HCEMM-SU In Vivo Imaging ACF
Location and Equipment
The IVI ACF is located in three sites, the headquarters being the Basic Medical Sciences Building at the Semmelweis University, 37-43 Tűzoltó utca, 1094 Budapest. The ACF HQ comprises two isotope laboratories and three imaging laboratory rooms. Permissions are in place to handle more than 80 isotopes for tracing, imaging and therapeutics and X-ray appliance. In addition, a local isotope-containing animal facility is also associated with the ACF. Two other sites are also involved, the Cardiovascular Centre (68 Városmajor u, 1122 Budapest) and the Basic Sciences Centre (4 Nagyvárad tér, 1082).
- Available Equipment for Molecular Imaging
- Small Animal Ultrahigh Resolution Optoacoustics and Doppler Ultrasound Imaging System: Iconeus ONE (Iconeus, France)
- Small Animal Ultrahigh Resolution Ultrasound System: Vevo 3100 (Fujifilm, Canada)
- Translational High Resolution Ultrasound System: Vivid E95 (GE, US)
- Dedicated Clinical SPECT/PET/CT System for large animals: AnyScan TRIO (Mediso, Hungary)
- Optical Imaging Systems: FOBI (Neoscience, Korea)
- Available Equipment for Structural Micro-Imaging
- Ex vivo Micro-CT System: Skyscan 1272 (Bruker, Belgium)
- Additional Equipment
- PET/MRI system: nanoScan PM 3T (Mediso, Hungary) installation in Q3 2020.
- SPECT/CT system: nanoScan SPECT/CT 4R (Mediso, Hungary) installation in Q1 2021.
The ACF provides services in a broad range of in vivo imaging applications using an already existing infrastructure, which is continuously being expanded. For the usually needed small animal imaging studies presumably in the forefront of possible users, the ACF has an immediate access to multiple animal models (optimized at conventional and SPF animal facilities) such as e.g. inflammation models (including collagen-induced arthritis, autoantibody-induced arthritis, various dermatitis models), cardiac and metabolic disease models (including acute and chronic ischemia-reperfusion, heart failure, hypertonia, atherosclerosis, diabetes, hyperlipidemia, obesity, uremia, cardiogenic shock) as well as various transgenic and humanized mice. Tumour xenografts including a variety of cell lines are also available based on user-borne cost and maintenance models.
The ACF offers a comprehensive service to its users. In this section we outline options users are able to utilize in their projects.
- Choice of imaging measurements and dimensions for the users’ given experimental questions.
- Choice of one or multiple imaging methods, selection of the applicable animal models and proposed imaging contrast materials.
- Experimental and statistical planning of image acquisition measurements.
- Planning of data analysis and if needed, radiomics generation methods.
- Short-time (up to 30 days) hosting of rats or mice in a separate animal house in the Facility.
- Generation of the animal model(s).
- Application of contrast materials or experimental treatments in all routes.
- Animal anaesthesia and image acquisitions.
- Storage and export/import of image data into common biomedical file formats: DICOM 3.0, RAW, Analyze 7.5, NIfTI, and different image file formats such as png, jpg, img, or bmp.
- Analysis of data files by creating quantitative Volume of Interest (VOI) data and exporting them to .csv or other spreadsheet readable formats.
- Biomedical scientific interpretation of visualised images and time series, data sorting and interpretation
- When specifically agreed, statistical methodology and evaluation of the generated data.
- The Facility obtains an imaging ethical permission for use of rats and mice. Mild-level animal imaging experiments (only anaesthesia and dosing routes of contrast agents considered mild) are readily available.
Essentially, the complexity of the above technological platform enables the ACF to offer an unprecedented and highly flexible solution to statically, dynamically, and functionally image all organ systems of the given organism. Moreover, it also provides an excellent technological arsenal for applications in multiple scientific fields of e.g. pharmacology, molecular oncology, histology, inflammation research, biomarker identification and detection – in parallel with assessing physiological and pathophysiological phenomena.
Therefore, the list below reflects rather examples of the specific services offered by the ACF and introduced following the specific features of the infrastructural repertoire:
- High Resolution MicroCT imaging Ex Vivo:
- The platform provides resolution not possible with live imaging due to the confounding effect of even minute movements in live animals.
- Microscopy resolution imaging on all hard and soft tissues
- Assessment of disease progression
- Fast reconstruction of CT images
- SPECT/CT applications
- Biomarker identification
- Spatial and temporal measurement of thyroid, cardiac, hepatic and kidney functions
- Bio-distribution and -availability of isotope-labeled theragnostic molecules already in clinical use or under development
- Imaging of stem cell functions
- PET/MRI multimodal applications
- High spatial resolutions: 100 μm (MRI), 700 μm (PET) – Cellular, subcellular, and molecular identification
- High sensitivity: femtoM/mg tissue
- Anatomical localization, exact morphology of metabolic foreground
- Radiolabeling-based assessment of tissue metabolic processes
- Special focus in neurotransmitter, oncology, regeneration and immune-system studies
- Fluorescent Imaging
- Very high throughput easy-to-use phenotyping of reporter animals without the need of external luciferase injection and ATP access to tissue
- Identification of autofluorescence-related processes
- Tumor and cell tracking and time-series imaging of tumor bio-distribution or cell-based therapies
- Ultra-high-frequency and 4D ultrasound imaging applications
- A platform with portable ultrasound unit operating from 10 up to 70MHz
- Applicable for mice, rats, and other larger animals
- Focused, but not limited to, cardiovascular applications
- fUS: High-resolution quantitation of cerebral blood flow in mice and rats for the fast, on-the-fly analysis of blood flow changes in stroke, inflammation, intestinal microbiome and cardiovascular model mice.
The IVI-ACF possesses a good status of being at the intersection of medical device development, medtech product application support, medical algorithm innovations and experimental biological or biomedical work.
Their extensive client experience ranging from alpha emitter nuclide therapy through detector development to optical imaging dye innovations opens several possible ways to create value.
Besides the status, their own scientific work can also be a center of profits in this regard.
Structuring this outlook, they present first the „static” potential of innovations and second, the „dynamic-research based” one.
STATIC (Algorithms, software, medtech)
STEP 1. PATENTING/INNOVATION
- Creation of disease-specific CT parametric radiomic sets and their validation
- Possibility of writing their own iterative platform-independent CT reconstruction algorithm (with appropriate technical support)
- Creation of 2D optical imaging filtering and detector-level denoising algorithms
- Collaboration in cell recognition and tracking algorithm developments and applications specialized in 3D plus time domain
- Collaboration in new targeted fluorescent and far-IR fluorescent dye developments for clinical surgeries – in very close collaboration with the Functional Cell Biology and Immunology ACF.
STEP 2. LICENSING/COMMERCIALIZATION
- Collaboration with specialised small animal imaging contrast material producers as their reference centre
- Collaboration with new, market entry level 3D optical imaging device vendors again both as a development centre with new application – level patent possibilities.
DYNAMIC (Radiopharmacy, radiochemistry, nanoparticle sensors/therapeutics)
STEP 1. PATENTING/INNOVATION
- Creation of a new polysaccharide-based nanoparticle shell material
- Creation of a new fluorescent dye application for SARS-CoV-2 or other viral effect detection based on metal ion sensing
- Creation of zero valent ferromagnetic Fe nanoparticles for both MR and magnetic particle imaging, coupled to a special magnetometric detector partnership with Wigner Research Centre for Physics (Institute of Eötvös Loránd Research Network) and University of Pannonia at Veszprém.
STEP 2. LICENSING/COMMERCIALISATION
- Collaboration with alpha – emitter development in Roche for oncotherapy
- Collaboration with the deep, therapeutic field modulation-level developments of Oncotherm Ltd. based on fractal nature signal analysis of the full detectable electromagnetic spectrum of tumorous tissue. Oncotherm is a world market leading niche company in a special type of adjunctive tumor therapy, very widespread in Germany and Asia
- Collaborations and cell tracking method developments with leading German start-up companies in CAR-T cell therapy
- New method of calcium activation measurements in the living whole brain using Pb isotopes and Mn ions (already discussed years ago with Richter).
HCEMM-BRC Singe-cell Omics ACF
Location and Equipment
The SCO ACF is located at the Biological Research Centre, 62 Temesvári körút, 6726 Szeged.
The involved facilities and available equipment are:
- Mass spectrometry unit
- ACQUITY UPLC M-Class-Orbitrap-Fusion-Lumos-Tribrid nanoLC/MS/MS system
- nanoACQUITY-LTQ-Orbitrap Elite LC/MS/MS system
- NanoMate – for MS sample introduction
- Analytical HPLC for peptide separations
- Sequencing unit (in cooperation with the Sequencing Platform of BRC)
- Single cell isolation unit (in cooperation with the Laboratory of Microscopic Image Analysis and Machine Learning of BRC)
- Zeiss PALM Microbeam laser microdissection microscope
This ACF is building on the existing but limited service capabilities of the Biological Research Centre (BRC) in the related field. The new ACF will provide proteomic, lipidomic, genomic and transcriptomic analysis with high sensitivity in addition to single cell isolation.
On the path of becoming an ACF the major improvement was the acquisition of a new mass spectrometer (Orbitrap Fusion Lumos Tribrid Mass Spectrometer) that has highly improved detection sensitivity and data quality, and thus, enables the analysis of limited sample amounts as small as only a few cells in case of lipidomics.
The main goal of the ACF is to provide high-sensitivity molecular analyses of limited sample amounts, including lipidomic, proteomic, genomic and transcriptomic analyses and to develop or adopt methods suitable to study a few hundred cells and below.
The ACF offers the following specific services:
- Protein identification from 1D- and 2D-gels, cell lysates, tissue or body fluids
- High mass accuracy, high resolution LC-MS/MS for the identification and relative quantification of protein mixtures of different complexity
- Post-translational modification analysis (mainly phosphorylation)
- Multi-dimensional peptide separation using liquid chromatography
- Protein-complex characterization: immunoprecipitation, on-bead proteolysis and LC/MS/MS analyses for the qualitative and semiquantitative characterization of immune complexes
- Membrane and storage lipid composition analysis on a basis of a few cells
- High mass accuracy, high resolution shotgun lipidomics for the identification and quantitation of cellular lipids at molecular species level
- Data presentation and interpretation
- Full genome sequencing of >1ng input DNA
- Targeted genome sequencing of single cells
- Full RNA and miRNA sequencing
- Transcriptome sequencing of few – as low as a single – cell/s
- Single-cell isolation, phenomics (support)
- Laser micro-capture and patch-clamp single cell extraction techniques assisted by machine learning. Morphology- or fluorescent based isolation of larger cell populations for less sensitive downstream analysis is also available.
Mass spectrometers used for proteomics and lipidomics analyses are operated in alternating 2-3 weeks intervals for the respective measurement types. Only trained personnel can operate the instruments, customers do not have direct access to the mass spectrometers. Data evaluation is usually performed by the ACF personnel, therefore raw data are typically given out solely for publication purposes.
The genomics and transcriptomics workflows are performed by the personnel of the NGS Platform of the BRC. The users provide the starting material (DNA, RNA, miRNA, ChIPped DNA, cDNA etc.), preparation of the sequencing libraries and the sequencing is performed by experts working at the NGS Platform. The resulting raw sequence reads are handed over to the users either by downloading from ftp/cloud or deposited on external hard disks.
Currently, the Single Cell Omics ACF offers mass spectrometry-based proteomics and lipidomics analysis. The vast majority of the facility’s current activity is based on standard published protocols for both sample preparation and downstream MS analysis to aid HCEMM and host institution research groups to pursue their scientific interests. Therefore, development of commercial products or know-hows is primarily collaboration project dependent. One such collaboration was recently initiated with the Laboratory of Bacterial Physiology and Strain Engineering (Institute of Biochemistry, BRC) based on our extensive experience in MS based de novo peptide sequencing.
The overall goal of this project is to identify novel antimicrobial peptides from various natural sources that may be used as new antibiotic drugs or may serve as templates for further drug development.
This capacity can be further extended to any activities requiring de novo peptide sequencing skills such as biosimiliar antibody characterization. However, this approach is low throughput, therefore a limited number of such projects can be performed by the ACF.
Capacity permitting, mass-spectrometry based comparative proteomics and shotgun lipidomics measurements can be offered as service for outside academic and industrial partners as well.
HCEMM-USZ Functional Cell Biology and Immunology ACF
Location and Equipment
The FCBI ACF is located at the Faculty of Medicine, Department of Dermatology and Allergology at the University of Szeged, 6 Korányi fasor, 6720 Szeged. The major available techniques include conventional (wide-field, confocal) and advanced (super resolution) light microscopy, scanning electron microscopy optimized for biological samples (including array tomography and correlative light and electron microscopy), an advanced cell sorter facility and a molecular biology facility.
- Light and Electron Microscopy facility
- Light Microscopy
- Zeiss LSM880 laser scanning confocal microscope
- Olympus FV10i self-contained confocal microscope
- Olympus high throughput fluorescence cell analysis system
- Oxford Nanoimager super resolution microscope
- Zeiss AxioImager fluorescence microscope
- Electron Microscopy
- Zeiss Sigma 300 scanning electron microscope
- RMC Powertome with ATUMtome tape collection
- Quorum Q150R Plus rotary pumped carbon coater
- Zeiss Axioscope fluorescence microscope
- Cell Sorter Facility
- Molecular Biology Support
The main goal of this ACF is to provide for high-throughput and high-resolution imaging, cell surface and intracellular marker detection as well as the possibility for sorting cells based on their expressed protein markers for cellular and immunology studies.
The ACF offers the following specific services:
- Static and dynamic imaging applications
- standard widefield and confocal imaging of fixed samples and live cells (up to 4 wavelengths simultaneously)
- two-colour super resolution microscopy based on STORM and PALM techniques
- single particle tracking
- TIRF microscopy
- Array tomography with ATUMtome – semi-automated cutting and imaging of 100-2000 slices with a z resolution of 30-50 nm and 3D rendering of the images
- Correlative light and electron microscopy (CLEM) – A comparative dual (light and electron microscopy) analysis of the same specimen, which is essential for the analytical interpretation of high-resolution electron microscopy images at the cellular level.
- High-performance cell sorting and marker identification applications
- Cell sorting and clustering from fluids and tissues
- Marker identification.
The major available techniques at the Functional Cell Biology and Immunology Advanced Core Facility (FCBI ACF) include conventional (wide-field, confocal) and advanced (super resolution) light microscopy, scanning electron microscopy optimized for biological samples (including array tomography and correlative light and electron microscopy), an advanced cell sorter facility and a molecular biology facility. This wide variety of advanced tools empower the facility and the researchers using it to develop patentable technologies and to create licensing and commercialization opportunities. Examples of such are the following:
- New fluorescent probes and fluorescent proteins with enhanced optical and chemical properties
- Nanotechnology-based new drug delivery methods and their novel applications in translational medicine. (Nanopharmaceutics, quantum dots, synthetic polymers, dendrimers, lipoproteins, liposomes, micelles, nanoparticles, microcapsules)
- Novel diagnostic applications ( g. Disease-specific novel cell surface markers and intracellular markers)
- New optical imaging techniques and patentable hardware improvements ( g. development of a specific sample holder or stage insert)
- Development of new microscopy imaging and image analysis software modules, apps, algorithms and plugins.
As a more specific example, FCBI ACF is planning to commercialize its intracellular antibody localization expertise as a service for antibody manufacturing companies (e. g. Agrisera).