Experimental Imaging

As technical methods continue to be advanced, new imaging procedures and evaluation methods used to depict structures and processes are always available for biological investigations. However, due to a parallel specialization of the majority of research areas, most scientists are lacking in the time, experience and in-depth knowledge that would enable them to really get to grips with the limits and possibilities of these innovative procedures. This means that the potential of modern imaging procedures often remains untapped.

The field of experimental imaging therefore unites the knowledge of various imaging procedures with the profound knowledge of applied life sciences. The unit is focussing here on developing imaging solutions to scientific tasks both with and for partners from the fields of research and industry.

Furthermore, considerable expertise and resources are required in order to operate and maintain the equipment pool and to secure modern quality assurance procedures; this need can be secured by the unit. The technologies available at the Fraunhofer IZI for experimental imaging include:

  • Specialized fluorescence and laser scanning microscopy
  • High-field (7T) magnetic resonance imaging in the small-animal scanner
  • Bioluminescence and biofluorescence imaging using the BioImager
  • Stereological and object-based analysis systems

Offers of the Experimental Imaging Unit

High-field MRI examinations in the small animal

The magnetic resonance imaging examination (MRI) is one of the most important diagnostic measures in the examination of living animals. Working together with the Diagnostic Radiology Clinic at the University of Leipzig, the unit carries out examinations using 1.5 and 3 Tesla systems. A 7T small-animal scanner can be used for high resolutions. The range of methods is extensive and includes most standard sequences. They can be adjusted to the task if necessary.

Bioluminescence and biofluorescence imaging

This technology is based on capturing photochemical light emissions of self-luminous or stimulated fluorescent dyes in the living animal. The most common way of achieving this is to genetically introduce a vector for the luciferase enzyme into the cell being examined and then stimulate this cell to emit light by adding luciferin. If the respectively marked cells are now applied to a small animal, they can be detected in vivo by a highly sensitive CCD camera from a given concentration, even over longer periods of time. The IVIS spectrum imaging system used for this purpose shows a high level of sensitivity in the examination range of 400 and 900 nm.

Confocal laser scanning microscopy

3D image of scar formation after stroke.
© Photo Fraunhofer IZI

3D image of scar formation after stroke.

In this unit, the qualitative and quantitative analysis of fluorescence-dyed samples is conducted using a confocal microscope (Zeiss LSM710). This microscopic technology allows non-overlapping recordings of specific signals in tissues or cell cultures. For further processing and 3D image analysis, the acquisition of images is complemented by the complex software package IMARIS.

Stereology

Stereological counts are the gold standard for error-free quantitative analyses in tissues. By using different algorithms it is possible to quantify object frequencies, surfaces and volumes as well as lengths and branching degrees of structures.

Imaging examination with clinical scanners and their evaluation

3D Reconstruction of skin and bone tissue of a sheep's head based on a CTI data set.
© Photo Fraunhofer IZI

3D Reconstruction of skin and bone tissue of a sheep's head based on a CTI data set.

Together with partners from the University of Leipzig, the majority of imaging techniques established in human medicine were adapted in order to examine large-animal models. This concerns both anatomical (computer tomography) and anatomical-functional (magnetic resonance imaging) examinations. Beyond this, metabolic processes can be visualized by means of positron emission tomography. The most varied quantifying evaluation routines permit an exact assessment of the change.

Magnetic resonance imaging description of infarct growth following a stroke in the rat

Various surrogate parameters are required to scientifically describe an effective stroke treatment. One of the quantification procedures frequently used in the literature defines the change in the spread of the stroke over time in the living animal. In addition, magnetic resonance imaging T2-weighted sequences are primarily surveyed and a volumetric evaluation of the affected brain area then carried out. Until now, these analyses were carried out using a 1.5T MR scanner. However, only a limited resolution could be achieved in examinations on small animals. Using the high-field MRI with up to a 7 Tesla field strength can significantly improve this resolution restriction. Besides examinations into the development of the stroke, further surrogate parameters can, of course, also be determined on the living animal.

Stereological evaluation of diaschisis following stroke in the rat

Following a stroke, damage occurs not only in the primary infarct area, but also in more distant regions of the brain – an occurrence referred to as diaschisis. This is how, for example, following ischemic damage in the primary sensorimotor cortex through axonal fibre connectivity, a selective die-off of cells takes place in the thalamus' more distant, ventral, posterior nucleus. The number of secondarily damaged neurons in this core region was determined as part of the project. Precise quantification took place via a stereological analysis using the program Stereo Investigator from MicroBrightField. This program allowed statements to be made on the total number of mortified neurons in the defined region. A stereological analysis is being demanded from an increasing number of scientific publishers as the gold standard for quantifications.

Investigations into distribution kinetics following cell transplantation in the rat

One of the main issues following the application of cells relates to the fact that these cells remain in the recipient organism. In order to shed light on this issue there are currently a range of procedures available, the majority of which involve the examined cells being marked. For this project, tumor cells were marked using a luciferase vector. After administering luciferin, a photochemical reaction occurs in the affected cells, which can be illustrated in the bioluminescence imager. This showed that the affected cell population builds up in the lung following intravenous administration and later likely continues to be redistributed.

Quantification of glial cells following brain tissue damage

Immunohistochemical staining of astrocytes.
© Photo Fraunhofer IZI

Immunohistochemical staining of astrocytes.

Rendered 3D models of immunohistochemical stained astrocytes.
© Photo Fraunhofer IZI

Rendered 3D models of immunohistochemical stained astrocytes.

Determination of colocalization.
© Photo Fraunhofer IZI

Determination of colocalization.

Brain tissue damage caused by trauma or hypoxia results in far-reaching changes in the affected areas of the brain. The rebuilding processes do not only affect the vulnerable nerve cells, but also the brain‘s connective and supporting tissue. These cells, referred to by Rudolf Virchow as glia (Greek for ”glue”) have extremely varied tasks to fulfill. They surround the nerve cells and provide them with nutrients, thus con­tribute to the forwarding of information and maintenance of homeostasis in the brain.

Following brain damage, some glial cells experience an en­largement of cells (hypertrophy) and an increase in the number of cells (hyperplasia). This can go so far that it becomes impossible to differentiate between certain glial cells (such as so-called astrocytes) on histological stainings, as they form a tight network of cell bodies and overlapping processes. In spite if this, in order to be able to describe the cells, processes are applied at the Fraunhofer IZI which transform them into definable three-dimensional objects. This makes it possible to quantitatively describe the number of cells, their morphology, interaction with other cells, and their changes over the course of time. For this purpose, the affected tissue is immunohistochemically stained and scanned using confocal laser scanning microscopy. The resulting dataset is processed and rendered into a 3D structure. Overlaps (colocalization) of selectively stained cells can then be projected on top of each other, allowing individual cells and cellular components to be segmented. This allows the subsequent count to determine exactly which segments should be recorded and which should be excluded.

This process enables a precise quantification of pathological changes following brain damage and is, for that reason, suitable for verifying the efficacy of new therapeuticprocedures. Additionally, not only the above-mentioned astrocytes, but also any desired cell in any desired histological section can be analyzed. The procedure is currently being adjusted to be able to describe microglial cells and nerve cell inter­actions in more detail.

  • Nuvo Research GmbH
  • University Hospital Leipzig, Department for Internal Medicine, Neurology and Dermato­logy, Clinic and Polyclinic for Derma­tology, Venerology and Allergology, Research Group Skin
  • University of Leipzig, Clinic for Nuclear Medicine
  • University of Leipzig, Clinic for Radiology
  • University of Massachusetts

  • Kaiser D, Weise G, Möller K, Scheibe J, Pösel C, Baasch S, Gawlitza M, Lobsien D, Diederich K, Minnerup J, Kranz A, Boltze J, Wagner DC. Spontaneous white matter damage, cognitive decline and neuroinflammation in middle-aged hypertensive rats: an animal model of early-stage cerebral small vessel disease. Acta Neuropatholica Communications. 2014 Dec 18;2(1):169. DOI dx.doi.org/10.1186/s40478-014-0169-8.
  • Pösel C, Scheibe J, Kranz A, Bothe V, Quente E, Fröhlich W, Lange F, Schäbitz WR, Minnerup J, Boltze J, Wagner DC. Bone marrow cell transplantation time-dependently abolishes efficacy of granulocyte colony-stimulating factor after stroke in hypertensive rats. Stroke. 2014 Aug;45(8):2431-7. DOI dx.doi.org/10.1161/STROKEAHA.113.004460.
  • Weise G, Lorenz M, Pösel C, Maria Riegelsberger U, Störbeck V, Kamprad M, Kranz A, Wagner DC, Boltze J. Transplantation of cryopreserved human umbilical cord blood mononuclear cells does not induce sustained recovery after experimental stroke in spontaneously hypertensive rats. Journal of Cerebral Blood Flow and Metabolism. 2014 Jan;34(1):e1-9. DOI dx.doi.org/10.1038/jcbfm.2013.185.
  • Boltze J, Kleinschnitz C, Reymann KG, Reiser G, Wagner DC, Kranz A, Michalski D; the meeting contributors. Neurovascular pathophysiology in cerebral ischemia, dementia and the ageing brain - current trends in basic, translational and clinical research. Exp Transl Stroke Med. 4 (2012), 1:14. DOI dx.doi.org/10.1186/2040-7378-4-14.
  • Wagner DC, Bojko M, Peters M, Lorenz M, Voigt C, Kaminski A, Hasenclever D, Scholz M, Kranz A, Weise G, Boltze J. Impact of age on the efficacy of bone marrow mononuclear cell transplantation in experimental stroke. Exp Transl Stroke Med. 24(2012), 1:17. DOI dx.doi.org/10.1186/2040-7378-4-17.
  • Wagner DC, Deten A, Härtig W, Boltze J, Kranz A. Changes in T2 relaxation time after stroke reflect clearing processes. Neuroimage.61 (2012), 4, S. 780-5. DOI dx.doi.org/10.1016/j.neuroimage.2012.04.023.
  • Boltze J, Kranz A, Wagner DC, Reymann K, Reiser G, Hess DC. Recent advances in basic and translational stroke research. Expert Rev Neurother. 2011 Feb;11(2):199-202.
  • Boltze J, Schmidt UR, Reich DM, Kranz A, Reymann KG, Strassburger M, Lobsien D, Wagner DC, Förschler A, Schäbitz WR. Determination of the therapeutic time window for human umbilical cord blood mononuclear cell transplantation following experimental stroke in rats. Cell Transplant. 2011 Dec 13. DOI dx.doi.org/10.3727/096368911X589609 [Epub ahead of print]
  • Wagner DC, Riegelsberger UM, Michalk S, Härtig W, Kranz A, Boltze J. Cleaved caspase-3 expression after experimental stroke exhibits different phenotypes and is predominantly non-apoptotic. Brain Res. 2011 Mar 24;1381:237-42.
  • Riegelsberger UM, Deten A, Pösel C, Zille M, Kranz A, Boltze J, Wagner DC. Intravenous human umbilical cord blood transplantation for stroke: impact on infarct volume and caspase-3-dependent cell death in spontaneously hypertensive rats. Exp Neurol. 2011 Jan;227(1):218-23. Epub 2010 Nov 16.
  • Kranz A, Wagner DC, Kamprad M, Scholz M, Schmidt UR, Nitzsche F, Aberman Z, Emmrich F, Riegelsberger UM, Boltze J. Transplantation of placenta-derived mesenchymal stromal cells upon experimental stroke in rats. Brain Res, 1315 (2010), S. 128-36.