Aperio ScanScope AT (Leica)

The scanner performs high-throughput scanning of microscopical slides. It is based on line scanning and equipped with 20x/0.75 NA Plan Apo (40x scanning with 2x optical magnification changer). It provides a resolution of 50,000 pixels per inch (0.5 μm per pixel with 20x objective) with 0.25 μm per pixel when scanning at 40x with 2x magnification changer. The automated loading enables consecutive scanning of 200 slides.

 

Live Cell Observer (Zeiss)

The live Cell Observer system is based on an Axiovert 200M inverted fluorescence microscope equipped with standard filter sets for all common fluorescent dyes. The live Cell Observer system includes a heated microscope table, temperature controller, CO2 controller and perfusion system which guarantee constant culture conditions for the entire duration of the experiment.

The system is equipped with a HBO/XBO light source, the high range monochrome camera AxioCam MR and with the following lenses: A-Plan 5x/12 Ph0; A-Plan 10x/0.25 Ph1; LD Plan-Neofluar 20x/0.4 corr. Ph2; LD Plan-Neofluar 40x/0.6 corr. Ph2; Plan Apochromat 63x/1.4 Oil with DIC capability; Plan Apochromat 100x/1.4 Oil with DIC capability.

AxioVision 4.3 software provides 3D Deconvolution and 4D (Wavelength Dimension; the electively marking of cell or tissue components with different fluorescent dyes and storage in 8 independent channels of the image), 5D (Time Dimension; recording of cells and tissues during a predefined period of time) and 6D (Local Dimension; observing various positions on the culture plate) imaging. Data are routinely displayed as video.

 

Cell IQ V2 MLF Cell Imaging and Analysis System (Chipman Technologies)

The system allows documentation and label-free semi-quantification of cellular processes using Machine Vision Technology for the automatic identification, analysis and quantification of morphological features. The system can work with multiple plate formats; imaging using dishes, slides and flasks is also possible.

The CelLite™ Multi-Label Fluorescence capability enabled fluorescence recording for green fluorescence plus two additional colors. The software includes Imagen™ System control and imaging viewing software and Analyser™ software for the automatic identification and quantification of cell types and growth phases. This software also allows image processing, automatic stitching and easy conversion of single image files into high definition videos.

 

TissueFAXS System (TissueGnostics)

The workstation consists of on upright fully motorized AXIOIMAGER.Z1 (Zeiss), Maerzhaeuser Scanning Stage for up to 8 slides, and colour and monochrome camera. Slides can be scanned with an auto focus up to 63x oil magnification and DAPI, FITC, Rhodamine and Cy5 fluorescence filter cubes.

The Tissuefax system includes two software packages, TissueQuest and HistoQuest, for the analysis and scoring of chromogenic and fluorescent staining. The system allows fully automated slide scanning, preview scan with zoom function, multi-channel acquisition, image stitching, analysis of staining intensities, scattergrams and DotPlot operations, and backward gating and forward connection from DotPlot and images.

 

Motorized microscope with high speed camera (Zeiss/Visitron)

The system consists of an inverted Axiovert 200M (Zeiss) with xenon lamp and high-speed polychromator system (VisiChrome) for wavelengths between 300 - 650 nm. The filter sets allow imaging of a wide variety of fluorescent indicators such as Alexa-Fluors, Calcium-Green, Cy2, Cy3, Cy5, DAPI, Fluorescein (FITC), FM1-43, Fura-2, GFP, GFP, Hoechst 33258, Rhodamine, Texas Red and TRITC.

Image acquisition is controlled by VisiView software which also allows cell counting and ratiometric measurements.

The microscope is equipped with the following objectives: A-Plan 10x/0.25 Ph1; A-Plan 20x/0.45 Ph2; LD Plan-Neofluar 40x/0.6 Ph2; EC-Plan Neofluar 40x/1.3 Oil and Plan Apochromat 63x/1.4 Oil with DIC capability. The Axiovert 200M is also equipped with a sensitive monochrome camera (Roper CoolSnap HQ). A stage heater and Perfusion System ISMATEC IPC 4 Tubing pump is available for imaging living cells at a set temperature.

 

LSM510 META (Zeiss)

The LSM 510 META is mounted on an inverted Axiovert M200 microscope and contains an optoelectronic Z drive, two single channel detectors, one META detector for 32 channel collection and spectral analysis, and seven laser lines. Integrated lasers are UV 405nm, multiline Argon 458/477/488/514nm, Helium-Neon 543nm as well as Argon 514 and Helium-Neon 633nm. Light entering the channels are detected with high-sensitivity photo-multiplier. The META detection upgrade uses emission fingerprinting to generate a spectral fingerprint of each fluorescent molecule. Imaging acquisition and analysis is performed with ZEN software with the possibility of z-scan, optical sections, determination of co-localization coefficient, etc.

Available objectives are: EC Plan-Neo 10x/0.3 NA; Plan-Neo 20x/0.5; EC Plan-Neo 40x/1.3 Oil with DIC capability; Plan-Neo 63x/1.4 Oil with DIC capability; alpha Plan-Fluar 100x/1.45 Oil.

 

Nikon A1R Ultra-Fast Spectral Scanning Confocal Microscope, best for imaging live/fixed thick samples (Nikon)

The system consists of an inverted Ti Nikon microscope, Nikon XYZ Motorized stage, Nikon A1- SHR High Speed Scan Head & Controller (Resonant Scanner). Integrated lasers are Violet diode 405nm, multiline Argon 457-514nm, DPSS laser 561nm, Diode Laser system642nm. For signal detection 4 Standard Photomultipliers (PMT) detectors, Standard Detector and Nikon A1 Spectral Detector (32 PMT array; wavelength range: 400-770 nm) are available. The system enables acquisition of images at a maximum scanning speed of 230 frames/second and is operated by Nikon NIS Elements Acquisition Software.

The microscope is equipped with the following objectives: CFI Plan Apochromat Lambda 10x/, CFI Plan Apochromat Lambda 20x, CFI Plan Apochromat Lambda 40x/0.95, and CFI Plan Apochromat Lambda 60x/1.4 oil immersion, CFI Plan Apochromat VC 100x/1.45 oil immersion and CFI75 Apochromat 25XW/1.1.

 

Barrier Models

Respiratory barrier

The assay uses respiratory cells (Calu-3, A549) on transwell membranes cultured in an air-liquid interface. Cells can be exposed to liquids, to nebulized aerosols in the VITROCELL® system and to manually generated aerosols using MicroSprayerÒ IA-1C Aerosolizer and DP-4 Dry Powder Insufflator™. The models are used for Papp testing, uptake and transport rates, and cytotoxicity studies.

Blood-brain barrier

In addition to the existing static model based on transwell membranes a dynamic system using hollow fibre CELLMAX DUO® bioreactor with dynamic TEER measurement is being developed. The system uses hcMED/D3 microvascular epithelial cells in co-culture with primary astrocytes.

 

     

 

 

 

Cytotoxicity: Acute and Chronic and Mode of Action Studies

Interference with cell function can be identified by cytotoxicity screening assays, such as MTS, MTT, WST-1, ATP-content, sulforhodamine B, neutral red, as well as by microscopy-based techniques (cell monitoring).

For testing of medical products detection of endotoxin and testing of cytotoxicity following ISO 10993-5 guidelines is offered.

More detailed information on the mode of interference with cell function is studied by assays for necrosis, apoptosis, and proliferation. These assays include LDH, AK, Calcein AM/PI, caspase 3/7 activation, TUNEL, M30, cleaved caspase 3, 3H-thymidine, BrdU, and cell cycle analysis.

Alterations of cell metabolism can also be due to interference with organelle function, which can be identified by Mitotracker, TMRM, JC-1, LysoTracker, LysoSensor, acridine orange, Lyso-ID, lysosomal sulfatase, and cathepsin B staining.

Oxidative stress as frequent cause for cell dysfunction is identified by using dihydroethidium, dihydrochlorofluorescein acetate, heme oxigenase-1 staining and depletion of GSH as indicators.

 

Effects of prolonged exposure over 14-28d can be studied by culture on microcarriers in the benchtop bioreactor BioLevitator™ 3D cell culture system. This system is also suitable to propagate cells to high densities.

Membrane-based transwell systems are available for long-term observation of respiratory cells in air-liquid interface culture.

 

     

 

 

Hemocompatibility and Immunotoxicity Testing

Adverse effects on blood, hemolysis and changes in hemostasis, can be identified using Hb-release and interference with plasmatic clotting and platelet activation is identified by F1.2, TAT, D-dimer, CD62P/CD42b, b-thromboglobulin detection and platelet aggregation.

For identification of interaction with the cellular (monocytes, macrophages, neutrophilic granulocytes) and the acellular unspecific immune system complement 3a and 5a, NO-detection, oxidative burst, E. coli K12-bioparticle phagocytosis, phagoburst, CD11b/CD15 and chemotaxis is performed. Mitogen-activated lymphocyte proliferation is used to assess lymphocyte function and cytokine release, either in conventional ELISA or as multiplex assays to study inflammation.

 

 

     

 

 

 

 

 

Histology

 For automated embedding, sectioning and immunhistochemical staining the following instruments are available:

  • Tissue-Tek® VIP tissue processor (Miles Scientific)
  • Tissue-Tek® tec 5 embedding console system for specimen orientation and paraffin embedding (Miles Scientific/Sakura) and cooling plate COP 30 (Medite)

Microtomes:

  • Sliding microtome HM 430 (MICROM) equipped with stretching bath (Medax Nagel)
  • Motorized rotary microtome HM 360 (MICROM) equipped with stretching bath (Medax Nagel)
  • Cryostat microtome HM 560M (MICROM)

 

Tissue-Tek® DRS 200 automatic slide stainer (Sakura)

 

Conventional microscopes:

  • Advanced routine stereo zoom microscope SZX12 (Olympus): maximum magnification (144x), maximum working distance (171 mm), SZX10 (Olympus): maximum magnification (63x), maximum working distance (81 mm) equipped with DP12 Camera
  • BX41 standard upright laboratory microscope (Olympus): brightfield
  • BX51 upright microscopes (Olympus): especially suited for DIC microscopy brightfield; brightfield and fluorescence
  • IX51 inverted microscopes (Olympus): brightfield and fluorescence, equipped with CellD Software. Cameras: Color View III and FView II

 

Labelling

Assistance is provided for:

  • Identification and quantification of cellular processes (e.g. proliferation, discrimination live/dead; organelles) by vital labelling of cells and microscopic and photometric evaluation
  • fixation and embedding protocols
  • immunohistochemistry with various detection methods (HRP-, AP-, fluorescence-based)

 

   

Prof. Dr. Eleonore Fröhlich

Core Facility Imaging Medical University of Graz

Stiftingtalstr. 24

8010 Graz

Tel: (+43) 31638573011

Fax: (+43) 31638573009

Email: Eleonore.froehlich@medunigraz.at

   

Claudia Meindl

Tel: (+43) 0316 385 73518

Email: claudia.meindl@medunigraz.at

   

Markus Absenger Novak

Tel: (+43) 0316 385 73503

Email: markus.absenger@medunigraz.at

   

Kristin Öhlinger

Tel: (+43) 0316 385 73522

Email: kristin.oehlinger@medunigraz.at

 

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