Medical Imaging 101 pt 6: Optical

It’s been a while since I last added to my #CHMedicalImagingSeries . I’ve been working on a project the past few weeks using our IVIS Spectrum at work. So I figured it’s time to write a post about optical imaging and talk about the IVIS Spectrum.  *Optical* imaging in medical imaging is either fluorescence (FI) or bioluminescence imaging (BLI). A near-field image (think standard photograph) is used for anatomic reference. Some newer systems are starting to use CT instead of the near-field image.  FI requires tagging a ligand with a fluorophore. A fluorophore can be a drug or compound that has been labeled with a fluorophore. More recently, fluorophores can be engineered from light emitting organisms. BLI relies on light emitting organisms, more specifically, it uses the enzymes from bioluminescent organisms. The most common technique is to genetically engineer an organism/cell to be bioluminescent via introduction of luciferase (think firefly). Optical imaging is typically 2D but there are some systems that are giving either 3D or “synthisized” 3D. Optical imaging has high sensitivity and relatively poor resolution. Its strength is relatively low cost, small size, and ability to visualize cell signaling. The last part, cell signaling, is huge.

❂ Physics for raw data acquisition:

The physics are relatively simple. You need a camera and a black box for optical imaging. In the case of the IVIS Spectrum, it has a cryogenically cooled CCD camera (-90 °C).  Cooling the camera maximizes sensitivity and reduces noise. There are two images in the album below to show you how the IVIS is set up. Other systems would be similar. It is capable of both FI and BLI. For FI, various combinations of emission and excitation filter wheels allow you to focus on particular wavelengths which translates to particular fluorophores. For fluorescence imaging, you excite the sample at one wavelength (in this case with white light and filter wheels) and you detect the emitted light at a different wavelength (typically longer wavelength, lower energy). For BLI, excitation is not required. You only need the emission filters.

❂ Contrast:

For FI, autofluorescence can be a problem. In fact sometimes a special diet is used so that you reduce the fluorescence from fecal matter. However, with enough signal, you can get really good contrast. You can use fluorescent dyes or produce a number of fluorescent proteins. There is some interesting physics with quantum dots in FI but I’ll leave that for you to Google. I’ll focus on one example, green fluorescent protein (GFP) from Aequorea victoria and Renilla reniformis, a jellyfish and sea pansy respectively. The importance of GFP was recognized by awarding the 2008 Nobel Prize in Chemistry to Martin Chalfie, Osamu Shimomura, and Roger Y. Tsien for their discovery and development of GFP. A model of the GFP and fluorophore are shown in the album.

For BLI, especially firefly luciferase (from Photinus pyralis), there is excellent contrast because you only get signal where the luciferin can react with luciferase. From the Wiki: the name is derived from Lucifer, the root of which means ‘light-bearer’ (lucem ferre). Firefly luciferase has a range between yellow-green (500 nm) and red (620 nm). The sea pansy gives us both GFP and renilla luciferase (Renilla-luciferin 2-monooxygenase). In a live sea pansy, luciferase light transmission is coupled to GFP so that it emits green light. In biomedical research, renilla luciferase is used alone and light is transmitted in the blue range of the spectrum (near 482 nm).

The genes for both GFP and luciferase can be inserted into a cell or organism for the purpose of cell signaling. This is typically done using a virus to transfect the cell. If you want to detect activation of a certain cell signaling pathway, you can insert the DNA for either GFP or luciferase upstream or downstream of the gene of interest and you will visually see if you have turned the gene on or off. It’s really brilliant, if you have photons the answer is yes. If there are no photons, you didn’t get either gene to express.

❂ How is the 3D image made?

Well there really isn’t 3D optical imaging. There are tricks to make pseudo-3D but I’ll skip that.

❂ Strengths:

Optical imaging’s strength, in general, is high sensitivity, much higher than MRI or CT. You can detect a handful of labeled or transfected cells, say maybe 20 cells. FI does not require a substrate which is a strength. BLI has greater depth of penetration relative to FI. As mentioned already both GFP and luciferase can be used in cell signaling studies. That makes optical imaging a tremendous  tool. In cells, renilla luciferase can have 3-5 times the photon flux compared to firefly luciferase. However, luciferin for firefly luciferase does not have autoluminescence.

❂ Weakness:

Optical imaging has relatively low spatial resolution, is 2D, and has issues with depth of penetration (especially for FI). If the signal is weak, as it passes through the body it can be scattered so that you don’t know the true origin. Let’s say that the signal is coming from the spleen and there is a tumor on the flank of a mouse. As the signal goes from the spleen, it hits the tumor and scatters. The resultant signal may look diffuse or may look like it is coming from multiple points in the abdomen. For renilla luciferase, the substrate, coelenterazine, can oxidize on its own and release a photon, i.e., it has autoluminescence. Just like some objects have fluorescence, the contrast is poor with renilla luciferase when the coelenterazine oxidizes. Apparently renilla luciferase is great with cells but not good in vivo. For BLI, one potential weakness, is access to the substrate. If the transfected cells cannot get the substrate, e.g. luciferin, then the signal will be lost.

❂ Something unique:

Although you can do cell signaling studies with other modalities, you cannot directly detect the status of the signaling pathway as you can with optical imaging. In other modalities, you would detect a byproduct or surrogate.

❂ Example:

In the example in the album, a mouse’s heart is injected with breast cancer cells that have been transfected using a lentivirus such that the cells produce firefly luciferase. It uses human ubiquitin C promoter, which basically means that the cells are producing firefly luciferase constitutively. So in this example, the cell signaling pathway isn’t of interest. The purpose is to track the cells with high sensitivity  in a model for breast cancer metastasis. Twenty days after injection, the cells are growing in the mammary fatpads of the mouse. The key is that you can see the metastasis over time, without sacrificing the animal.

There is a lot more to write about but this is a quick overview of optical imaging in biomedical research.

#ScienceSunday

Sources and references:

GFP

http://goo.gl/JAAkrZ

Aequorea victoria

http://goo.gl/7E3JKm

http://en.wikipedia.org/wiki/Green_fluorescent_protein

Renilla reniformis

http://goo.gl/kVEf60

http://goo.gl/6Quk6e

http://en.wikipedia.org/wiki/Luciferase

IVIS Spectrum

http://goo.gl/Wkobfb