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Keywords:

  • molecular imaging;
  • optical imaging;
  • tumor imaging;
  • fpVCT;
  • near infrared range imaging;
  • renal cell carcinoma;
  • in vivo cytometry;
  • fluorescence in vivo imaging

Abstract

  1. Top of page
  2. Abstract
  3. Molecular and Optical Imaging––Definitions
  4. Molecular Imaging––A Short Overview
  5. Molecular and Optical Imaging in Preclinical Tumor Research
  6. Cytometry in Whole Animal Tumor Imaging
  7. Conclusions
  8. Literature Cited

The multiparametric molecular cell and tissue analysis in vitro and in vivo is characterized by rapid progress in the field of image generation technologies, sensor biotechnology, and computational modeling. Fascinating new potentials in unraveling the detailed functions of single cells, organs, and whole organisms are presently emerging and permit the close monitoring i.e. tumor development or basic cell development processes with an unprecedented multiplicity of promising investigative possibilities. To answer basic questions of in vivo tumor development and progression fluorescence based imaging techniques provide new insights into molecular pathways and targets. Genetic reporter systems (eGFP, DsRED) are available and high sensitive detection systems are on hand. These techniques could be used for in vitro assays and quantified e.g. by microscopy and CCD based readouts. The introduction of novel fluorescent dyes emitting in the near infrared range (NIR) combined with the development of sensitive detector systems and monochromatic powerful NIR-lasers for the first time permits the quantification and imaging of fluorescence and/or bioluminescence in deeper tissues. Laser based techniques particularly in the NIR-range (like two-photon microscopy) offer superb signal to noise ratios, and thus the potential to detect molecular targets in vivo. In combination with flat panel volumetric computed tomography (fpVCT), questions dealing e.g. with tumor size, tumor growth, and angiogenesis/vascularization could be answered noninvasively using the same animal. The resolution of down to 150 μm/each direction can be achieved using fpVCT. It is demonstrated by many groups that submillimeter resolutions can be achieved in small animal imaging at high sensitivity and molecular specificity. Since the resolution in preclinical small animal imaging is down to ∼10 μm by the use of microCT and to subcellular resolutions using (∼1 μm) microscope based systems, the advances of different techniques can now be combined to “multimodal” preclinical imaging and the possibilities for in vivo intravital cytometry now become within one's reach. © 2007 International Society for Analytical Cytology


Molecular and Optical Imaging––Definitions

  1. Top of page
  2. Abstract
  3. Molecular and Optical Imaging––Definitions
  4. Molecular Imaging––A Short Overview
  5. Molecular and Optical Imaging in Preclinical Tumor Research
  6. Cytometry in Whole Animal Tumor Imaging
  7. Conclusions
  8. Literature Cited

Molecular Imaging is a rapidly expanding field in which the modern tools of molecular cell biology are being married to state of the art technology for noninvasive imaging.

Within the last years, crucial improvements, notably in optical resolution, have been made. This is a very promising field which actually aims at developing different tools, reagents, and methods to image specific molecular pathways in humans and animals, e.g. those that are key targets in tumor development.

Many of these novel imaging techniques could be used for preclinical small animal imaging, for example tumor research, today. Next to fascinating high resolution, these techniques offer the opportunity to precisely measure tumors within small animals in 3D even deep in the tissues. This “anatomical” approach of visualization may in future be combined with the virtue of cytometric analysis i.e. the stochimetric analysis on the single cell level in live animals. Recent progress in whole animal fluorescence imaging makes it possible to quantitatively analyze protein expression, DNA content, or drug uptake.

The term molecular imaging could be defined as the visualization and characterization of biologic processes in living animals at the cellular and molecular level. In contradiction to classical diagnostic optical imaging, it sets forth to probe the molecular abnormalities that are the basis of disease rather than to image the final effects of theses molecular alterations (1).

Molecular Imaging––A Short Overview

  1. Top of page
  2. Abstract
  3. Molecular and Optical Imaging––Definitions
  4. Molecular Imaging––A Short Overview
  5. Molecular and Optical Imaging in Preclinical Tumor Research
  6. Cytometry in Whole Animal Tumor Imaging
  7. Conclusions
  8. Literature Cited

Molecular Imaging is not targeting the classical cross sectional imaging techniques (such as magnetic resonance imaging (MRI), CT, and ultrasonography), based on physical properties like absorption or proton density. Molecular Imaging is based on different imaging techniques using specific molecules as source for imaging contrast. Merchant-S et al. noted that this “…paradigm shift from nonspecific physical to specific molecular sources is the underlying tenet for many of the current molecular imaging research efforts” (2).

Today, molecular imaging has often become a catchword-character and is mixed with classical optical imaging techniques. In principal all modern imaging technologies are physically based on optical imaging (either by bioluminescence or fluorescence), on tomography, on fiber based microscopy, on radionuclide imaging (e.g. positron emission tomography, PET; single photon emission computed tomography, SPECT; on X-Ray volumetric computed tomography, VCT), on magnetic resonance imaging (MRI), or on ultrasound.

Each of these physically different technologies have their own particular advantages and disadvantages and the use is dependent on question and hypothesis to be tested. As Weissleder et al. pointed out that there are four conspicuous areas in which extensive endeavors will be necessary to develop (3):

  • Feasible molecular probes—in vivo affinity ligands.

  • Efficient organ and intracellular targeting strategies.

  • Amplification strategies (typical target concentrations are in the pico to nanomolar range).

  • Imaging systems with high spatial resolution and sensitivity.

Molecular and Optical Imaging in Preclinical Tumor Research

  1. Top of page
  2. Abstract
  3. Molecular and Optical Imaging––Definitions
  4. Molecular Imaging––A Short Overview
  5. Molecular and Optical Imaging in Preclinical Tumor Research
  6. Cytometry in Whole Animal Tumor Imaging
  7. Conclusions
  8. Literature Cited

Referring to questions dealing with basic tumor research, it often makes sense to combine different technologies from both areas, molecular and optical imaging.

For example, tumor growth, vascularization, and metastasis screening could be studied noninvasively using flat-panel volumetric computed tomography (fpVCT). Molecular targets (tagged by any kind of fluorescence) could then be observed on cellular and subcellular level and analyzed using near infrared laser based total body scanners like the eXplore Optix system (General Electric Healthcare, London, Canada) or macro-zoom and HBO/XBO lamp-based systems like the OV100 (Olympus Optical GmbH, Hamburg, Germany).

Refer to Table 1 for a summary of the recent technologies. Since the availability of Micro-CT (μCT)—a further development of CT technology, tumor volumes could be determinate in 3D also if located deep in the tissues of treated animals—nowadays these measurements can be done also using the fpVCT within seconds. These technologies are outlined in more detail below.

Table 1. Comparison between different preclinical in vivo imaging techniques
 Flat-panel volumetric computed tomography (fpVCT)Microcomputed tomography (μCT)Two-photon microscopy (2PM)Whole body small animal imaging system by Olympus (OV100)Near infrared fluorescence (NIR) animal imaging i.e. eXplore optix (GE Healthcare)
LabelingContrast agentContrast agentFluorochrome, Dye, VectorFluorochrome, Dye, VectorFluorochrome, Dye, Vector; all above 620 nm
Spatial resolution/resolutionHigh, ∼150 μm∼10 μmVery high, ∼1 μmDepending on the used objective;Low, ∼3,000 cells/area
Macrozoom-objective: resolution = 0.7 μm, NA = 0.43
0.14× objective: resolution = 7.6 μm, NA = 0.04
Discrimination e.g tissue/vessels/bonesExcellentExcellentExcellent (subcellular level)ExcellentPoor
Time/whole body scan∼20 s∼5 min (maximum object size of reconstruction ∼70 mm)∼2 min/frame (1024 × 1024 pix); Whole body scan unpossible,Depending on signal intensity and CCD-camera normally ∼200 μsDepending on the scan-area and resolution (∼15 min/total body scan/high resolution)
Noninvasive++
Normally used for dead specimens only
Frequent measurements++±±+
Depending on toxicity of contrast agentsNormally used for dead specimens onlyDepending on anesthesia and operation procedures (observation area must be accessible by optics)Depending on anesthesia and/or operation procedures for higher magnifications (observation area must be accessible by optics)Local temperature could be increased by NIR–laser (eye protection necessary, if whole body scan includes head area)
Time kinetics± (only size, morphology)± (only size, morphology)+++
Measurement functions3D3D3D2D only2D (pseudo 3D)
Fluorescence detection+++
Fluorescence lifetime++
Biological processes+++
Anatomical structures+++±+
Multiple detectable fluorochromes+ depending on dyes and lasers+ depending on dyes and filter setup+ depending on dyes and lasers (all >630 nm)
Tissue penetration depthTotalTotal (maximum object size of reconstruction ∼70 mm)Within μmWithin μmUp to several cm

Flat-Panel Volumetric Computed Tomography and μCT

fpVCT is a powerful new tool for noninvasive imaging of different organ systems in preclinical research. As Greschus et al. pointed out that this technique permits the acquisition of a large volume of—rather than limited—slices per rotation, with intrinsically higher resolution than is achievable with conventional CT. The system provides isotropic voxels at high resolution, which facilitates 3D visualization of the imaged anatomy, and slices reformatted at arbitrary orientations with consistently high resolution. In comparison to small animal microcomputed tomography (μCT), the fpVCT technology offers a larger field of view (∼30 cm) and dramatically shorter scanning times (2–8 s per rotation), which is desirable for animal imaging (4). fpVCT fills the gap between clinical multislice computed tomography (MSCT) and preclinical μCT systems and is highly suited for studying orthotopic and metastasizing tumor models. See the precise fp-VCT tumor measurement functions in Figure 1.

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Figure 1. fpVCT images of a human RCC (ACHN) in SCID mice. Left picture: overall tumor volume is measured with 0.790 cm3. Middle picture: Horizontal measurement of width and length. Right picture: Measurement of depth (rotated tumor). Rough calculation based on formular ((a × b × c) × 0.5) results in 0.622 cm3 and 3D fpVCT measurement results in 0.790 cm3.

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Flat-Panel Volumetric Computed Tomography and μCT in Preclinical Tumor Imaging

Up till now, computed- (CT) and magnetic resonance tomography (MRT) were used to visualize tumors and their connecting blood vessels (angiogenesis) within the body. However, the resolution of these established technologies is often not high enough to look in detail at very small vessels, supplying the tumor. Microcomputed tomography (μCT) in animal research becomes possible, but still long scan times and higher radiation doses—compared to fpVCT—are mandatory. Based on long scan times and very high radiation doses this technology is mainly applied for specimens. The main field of application for μCT is therefore the analysis of defined specimen areas instead of in vivo whole-body imaging. Even if the spatial resolution of new μCT-systems is down to ∼10 μm, fpVCT is an excellent alternative method (5) and represents an implementation of conventional computed tomography with increased isotropic resolution. The way of manufacturing of the applied detectors makes it possible to diminish the size of single detector elements. As the size of the detector elements is a significant influencing factor for the spatial resolution of computed tomography, fpVCT comes along with clearly enhanced resolution and minimized metal artifacts in comparison to modern multislice-computed tomography systems. The reduction of detector elements as well as new detector attributes enhances the graininess and diminishes the contrast resolution of soft tissues. These attributes of fpVCT are very interesting for diagnostic questions dealing with higher spatial resolution than for soft tissue resolutions. Kiessling et al. have described the advantages of fpVCT in the detection of smallest tumors and estimation of tumor growth kinetics or tumor angiogenesis (6). Our group has recently developed both an orthotopic and subcutaneous murine model of human renal clear cell carcinoma (hRCC) in SCID mice to study angiogenesis and tumor growth in hRCC. To study e.g. anti-angiogenetic effects, mice were anesthetized throughout the imaging session (∼10 min) and centered on the fpVCT gantry axis of rotation. 200 μl of an iodine contrast medium (Isovist) were applied intravenously in tail vein 30 s before imaging. We used fpVCT prototype which was developed and constructed by General Electric Global Research, Niskayuna, NY. It consists of a modified circular CT gantry and two amorphous silicon flat-panel X-ray detectors each of 20.5 × 20.5 cm2 with a matrix of 1024 × 1024, 200 μm detector elements. The fpVCT works with a step-and-shoot acquisition mode. Standard z-coverage of one step is 4.21 cm. All data sets were acquired with the same protocol: 1,000 views per rotation, 8 s rotation time, 360 used detector rows, and 80 kVp and 100 mA. A modified Feldkamp algorithm was used for image reconstruction resulting in isotropic high resolution volume data set (512 × 512 matrix, resolution about 150 μm).

What are the major advantages of this technology in preclinical tumor research? First, the fpVCT based imaging is noninvasive and can therefore be used for the initial accurate determination of tumor growth over time. Even very small tumor sizes less than ∼1 mm3 can be observed and frequent observation is possible. The limitation for frequent measurements is the toxicity of contrast media even if less toxic agents are on hand. The noninvasive measurement of tumor volumes is a milestone in animal research because the precise measurement of an initial tumor size is essential looking at pharmacological animal studies to guarantee simultaneous treatment onset (by overcoming a certain tumor volume) in all animals. Without the exact knowledge of tumor size before starting pharmacological treatment, comparable statistics between different animals (and therefore different tumor sizes but same amounts of pharmaceuticals) casts statistics into doubt.

Compared to other technologies fpVCT is very fast. Within about 20 s the animal is completely scanned. Using μCT instead of fpVCT would increase the spatial resolution, but would also increase the scan times from seconds to minutes. Using specific fpVCT-software any interesting section can be calculated and enlarged as seen in Figure 2.

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Figure 2. fpVCT images of a human RCC (ACHN) in SCID mice. Side view (A,B) and ventral view (C,D), calculated from the same image stack. See high resolution in D. Even vessels down to approximately 150 μm can be clearly observed by this method. (see red 1 mm scale bar on right side).

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Real-Time Subcellular Imaging in Live Animals by Variable-Magnification Whole-Mouse Imaging System OV100

A new highly sensitive whole-mouse imaging system OV100 is available from Olympus Optical (Hamburg, Germany) since 2006. This imaging system is equipped with both macro-optics (microZoom, magnification 16×–1.6×, Observation area in mm: 0.69–69, NA: 0.43–0.07, Resolution: 0.7–4.4 μm) and micro-optics (from 0.8× to 0.14× magnification, Observation area from 78.5 to 13.8 mm, NA from 0.04 to 0.22, and resolution from 7.6 μm down to 1.4 μm). Five objective lenses (0.14×, 0.27×, 0.4×, 0.8×, and zoom 1.6×–16×), parcentered and parfocal and a sensitive CCD-camera (Olympus DP71, Olympus Optical, Hamburg, Germany) enables imaging from macrocellular to subcellular structures. Based on the size of the region of interest identified on-screen by the user, the best objective is automatically chosen and the image displayed (see Fig. 3). Eight arbitrary specific emission filters (25 mm) and six excitation filters (35 mm) allow to detect different fluorochromes/dyes either as single channels or as combination e.g. eGFP and DsRED simultaneously. The OV100 integrates many basic needs of whole animal imaging including e.g. the removal of auto-fluorescence background and high performance in the near infrared region (NIR) range up to 1,000nm for fluorescence detection, heated stage plate, and anesthesia system next to other useful features. Next to animal tumor research this system could also be used for preclinical models e.g. of drug response and direct studies of biodistribution of different labeled compounds, for stem cell research or cardiovascular experiments. By the use of this system, Yamauchi et al. have reported multicolor imaging of cancer-cell trafficking in live mice (7). Tumor cells genetically labeled with GFP in the nucleus and RFP in the cytoplasm to study cellular dynamics are reported by Jiang and coworkers (8). A review by Robert Hoffman on real-time subcellular imaging in live animals clearly pointed out the benefits of systems like the OV100 if working with genetic labeling via eGFP and/or RFP (9, 10). Noteworthy is the plain fact, that for subcellular observations e.g. to study cancer cells within vessels, the anatomic structures must be accessible by operation even if the working distance (objective to animal) is still about 4 cm.

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Figure 3. Tumor imaging by OV100 (Olympus) from whole body overview mode (left image) to single cell level (right picture) in living animals. A dsRED marked subcutaneous transplanted human renal cell carcinoma could be detected on the left flank (left) and dsRED-positive tumor cells could also be detected within blood vessels. To visualize these tumor cells within the vessels, an arc-shaped incision was made in the abdominal skin in order to prepare a skin flap. Care was taken to avoid injury of the epigastrica cranialis vein and artery. The skin flap was spread and fixed on a flat stand. Cancer cell trafficking was carried out real time within the skin flap. Vessel structures at the inside of the skin flap were directly imaged. A high magnification range of ×1.6 to ×16 and a field of view ranging from 6.9 to 0.69 mm was used.

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Correlation Between Whole-Body Fluorescence Protein Imaging and Magnetic Resonance Imaging

In 2005, Bouvet et al. have reported the high correlation of whole-body red fluorescent protein imaging and MRI on an orthotopic model of pancreatic cancer (11). The authors pointed out the strong correlation between images taken with fluorescence protein imaging (FPI) and MRI. FPI permitted rapid, high throughput imaging without the need for either anesthesia or contrast agents. Both FPI and MRI enabled accurate imaging of tumor growth and metastasis, although MRI enabled tissue structure to be visualized as well (11).

Correlation Between fpVCT and Classical Histology in Preclinical Tumor Imaging

Noteworthy is the remarkable macroscopic correlation between invasive post therapeutic immunhistochemistry (IHC) and noninvasive fpVCT imaging as seen in Figures 3a and 3b. Even if the macroscopic correlation is outstanding the microscopic correlation is not ranking behind. In a comparison between noninvasive fpVCT and invasive classical HE-staining (Figs. 4c and 4d), the same area (pseudocyste) was measured with a discrepancy of only 100 μm. Despite of the excellent resolution by fpVCT here, this technology can not distinguish between benign oncocytoma and the malignant chromophobe RCC. For diagnosis still a histological analysis of the cellular morphology is essential.

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Figure 4. (A,B) “Surgical view” on the prepared tumor versus the noninvasive fpVCT image. See the clear correlation to the fpVCT images. The contours of the two segments within this hRCC could be detected also on the fpVCT image. (C,D) Correlation between histology and fpVCT images. The same distances are measured with 31 mm (Histology) and 30 mm (fpVCT).

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Near Infrared Range Fluorescence Imaging in Preclinical Tumor Imaging

Another noninvasive imaging technique can now be combined with the fpVCT technology. The main advantage of the fpVCT imaging is, as pointed out, the fast access to anatomic and morphological information in high resolution by the use of contrast agents. To answer questions dealing with molecular pathways or pharmacological treatments, other complementary near infrared based techniques and respective fluorochromes/dyes (CY5.5, CY7 and others) are available at present (12–14). All near infrared based animal imaging technologies rely on nonionizing radiation, typically produced by a low-intensity laser, which interacts with the tissue to emit a signal captured by a high-sensitivity photon detector or by HBO/XBO excitation in combination with CCD camera–based readout. The NIR is favored because of the tissue's low absorption properties in the spectral range between ∼650 and 900 nm (15). This spectral band is necessary to penetrate several centimeters of tissue by light (16). Near infrared photons traveling through tissue are highly scattered before either being totally absorbed by the tissue or emerging at the surface where they are detected. Since scattered photons have no preferential direction or orientation, it is possible to statistically differentiate one from another by observing the time at which they emerge from the scattering medium; this is known as Time Domain (TD) imaging [Art Technology, Handbook eXplore Optix]. The eXplore Optix® (NIR) System (General Electrics Healthcare, London, Canada), as well as comparable systems like the Xenogen IVIS system (Xenogen Corporation, Hopkinton, USA) allows longitudinal studies to be conducted in the same animal. The main advantage of this technology is the opportunity to more accurately measure the effect of intervention, disease progression, and outcome in living animals. In reference to preclinical tumor imaging, the in vivo NIR fluorescence imaging systems offer quantitative measures of intensity, localization, and reconstruction of molecular targets in 3D next to fluorescence concentration estimation and fluorescence lifetime.

Two Photon Fluorescence Microscopy

Multiphoton fluorescence microscopy is a relatively novel technique to study cell biology also in living animals. This technology is based on the quasi-simultaneous absorption of two or more photons. Multiphoton absorption was predicted in 1930 (by Maria Göppert-Mayer) and the proof-of-principle was performed in the 1960s using continuous-wave laser sources. In the 1990s the technique was further developed for biological applications (17), and in 1996 the first intravital tissue imaging using green fluorescence protein (GFP) was published by Potter et al. (18). For cell biology pulsed lasers (pulse width typically ∼100 fs) with high repetition rates from 10 to 100 MHz are used in multiphoton fluorescence microscopy. The two-photon fluorescence microscopy (2PFM) is the most common multiphoton fluorescence application in cell biology because on the one side the best performing commercially available lasers of today covers the necessary NIR spectral region 700–1,200 nm, and on the other side well characterized NIR-dyes are available. 2PFM in NIR also enhances the tissue penetration depth. By the use of two-photon laser-scanning microscopy e.g. Schwickert et al. could show in mice that germinal centres reveal a dynamic open structure (19). In a recently published paper, Boissonnas et al. could show in vivo imaging of cytotoxic T cell infiltration and the elimination of a solid tumor by the use of two-photon intravital microscopy (20). The integration of fiber optics into an imaging system for convenient delivery and collection of light has resulted in many hybrid forms of novel biomedical optical instrumentation. Bird et al. have recently described optimal temporal response in multichannel two-photon fluorescence lifetime-microscopy using a photonic crystal fibre (21). The motility and invasion of cancer cells have been investigated by live intravital tumor imaging in small animals (22).

Also the quantitative linear unmixing of the spectral variants of green fluorescent protein CFP and YFP from spectral images acquired with two photon excitation now becomes possible as recently reported by Christopher Thaler and Steven Vogel (24). Because the emission spectrum of YFP is a subset of the emission spectrum of CFP, emission filters are not capable of completely separating their fluorescent signals. The authors show that 2P excitation in conjugation with linear unmixing of spectral images can separate and accurately quantify the fluorescent signals emanating from mixed populations of CFP and YFP.

Cytometry in Whole Animal Tumor Imaging

  1. Top of page
  2. Abstract
  3. Molecular and Optical Imaging––Definitions
  4. Molecular Imaging––A Short Overview
  5. Molecular and Optical Imaging in Preclinical Tumor Research
  6. Cytometry in Whole Animal Tumor Imaging
  7. Conclusions
  8. Literature Cited

Since both, photomultiplier (2PI) and CCD-based (OV100) imaging techniques have been shown to facilitate single cell or subcellular resolution, in vivo cytometry in living animals seemed to be within one's reach. Using dual color cell labeling i.e. GFP for nucleus and RFP for cytoplasm, the OV100 now open new possibilities to measure size, nucleus–cytoplasmatic ratio, and to observe clasmocytosis (destruction of the cytoplasm) as Bouvet et al. has recently shown in an in vivo color-coded imaging of the interaction of colon cancer cells and splenocytes in the formation of liver metastases (23).

Conclusions

  1. Top of page
  2. Abstract
  3. Molecular and Optical Imaging––Definitions
  4. Molecular Imaging––A Short Overview
  5. Molecular and Optical Imaging in Preclinical Tumor Research
  6. Cytometry in Whole Animal Tumor Imaging
  7. Conclusions
  8. Literature Cited

Since different imaging technologies now become available and the images can be combined or merged, many open questions may be answered. These technologies will assist us to e.g. identify the molecular basis, fundamental pathways, and genetic origins of tumor development. In case of basic tumor research, the combination of different optical and molecular imaging technologies is absolutely reasonable. The increased interest in animal models of human cancer for drug evaluation and the deep tissue locations of tumors in these animals makes the assessment of anticancer activity difficult without the use of anatomical and morphological imaging techniques. The described advanced imaging techniques not only enable measurements of anticancer activity by determination of tumor size, tumor volume, and vascularization, but also provide information at the level of the molecular targets on cellular and subcellular levels. By optimized diagnostics e.g. with regard to initial tumor volume and a possible combination of multiple technologies within one anesthesia, thus the number of experimental animals can be probably effectively reduced–without any constriction in diagnostic or therapeutic quality. For the simple reason that some of these new and highly innovative technologies have now reached the level of single cell resolution in living animals, it should be investigated in specified activities to enable intravital cytometry. Once intravital cytometry becomes possible, this will lead to better understanding of i.e. regenerating processes and to develop new ways to corroborate the regenerative therapy. A careful estimation concerning the imaging techniques to be used to answer the raised questions is essential. Since these technologies are expensive in acquisition and the operation needs specialized staff, reasonable appropriate technologies should be fused in locally coherent areas like core facilities or competence centers.

Literature Cited

  1. Top of page
  2. Abstract
  3. Molecular and Optical Imaging––Definitions
  4. Molecular Imaging––A Short Overview
  5. Molecular and Optical Imaging in Preclinical Tumor Research
  6. Cytometry in Whole Animal Tumor Imaging
  7. Conclusions
  8. Literature Cited
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