2 Department of Biomedical Engineering, Technical University of Eindhoven. Den Dolech 2, P.O.Box 513, 5600 MB Eindhoven, the Netherlands

The Department of Biophysics takes part in the Vital Imaging Unit CARIM / BME in Maastricht. In this unit, several groups of both the University of Maastricht (UM) and the Technical University of Eindhoven (TUE), involved in various aspects of vital imaging, cooperate to strengthen and facilitate the techniques available. Here we mention confocal fluorescence microscopy, intravital microscopy, atomic force microscopy, and FIMS. The research carried out in Maastricht centers around the various aspect related to cardiovascular diseases, where at the Department of Biophysics we focus on the technical developments and physical aspects of imaging methods.

One of such methods is the vital imaging of processes of vascular occlusion at the cellular level, such as the interactions of blood cells with the vessel wall. Examples are platelet activation after microvascular wall puncture and leukocyte rolling and adhesion after inflammation or ischemia. Next to many other (biochemical) techniques, traditionally various microscopy techniques have been developed, with special attention to the possibility to visualize these processes in living animals, so-called intravital (fluorescence) microscopy. The department of Biophysics has a longstanding interest in those microcirculatory aspects in the person of Prof. Dick W. Slaaf.

Recently, there has been a shift of interest to the visualization of interactive processes in larger vessels, since these vessels are the ones that are prone to vascular diseases such as atherosclerosis. However, the traditional fluorescence microscopy techniques lack sufficient penetration depth and are thus not able to visualize structures and processes in vessels with diameters over 50mm. Furthermore, in such dense tissues analysis of structures is hindered by the mixing of probe fluorescence with tissue autofluorescence. This autofluorescence cannot be separated from the probe fluorescence, since it overlaps spectroscopically. Finally, the quantification of various processes, such as the Ca²⁺ flux or NO transients, is impossible using traditional fluorescence techniques or at least requires a cumbersome calibration method.

Since a few months we have the availability of a so-called Two Photon Lifetime Scanning Microscope (TPLSM). This commercially available microscope (BioRad, Nikon/Uvikon and SpectraPhysics) has the capacity of solving most of these problems. The two photon excitation principle results in larger penetration depths (up to 200mm in blood vessels), large enough to visualize, e.g., the carotid artery of a mouse. Furthermore, the application of the fluorescence lifetime detection method using the gating technique, results in easy to use calibration procedures for ion flux quantification and adds a new contrast mechanism. Recently, we have carried out some experiments centered around the latter issue, by imaging the adhesion of Oregon Green Bapta 488 labeled blood platelets on a layer of collagen. Oregon Green Bapta 488 is a calcium probe and as such images the activation state of blood platelets. A typical image is given in Figure 1.

Figure 1: Fluorescence intensity (left) and lifetime (right) image of blood platelets labeled with Oregon Green Bapta 488. In the intensity image, non-attached platelet can be discriminated from attached ones by image streaking. However, contrast is low and the activation state cannot be uniquely related to fluorescence intensity. In the lifetime image contrast is much higher and activated platelets exhibit a longer lifetime (blue, 3.5 ns) than the nonactivated, moving platelets (orange, 2.3 ns).

Having the set-up mentioned above, one enters the area of image analysis. Many aspects have to be considered here. As examples, we mention the correction for refractive index mismatch in images of deeper lying structures, the correction of motional artifacts in in vitro flow or in vivo experiments and, finally, the analysis of lifetimes per pixel requiring the use of exponential fitting procedures. While for many of those problems dedicated software exists, we wanted to create a more versatile image processing facility to be able to integrate the solutions of the various aspects mentioned above. Therefore we decided to make use of the IDL 5.4 software on a Pentium III, Windows 98 computer. With its easy-to-program blocks and modular structures it allows the simple addition of new functions in a transparent way. Until now it has helped us to fastly obtain beautiful images demonstrating the new possibilities of TPLSM in combination with adequate image analysis software. For an example see Figure 1, where the lifetimes were obtained by a simple exponential fitting procedure (Dr. C.J. de Grauw, University of Utrecht) plugged in the analysis software. From our experiences so far we feel convinced that any new desires arising in the future can be easily fulfilled with IDL.