Fluorescence tomography

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Measurement of STL-6014, a potential bacterio chlorophyll (BChl), which has been supplemented with an RGD sequence and accumulates in the necrotic parts of a breast cancer tumor of the type MDA-MB-231 -RFP. The tumor was previously implanted in a female CD-1 nude mouse .

The fluorescence tomography is in the in vivo diagnosis used imaging technique . It is a special form of diffuse optical tomography . With fluorescence tomography, the distribution of fluorophores in biological tissue can be recorded and quantified in three dimensions. The high sensitivity of the method enables it to be used for molecular imaging . The method is mainly used in research and in preclinical studies .

Other names for fluorescence imaging, such as in the literature fluorescence imaging (Engl. Fluorescence imaging ), common. In the English-language specialist literature, there is as yet no uniformly used name for this method. The terms fluorescence molecular tomography, fluorescence tomography , fluorescence (-enhanced) optical tomography , or fluorescence optical diffusion tomography , among others, are used.

Procedure

The types of radiation transport in body tissue
Basic sketch for the construction of a fluorescence tomograph for small animals.
A device for in vivo fluorescence imaging for small animals.
Fluorescence imaging of an orthotopic implant of a pancreatic carcinoma of a mouse. The imaging is used to determine the tumor volume. The mouse was photographed two weeks after the injection of human pancreatic tumor cells of the type XPA-1. Red fluorescent protein (RFP) was used as the dye. Series A – C shows the anesthetized mouse. Image A is a fusion image of visible light and fluorescence image. Panel B shows the fluorescence of the tumor marked with RFP (not quantitative). Image C is a monochrome quantitative recording of the fluorescence of the tumor from image A + B. In rows D-F, the abdominal wall of the mouse was opened and the recordings from A to C were repeated. The better resolution of the images can be clearly seen, since the disturbing influences of the abdominal wall are missing.
The accumulation of STL-6014 in the necrotic tissue of orthotopic breast cancer tumors implanted in CD-1 nude mice. Line A: Fusion image of daylight / “NIR” image (575–650 nm) of the mouse, shows the entire red fluorescent tumor.
Line B: Fusion image of daylight / NIR image (810–875 nm) of the mouse, shows only the necrotic areas of the tumor
Row C: Fusion image daylight / NIR image of the excised tumor
Row D: Daylight image of the excised tumor
Row E: Histological section through the stained tumor ( HE staining )

Fluorescence tomography is usually carried out in the near infrared range (NIR). In the range from about 700 to 900 nm wavelength, the body tissue has only a low level of light absorption . The most important thing here is the low absorption of hemoglobin and water . In “typical” tissue with 29 percent fat and 8 percent blood, hemoglobin is responsible for 39 to 64 percent of the absorption of the NIR and is therefore the determining factor. In this “spectral window” of 700 to 900 nm, the radiation from fluorescent dyes that emit in the near-infrared region of the spectrum can penetrate the tissue relatively well. The residual absorption, together with the scattering effects of the tissue, is the limiting factor of the method, which currently limits the application to small tissue volumes, superficial fluorophore concentrations and small animals without fur (for example nude mice ). The scattering effects are caused by different refractive indices of extra- and intracellular structures. The scattering of photons on cell membranes and cell organelles is one of the main problems of all optical imaging methods. Due to the development of time-selective processes, it is now possible to separate the strongly scattered photons from the less strongly scattered photons for imaging. Further advantages of the NIR range are the low level of autofluorescence of the body tissue and - compared to X-rays , computer tomography (CT) and the nuclear medicine methods positron emission tomography (PET) and single photon emission computer tomography (SPECT) - the safe form of the non- ionizing Radiation .

The resolution of fluorescence tomography in small animals can ideally reach down to the submillimeter range. The penetration depth is limited to a maximum of about 50 mm.

Before the examination, the test animal is given a fluorescent marker - usually intravenously . The process of dye distribution and accumulation in the target tissue can be observed in a time-resolved manner. The animal's body is irradiated with a NIR light source. This is a NIR usually laser , nm, for example, with an emission wavelength of 780, which scans the surface of the animal (scan) . The irradiated object is recorded with an NIR camera, for example a CMOS camera with a corresponding filter. The camera only records the longer-wave infrared radiation emitted ( Stokes shift ) and not the light from the laser (excitation source) absorbed by the filter. Recordings can be made of the animal from different directions. For this purpose, the animal is usually rotated around the fixed camera. In a data processing system, the various recordings can be combined to form a 3D film . In addition, the volume of the tissue marked with the NIR fluorescent dye - for example a tumor - can be recorded quantitatively.

In many cases, recordings in visible light are also made to better localize the position of the fluorescence. These can then be superimposed together with the fluorescence images to form fusion images.

Fluorescent biomarkers

Fluorescence biomarkers, consisting of a ligand and a fluorophore, are mostly used for fluorescence tomography . In special cases, non-conjugated fluorophores can also be used as “contrast media”, for example in angiography for burns. With indocyanine green (ICG), a NIR fluorescent dye for use as since 1959 diagnostic in man admitted . Every conjugation with a ligand leads to a new non-approved substance, a new chemical entity (NCE). No conjugated fluorescent biomarker is currently approved for use in humans.

Ligands

In principle, the compounds that are also used in nuclear medicine are suitable as ligands. For example, peptides , proteins (for example monoclonal antibodies or their fragments) or aptamers can be used for conjugation with a fluorophore for fluorescence tomography.

Fluorophores

The fluorophores used in model organisms are essentially NIR fluorescent dyes, especially from the group of polymethines , such as cyanines . However, these organic dyes have some intrinsic disadvantages in their application. The quantum yield in water is usually below 15 percent. As a rule, only one dye molecule can be attached to each ligand molecule and the dyes tend to degenerate ( photobleaching ) after prolonged exposure . These disadvantages limit the use of organic dyes, in some cases considerably. An alternative to this are quantum dots (Engl. Quantum dots ) of semiconductor materials that do not have these disadvantages, but very questionable elements such as arsenic , selenium or cadmium may contain, which rule in vivo use in humans in principle.

The plasma half-life for indocyanine green is only 3 to 4 minutes. For many applications this is too low a value. Encapsulation in micelles can significantly increase the plasma half-life of ICG.

Potential uses

In addition to the versatile pre-clinical use of fluorescence tomography, intensive work is being carried out on the application of this method in human diagnostics. One focus is the in-vivo diagnosis of cancer, especially breast cancer . The good accessibility of the breast for imaging and the occurrence of tumors mostly near the surface are beneficial for fluorescence tomography. Since this is also a procedure without ionizing radiation, no long-term consequential damage is to be expected from this side, as is repeatedly discussed in the case of mammography, for example ( radiation exposure ). The fluorescence mammography has the potential for rapid and cost-effective screening method for breast cancer. The Schering AG introduced in 2000 a two- glucosamine modified molecules indocyanine green as a potential contrast agent for NIR mammogram before (NIR-1 designation). It is a non-specific binding contrast agent. No approval has yet been obtained for use in humans. A similar substance is KC 45 . In 2007, promising results were published with a special fluorescence biomarker with which microcalcifications , a typical deposit of malignant breast tumors, can be visualized. On the device side, prototypes of small devices for breast cancer diagnosis (hand-held probes) are now available.

In principle, fluorescence tomography is also suitable for imaging the lymph flow and assessing the sentinel lymph node .

Fluorescence tomography could also be used to stratify patients ( stratified medicine , stratified medicine ), especially in oncology. This determines whether a patient's tumor expresses certain stratification markers (for example HER2 / neu ) and whether the therapy (in the example trastuzumab ) is even indicated .

An elegant approach is the use of fluorophore-polymer conjugates, which are only activated to fluorescence through the catalysis of certain enzymes that are mainly overexpressed in tumor cells . The fluorescence had previously been extinguished .

Novel markers for fluorescence tomography are also being developed for the early detection of rheumatoid arthritis . With conventional X-ray diagnostics, this clinical picture is usually diagnosed at a very advanced stage. An earlier diagnosis can have a positive effect on treatment options and success.

Strengths and weaknesses of fluorescence tomography

Fluorescence tomography is a highly sensitive method with which even the smallest amounts of a suitable fluorophore can be detected. The sensitivity approaches that of nuclear medicine procedures such as PET or SPECT and is far superior to magnetic resonance tomography (MRT). The method is - compared to other tomography methods - comparatively inexpensive; in terms of equipment investments , equipment operation ( operating costs ) and the execution of a scan. The procedure does not require any radiation exposure and is suitable for the representation of structures and functions.

The disadvantage is the low information content, which is caused by scattering effects. This problem increases with increasing tissue depth and the achievable spatial resolution decreases drastically, with fatty tissue additionally intensifying the effect. In larger animals or even in humans, internal organs cannot currently be represented in a usable form.

See also

further reading

Individual evidence

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Web links

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