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Tutorials : Apr 15, 2008 ( )
Combining Microscopy with In Vivo Imaging
INphoton Uses Intravital Multiphoton Microscopy for Novel Drug Research!--h2>
Typical drug discovery processes utilize high-throughput methods to identify promising compounds followed by animal studies to examine efficacy and toxicity. This is a costly and high-risk venture, and the time spent on assessing compounds that ultimately fail toward the end of this process contributes to the rapidly escalating cost of bringing a drug to market.
As shown in Figure 1, in vivo MFI provides nanomolar sensitivity at submicron resolution. For the large number of drug compounds whose distribution in tissues cannot be preserved for histological evaluation, MFI provides a means of identifying the cells and subcellular compartments where experimental drugs accumulate. This data is frequently crucial to understanding drug efficacy and toxicity.
In addition, in vivo microscopy techniques capture images at 1–30 frames per second. The spatial and temporal resolution make in vivo MFI capable of characterizing the dynamics of drug transport at the cellular and subcellular level.
Figure 2 demonstrates the high-content, high-resolution capability of MFI as an in vivo imaging modality. It shows an in vivo multiphoton fluorescence image of the kidney from a healthy living rat collected minutes after intravenous injection of fluorescent-labeled compounds including gentamicin (red), a large molecular weight probe (green), and a probe to label the nucleus (blue). Important aspects of tubular and subcellular drug distribution are apparent in the glomerular filtration and subsequent tubular reabsorption of gentamicin via endocytosis by cells comprising the proximal tubule.
The resolution of this image shows not only that gentamicin uptake is limited to proximal tubule cells, but also, that gentamicin accumulates in endosomes and lysosomes of proximal tubule cells. By collecting images in time series, the kinetics of cellular accumulation of gentamicin can also be quantified (Figure 3).
Essential physiologic processes are also evident in Figure 2. These processes can be quantified and the alteration of these processes can serve as a marker of drug efficacy or toxicity. For instance, the unfiltered large molecular weight probe (green) is retained within the vasculature and effectively defines the vascular space (arrow). Microvascular perfusion rates can be determined by the geometry of red blood cell “shadows,” which form voids in the signal of the large probe coursing through the capillaries.
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