Optical Imaging (OI)

Molecular imaging by means of light detection


Optical imaging (OI) is a sensitive tool for molecular imaging, based on the detection of light emitted from vital cells or tissues. The two most often used optical imaging approaches rely on fluorescence or bioluminescence as a source of light. The emitted light is detected by sensitive, intensified and cooled charge-coupled device (CCD) cameras.

Advantages of OI compared to other imaging modalities:

  • relatively low cost
  • high sensitivity of signal detection
  • short imaging time (in order of seconds to minutes)
  • allows for longitudinal monitoring of a disease course within the same animal.

On its way to the surface light passes through different types of tissue thereby scattering and absorption of light take place. Therefore exact spatial localisation and quantification of signal intensities are impaired and the bioluminescent image obtained represents only a 2 dimensional skin reflection of the 3 dimensional, deeper located signal source.

Bioluminescence imaging (BLI) requires genetic engineering of the cells or tissues to image with a reporter gene that encodes one of a number of light-generating enzymes (luciferases).
For in vivo fluorescence imaging (FLI) fluorescent proteins or dyes are used, which need external excitation for light emission. The most commonly used fluorescent proteins are the green fluorescent protein (GFP) and the red fluorescent protein (RFP). A major advantage of those proteins is the independence of any substrate. The major disadvantage of fluorescence imaging is auto-fluorescence from endogenous molecules such as haemoglobin and cytochromes, causing significant background signals. 


Fluorescence detection of RFP (left) and GFP (right): Gli36dEGFR-cells were infected in culture with a virus allowing the expression of GFP (permanent) and RFP (regulated by doxycycline). The expression of RFP was induced by addition of doxycyclien in a part of the cells. Subsequently, induced and non-induced cells were implanted subcutaneously. Both cell types show permanent expression of GFP. However, RFP-expressing cells can only be detected after induction with doxycycline (Kodak Image Station 2000).

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Detection systems

Intensified detectors use photocathodes to convert captured photons to electrons that are amplified, converted back to photons using a phosphorscreen, and finally detected on the CCD. Dependent on the photocathode used, the light detection is limited to specific regions of the light spectrum (bialkali photocathodes are best at detecting light in the blue-green range but perform less well above 600nm; gallium arsenide photocathodes extend the spectrum into the red range). CCD cameras can be cooled down to -120°C to reduce the thermal noise, greatly improving the signal-to- noise ratio while preserving the spectral sensitivity of the CCD.

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Luciferases are enzymes that can generate visible light through the oxidation of an enzyme-specific substrate in the presence of oxygen (and sometimes other co-factors like ATP). Luciferases are present in certain bacteria, marine crustaceans, fish and insects. Because mammalian tissues do not naturally emit bioluminescence, the advantage of in vivo BLI is the inherently low background signal.

The most commonly used luciferases are the ones from the North American firefly (Photinus pyralis) and the sea pansy (Renilla reniformis). The firefly luciferase enzyme converts the substrate D-luciferin to oxyluciferin, resulting in green light emission at 562nm. The renilla luciferase enzyme uses the substrate coelenterazine to produce blue light at 482nm.


Technical equipment

An industrial cooperation with Berthold allows us to study certain applications by use of the Berthold Nightowl Camera. Furthermore, in collaboration with the Centre for Molecular Medicine Cologne (CMMC) we have access to the IVIS 200 system (Xenogen).



Bioluminescence imaging of luciferase expressing cells. Cells with regulated expression of luciferase show luciferase activity only in the presence of the inducer doxycycline (Dox). (top – Dox, bottom + Dox; Xenogen, IVIS 200)

Bioluminescence image of a growing glioma. The growth profile of a luciferase expressing tumour is displayed by means of bioluminescence after injection of the substrate D-luciferin.