REMEDI - News & Events
Meetings
27.04.2010: Kickoff-Meeting in Gräfelfing, Germany
31.03.2011: 1st Annual Meeting in Nijmegen, Netherlands (RUNMC)
Results of the first project period (01.01.2010 - 30.06.2011)
Valid diagnostic criteria should directly reflect functioning and malfunctioning of the patient’s organism. Since most diseases can ultimately be traced to failures of the molecular machinery underlying cellular processes, diagnosis would ideally be based on direct inspection of the relevant molecular events, instead on emergent organismic phenomena only. However, it is not merely the presence or absence of molecular components, but their interaction that determines cellular health. Thus spatial resolution constitutes an irreplaceable advantage in diagnostics.
Interactions relevant for cell-based diagnostics span a wide range of length scales: protein-protein interactions occur on a nanometer scale, protein complexes and clusters of proteins measure several tens to hundreds of nanometers and functional rearrangement of proteins to cellular substructures, can be observed at scales of hundreds of nano- to several micrometers.
Unfortunately, conventional microscopy techniques can only access length scales from 200 nm upwards (light microscopy), or are limited to observing a small number of different protein targets in fixed samples (electron microscopy). It is thus the goal of REMEDI to adapt recently developed optical single-molecule-imaging techniques for the use in medical diagnostics facilities in order to address the questions of protein presence, interaction, and spatial distribution in tissue samples and live cells with ultra high sensitivity.
Conventional microscopes are not well suited for the stability requirements of superresolution. Stability critically depends on the vibration susceptibility and the thermal behavior of all opto-mechanical components of the system, including immersion fluids connecting objective and sample.
The consortium is therefore exploring new materials with maximal vibration damping capabilities, minimal or matched thermal expansion capabilities and, where all these measures turn out insufficient, new concepts for feedback-controlled dynamic realignments.
Stability issues also arise due to the fact that the acquisition of single-molecule-imaging technique-based images takes very long. Every reduction of the image acquisition time will thus help reduce the stability requirements to levels, which can reasonably be expected from routine medical diagnostics facilities. This goal is addressed by employing novel sCMOS camera technology in combination with read-out schemes tailored especially for their application in single molecule localization imaging.
The consortium addresses resolution enhancement not only in 2 dimensions, but also in 3D. The large chip-size of the sCMOS camera allows projecting two images obtained at different focal positions next to each other onto a single chip, and by using suitable algorithms the differential-focus recordings allow reducing the vertical position accuracy down to 20 nm levels, too. Moreover, in order to increase selectivity, each of the two by their different z-position separated images can be split into two emission colors and projected, side by side, on the large sCMOS chip.
Technology development is rounded up by firm- and software development for FPGA-based image preprocessing. Goal is to extract superresolution images while thousands of localization images are being gathered. This is achieved with a novel single molecule localization software.
These novel superresolution technologies are being validated with two key microscopy applications: 1) diagnostic/experimental pathology using the example of important receptors in breast cancer and 2) tumour immunology using the example of lipid raft-associated signal transduction in lymphoma.
In the first reporting period, significant progress has been made by the industrial partners TILL I.D., TILL Photonics and Andor in developing the microscopy hardware and software for super-resolution and single molecule localization techniques. The application partners Niendorf and RUNMC have selected cases and established protocols ready to be used for validation of the REMEDI microscopy platform.
The novel super-resolution techniques determine the exact position of insular molecules within a cell with a precision of approximately 20 nanometres and combine many thousand such insular molecule localization maps into a global molecular map of the object under study. Since those techniques combine the advantages of optical microscopy (live-cell imaging, multi-colour imaging, 3-D imaging) with the resolution of electron microscopes, it is safe to assume that they will not only revolutionize microscopy in the coming years, but will also have a great impact on medical diagnostics, given that most diseases can ultimately be traced to failures of the molecular machinery underlying cellular processes.
The new technology will allow studying these molecular machineries on site, rather than in bulk on a chip. Thus, it will extend the field of vision of the light microscope from a macroscopic level, where organismic phenomena reflect a given disease, down to the molecular level, where disease-relevant molecular events take place.
Thus, this project will explore the potential of the super-resolution method with two pilot studies in the field of medical diagnostics. The first is focused on the molecular-resolution immuno-histochemical staining and imaging of solid tumour samples. The second looks at nanoscale protein and lipid structures in lymphoma cells as well as in cells infected by viruses. In both cases, the aim is to visualize individual protein molecules and their interaction with other proteins, all of which play a crucial role in the disease process under study (cancer, virus infection), and which hence should provide subtle, but valid, criteria for diagnosis, prognosis, and therapy monitoring.