotope analyses for analysing bio-geo-chemical and biological processes in biological experiments and at field scale. For physiological and metabolic analysis we have established the work-flow for high resolution metabolomics and proteomics. High resolution isotope analysis is used for characterising biogeochemical processes in the environment and comprised:

• A 12 tesla-Solarix 120 Hybrid FTICT MS system (Bruker) is equipped with different periphery instrumentation such as direct infusion, UHPLC and Nano-HPLC for analysis of a wide range of organic substances. The mass resolution up to 5 Mio. (MRP) allows for the analysis of atomic composition of each ion.
• An high resolution Orbitrap MS system (Fusion, Thermo Scientific) is equipped with different periphery instrumentation such direct infusion, UHPLC and Nano-HPLC for analysis of a wide range of organic substances. The mass resolution up to 340T (MRP) allows for the analysis of a wide range chemicals. The Orbitrap is mainly used for proteomics and for example stable isotope probing of proteins and lipids. 

The multi collector ICPMS is used for analysing the concentration and isotope composition of elements in environmental samples, geological material, and aqueous solution and can be connected additionally to HPLC, GC and LAS systems.

The high-end analytics for chemical microscopy and chemical analysis in one platform (ProVIS) is unique in Europe. The capabilities for correlative microscopy along with high resolution MS and isotope analysis provide complementary capabilities for analysis biogeochemical processes from a molecular level to the field scale.

SIMS techniques and a number of our equipment for chemical microscopy is fully remote controlled and can be used with clients via internet directly from their office. After introduction the clients can analyse their samples with assistance of our staff via internet from any place in the world. For collaborators we provide excess to our computer cluster via internet for correlative microscopy experiments.

The mission of ProVIS is to accelerate common research in Saxony, Germany and the European Union on a non-commercial basis. Thus ProVIS is open for national and international research collaborations. Collaborative research projects are invited and preference will be given to quality of projects and feasibility of the approach. ProVIS reserves space and capacity for visiting scientist. Visiting scientists are invited to send a request with a small project outline to contact person of ProVIS. Preference is given to joint projects.

The UFZ is accepting proposal for collaborative research. Proposals for research projects can be sent to Dr. Hans H. Richnow or Dr. Niculina  Musat. The proposal for 2015 will be evaluated by a scientific committee in the UFZ.

Basic running cost of ProVIS is covered by the UFZ. Preference for use will be given to collaborative research. Contribution of running costs is expected for collaborative research projects. The UFZ is a non-commercial research institution and calculation of costs for use will be made on real expenses related to the project. Cost for analytical service will be calculated on expenses related to the project. Please contact the staff of ProVIS for further information.

hans.richnow@ufz.de
niculina.musat@ufz.de
gregory.stryhanyuk@ufz.de