Intravital microscopy (IVM) enables the study of cellular and molecular events in living organisms. Confocal microscopy permits images to be collected from narrow focal planes without interference from out-of-focus regions, and multi-photon microscopy produces high-resolution images from deep (several hundred micrometers) within opaque organs and tissues. Quantitative IVM requires four components: A tissue preparation that allows visualization; A molecular probe that can be detected by a microscope; A microscope and detection system (for example, charged-coupled device camera and computer); Computer algorithms and mathematical models that can be used to extract parameters of interest from the optical information. Molecular imaging, Cellular imaging, Anatomical imaging, Functional (physiological) imaging, Therapeutic imaging can be done using intravital microscopy and tumour pathophysiology can be studied using this novel technique.

There is little difference between conventional microscopy methods and intravital microscopy in principle, but in practice there are significant differences in the way images are acquired and processed. The first and most obvious difference between a conventional microscope and one optimized for animal in vivo work is the way the 'sample' is handled: animals are thicker than a microscope slide.

Lasers are used in microscopy and are targeted through microscope objectives can injure individual micro blood vessels and cause thrombi which is an aggregation of blood factors, primary platelets and fibrin with entrapment of cellular elements, causing vascular obstruction at point of its formation. Thrombi can be studied in detail. The marriage of these technologies provides exciting possibilities for investigating the inflammation and coagulation that is associated with disseminated intravascular coagulation.

Lets understand what is actually a probe. It is a slender, flexible instrument designed for introduction into wounds, cavity or sinus tract for purpose of exploration. In molecular genetics, it is defined as a radioactive RNA or DNA sequence used to detect presence of complementary sequence by molecular hybridization. The tissue preparations fall into three broad categories: Chronic-transparent windows; acute (exteriorized) tissue preparations; and in situ preparations. Each of these preparations can be used to study normal tissue or an implanted tumour. The tumour source can be a suspension of cancer cells or a fragment of tumour tissue. Alternatively, a gel containing defined growth factor(s) or engineered cells can be implanted in these tissue preparations. As with other investigative tools, each preparation has its strengths and weaknesses, and a combination of approaches is normally warranted. Once the preparation is ready for observation, the animal is brought to the stage of a specially designed microscope. Depending on the experimental aim, an appropriate exogenous or endogenous molecular probe is used and either trans-illumination or epi-illumination is applied to visualize the whole tissue (for example, mesentery) or only the superficial regions (for example, cranial window). The optimal type of illumination is dictated by the thickness of the tissue and its optical properties. Increasing depth of imaging can be achieved by confocal laser-scanning microscopes (cslms) and multi-photon laser-scanning microscopes (MPLSMs).

Molecular imaging
In principle any molecule that can be detected by regions, despite active blood flow. Optical microscopy can be tracked by molecular imaging in vivo. The easiest, and hence most widely used, application of this concept is monitoring the delivery (micro-pharmacokinetics) of molecularly targeted therapeutics. The therapeutic agent can be naturally fluorescent (for example, Adriamycin) or tagged with a fluorescent tracer; for example, therapeutic antibodies can be tagged with fluorescein isothiocyanate (FITC). If the agent is delivered using a carrier, such as a liposome, then both the drug and the carrier can be monitored using different optical tracers. Such an approach led us to the conclusion that it is difficult to deliver therapeutic agents in optimal quantities to all regions of a tumour, and this spawned interest in understanding physiological barriers to drug delivery. Monitoring molecules that are present in a tissue, but not naturally fluorescent, requires molecular probes that change their optical properties as a function of the concentration of the test molecule and independently of the concentration of the probe. Molecular probes that respond optically to changes in pH and pO2 have been successfully used to map the metabolic microenvironment of tumours. These studies have shown that tumours can contain hypoxic Moreover, low pH and low pO2 can be independent of each other. This finding has significant implications for the efficacy of drugs and/or expression of genes that respond to low pH or hypoxia. Similarly, monitoring the activity of an enzyme requires a molecular probe that changes its optical properties by specific interaction with the enzyme. As the product of the reaction accumulates in the tissue, the optical signal increases (or, in some cases, decreases) in proportion to the enzyme activity. Such an approach has been successfully applied to monitor the activity of cathepsin B using nearinfrared (NIR) imaging and epi-fluorescence microscopy (D. Fukumura et al., unpublished observations). This area of molecular imaging is awaiting the development of new molecular probes to monitor different enzymes and receptors that are involved in tumour progression, and to assess their involvement in different signalling pathways.

Cellular imaging
Intravital microscopy has provided powerful insights into cell-cell interactions in vivo and especially in the movement of cancer cells and cells of the immune system - the former to understand various steps in metastasis and the latter to improve immunotherapy of cancer. Metastasis is the transfer of disease from one organ or part to another not directly connected with it. It may be due to either transfer of pathogenic microorganisms (ex-tubercle bacilli) or transfer of cells as in malignant tumours. Recently, a number of investigators have used cells that constitutively express GFP in combination with video-microscopy to monitor various steps of tumour growth, angiogenesis (formation of blood vessels) and metastasis. The key findings from these studies include the following. First, implanted tumour cells migrate towards host blood vessels before their proliferation and induction of angiogenesis, which is induction of growth of blood vessels from surrounding tissue into solid tumour by a diffusible chemical factor released by tumor cell. Second, the lining of tumour blood vessels has non-uniform expression of endothelial markers, and has therefore been dubbed 'mosaic' vessel; Third, tumour cells migrate, intravasate (entry of foreign material into blood vessels) and are carried to a secondary site at a rate that is proportional to the metastatic potential. Fourth, cells lodged in the secondary site frequently stay dormant. Unfortunately, IVM has not been applied to discern or distinguish various steps of lymphatic metastasis, presumably owing to a lack of suitable animal models. With the availability of engineered cancer cell lines and mice that over express or lack the genes that encode molecules involved in metastasis, IVM will continue to provide new insights into metastasis.

MPLSMs have provided new insights into the role of other host cells (for example, fibroblasts, endothelial precursor cells and embryonic stem cells) in tumour growth and angiogenesis. For example, by growing tumours in VEGF-GFP transgenic mice, it has been tracked that the movement of activated host stromal cells and discovered that these cells wrap around the angiogenic vessels, and that they might even guide the formation of angiogenic vessels in tumours. Although various invasive techniques can provide similar spatial information, they miss the temporal dynamics that are provided by IVM. So, IVM can be used to increase our understanding not only of tumour biology, but also of developmental biology, regenerative medicine and tissue engineering.

Anatomical imaging
Determining the size and architecture of a tumour and its vasculature is perhaps the most common application of IVM. In general, vessel diameter, length, intercapillary distance and branching patterns can be determined using RBCs as a natural contrast agent under conventional transillumination or linearly polarized light. Alternatively, a high-molecular-weight fluorescent tracer (for example, FITC-conjugated 2-MDa dextran) can be used to demarcate (marking off or ascertainment of boundaries) the blood vessels before it begins to leak appreciably into the extra vascular compartment. It is straightforward to calculate the vascular surface area and volume of a growing or regressing tumour using these parameters and stereological principles. A striking finding from such studies is that tumour vessels have an abnormal morphology: they are dilated, saccular, and tortuous and have abnormal branching patterns. Fractal analysis of normal and tumour vascular networks reveals that the former are optimally designed to provide nutrients by diffusion to all normal cells (so-called diffusion- limited aggregation), whereas the latter are restricted by the mechanical properties of the matrix (called invasion percolation). Interestingly, during the course of various direct and indirect anti-angiogenic therapies, tumour vessels begin to develop a normal appearance: their diameter begins to decrease, they become straighter and less tortuous, and their fractal dimension begins to decrease towards the diffusion-limited aggregation pattern. Fluorescence micro lymphangiography of tumours growing in the tails of mice has shown that the lymphatics in the tumour margin are hyperplasic (abnormal multiplication or growth of cells), similar to those in the skin of mice that are engineered to over express VEGFC - a growth factor believed to be involved in lymphangiogenesis - in their keratinocytes. The diameters of these lymphatics in the tumour margin increase even further in tumours that over express VEGFC. Surprisingly, over expression of VEGFC does not induce any functional lymphatics within these tumours. The lack of functional lymphatics within tumours is a key contributor to the interstitial hypertension that is measured in animal and human tumours. Owing to physical limitations of optical microscopy, it is not possible to directly measure the dimensions of submicron structures in vivo. However, such measurements can be made by monitoring the movement of submicron particles in the tissue. The approach of relating structure to function is likely to provide new insights into the molecular determinants of vascular permeability in tumours.

Functional imaging
IVM has enriched our understanding of the various physiological determinants of drug delivery to tumours. A blood-borne agent must be delivered via the vasculature, enter the tissue by transvascular exchange, move through the interstitial space by diffusion or convection, and be cleared by lymphatics in the tumour margin. Beginning with the seminal work of Intaglietta and co-workers, several groups have shown that blood perfusion in tumours is spatially and temporally heterogeneous. Furthermore, in some tumours, average RBC velocity is lower than that in host vessels and does not correlate with vessel diameter. The heterogeneous and chaotic microcirculation of tumours results, in part, from the abnormal architecture of the vascular network, and adversely affects both the delivery of drugs and the metabolic microenvironment. The latter, in turn, compromises the effectiveness of various therapies, and selects for more aggressive and metastatic cancer cells. Measuring the permeability of the tumour vascular network as a whole, as well as of individual tumour vessels, has revealed it to be an order of magnitude higher than that of most normal vessels. Vascular permeability of different tumours growing in the same site is different, and varies as the same tumour is grown in different sites or as it responds to therapy. Although, it is possible to lower the vascular permeability of a tumour by blocking VEGF signalling, increasing permeability by adding VEGF or PlGF is not always possible. But perhaps the biggest challenge in transvascular transport in tumours stems from the spatial and temporal heterogeneity in permeability, which restricts access to some regions of tumours. Even after a drug molecule has overcome these barriers to extravasation, it must negotiate the extra cellular matrix to reach cancer cells. By adapting fluorescence recovery after photo bleaching (FRAP) methodology to intravital applications, we have measured the interstitial diffusion, convection and binding of macromolecules in tumours in vivo.

Imaging therapeutic response
In general, the preclinical response to a new therapy is quantified in terms of reduction in (or stabilization of) tumour size and survival time of the animal. However, the ability to monitor several parameters simultaneously with IVM has provided integrated insight into a tumour's response to various therapies. This insight has led to new strategies for improving cancer detection and treatment. For example, we have recently shown that blocking VEGF or VEGF receptor 2 (VEGFR2) leads to a decrease in vessel diameter and vessel density (anatomical imaging) and a decrease in vascular permeability (functional imaging). This 'normalization' of tumour vessels can lower interstitial fluid pressure, improve perfusion and, in some cases, increase tissue pO2 (molecular imaging). Similar normalization has been observed intravitally following hormone withdrawal from a testosterone-dependent tumour and following treatment of an ERBB2 (also known as HER2/neu)-overexpressing tumour with trastuzumab (Herceptin) - a monoclonal antibody to ERBB2. This indicates that a cytotoxic therapy (such as radiation or chemotherapy) applied during this 'normalization'window might lead to synergistic (acting together or enhancing other effect) effects. Conversely, if vessels are obliterated (complete removal by surgery or degeneration) to the point that the microcirculation is compromised, the combination might lead to antagonistic results. The challenge is to develop imaging technology for clinical use that can provide molecular, anatomical and functional data with the same temporal and spatial resolution as animal-model imaging.

Future directions
IVM has provided useful insights into many aspects of tumour biology. However, there are several unanswered questions awaiting the development of new animal models, probes and microscopes. Virtually all IVM studies have used transplanted tumours. In principle, it should be possible to place 'windows' on spontaneously occurring tumours, but this has not yet been done. Various molecular probes are available for anatomical and functional imaging. However, there are few probes for molecular imaging. GFP and its variants have already allowed visualization of molecular processes that, until recently, could only be imagine. This might limit the number of reporters that can be used simultaneously. However; the rapid developments that are taking place in this area are likely to overcome these problems. Live reporters are needed to dissect protein-protein interactions and to investigate signal-transduction pathways in vivo. Unexpected discoveries might emerge when we can examine the interaction of multiple genes in an intact organism using new molecular probes. Concerted efforts are also needed to improve subsurface imaging technologies. Most of the current data come from techniques that emphasize surface features at the expense of information that is found from deeper within the tissue. Multi-photon laser-scanning microscopy can image structures up to 700 µm deep, depending on the tissues and probes used. Other optical techniques, such as optical coherence tomography, can image deeper regions, but its application to intravital microscopy has been limited owing to lack of intrinsic contrast. An alternative approach is fluorescence-mediated tomography. Other techniques such as positron-emission tomography, computed tomography, magnetic resonance imaging and near-infrared imaging have been developed for use in rodent models and can provide deeper imaging, but they lack spatial resolution. Image acquisition rate and speed of imaging also need to be improved. High-speed imaging is necessary to capture dynamic events such as blood flow, leukocyte-endothelial interactions, tumour cell-blood-vessel interactions and movement of small molecules. Such kinetic information is vital for understanding the biology of tumours and for optimizing therapeutic approaches. Most IVM set-ups are bulky bench-top devices, but there is an increasing effort to miniaturize the cameras and microscopes to make the whole unit hand-held. As these devices become more user-friendly, IVM will become a useful clinical tool for diagnosis and monitoring the response of cancer patients to various therapies.


(The authors Anantha Naik Nagappa and Faisal Khan are with Manipal College of Pharmacuetical Sciences, Manipal University, Manipal, 576104 and Ishita Arora is with Department of Pharmacy, Birla Institute of Technology and Sciences, Pilani.)