Molecular Imaging

Equipment: Xenogen - IVIS 100 used for for:

• Bioluminescence

• fluorescence imaging

   

Bioluminescence imaging

Commonly used for Reporter Gene imaging

How does it work?

Typically a gene encoding for an enzyme such as luciferase, is inserted into tissues of the animal model of interest. Cells expressing the gene are allowed to grow for the desired period of time. A substrate for the enzyme (eg. luciferin) is then administered to the animal. The reaction between the enzyme and substrate results in emission of light by the cells expressing the luceriferase. This light is imaged by the IVIS device- essentially a very sensitive CCD camera.

What can it be used for?

Essentially to monitor gene expression in-vivo. The most simple applications use the luciferase expression as a marker of cell viability. If, for example, expression is limited to tumour cells growing in the animal, the effect of therapeutic interventions on tumour size can be determined by measuring their effect on the amount of bioluminescence produced. If used as part of a multi-cistronic vector, then the signal can be used to monitor the function of control elements or other genes in the construct.

Pros and Cons

Relatively straightforward to use, high sensitivity, low resolution. Good for superficial tissues, less good for deep-seated organs.

Fluorescence imaging

Used in either direct imaging or indirect imaging modes.

How does it work?

Direct fluorescence imaging is similar to bioluminescence imaging expect that it uses a gene that codes directly for a fluorescent protein such as GFP. The difference is that the protein needs to be excited by external photons of the required wavelength. The downside is that this produces auto-fluorescence by non-target tissues which increases the level of background.

Indirect fluorescence uses a exogenous fluorescent marker that is administered to the animal prior to imaging. The marker will bind to target tissues (such as receptors) and produce an enhanced fluorescent signal from these tissues. However background fluorescence will also arise from non-targeted marker as well as from auto-fluorescence. This latter phenomenon can be reduced by using fluorophores which absorb and emit in the Near Infra-red range (NIR imaging).

What can it be used for?

Direct fluorescence is used to monitor gene expression in vivo. Indirect fluorescence imaging can be used to measure levels of expression of target molecules to which the fluorescent marker binds in vivo. It is also possible to design fluorophores which are normally quenched but become activated as a result of a molecular event in-vivo – eg enzymatic cleavage. This potentially permits changes in expression of many biochemical processes to be monitored in-vivo.

Pros and Cons.

Relatively straightforward to use, high sensitivity, low resolution. Good for superficial tissues, less good for deep-seated organs. High background can be a problem.

PET (ie positron emitting isotopes) 

-are normally very short-lived with half-lives of minutes or a few hours. They require specialised skills and facilities for their preparation. PET tracers can interact with trans-membrane transport systems such as glucose, amino-acid or nucleotide transporters and map their activity in-vivo. PET imaging is most often used to assess changes of the levels of these metabolic functions in disease and as a result of their treatment.

SPECT (ie gamma-emitting isotopes)

-have half-lives in the range of several hours, to days to weeks. They also require specialised facilities to produce but are somewhat easier to prepare than PET tracers. SPECT tracers can bind to extracellular receptors or markers on target tissues and also be used to measure regional organ function such as cardiac, hepatic or renal function. SPECT imaging is most widely used in pre-clinical radiopharmaceutical development (prior to clinical application) but can also be used to study changes certain biochemical functions during disease progression or treatment.

Both PET and SPECT can also be used for reporter gene imaging to monitor the expression of genes that encode for enzymes or transporters for which the radiotracers are substrates.