It's getting clot in here.
This is a blog about the biochemistry of blood clotting. The focus will be on drugs that influence the formation of blood clots in humans.
illuminated-shrooms
IMAGINE THAT: BIOLUMINESCENCE IN MEDICINAL IMAGING
Modern
medicine wouldn’t have gotten as far as it has without the development of medicinal
imaging. This technology is used every day for the diagnosis of illness,
cancers, and diseases for millions of patients around the world, and gives us
the amazing ability to detect and study diseases as they progress right down to
the molecular level - and it even helps to save lives.
So how can we make this technology better, cheaper, and more accessible? Bioluminescent imaging might just be the answer we’re looking for.
The birth of the GFP:
Using molecules that have a natural “glow” has been a staple in biological studies for decades. This all began in the 1960s (almost 60 years ago) when a marine biologist discovered a natural fluorescent protein derived from a crystal jellyfish off the coast of Japan. With its fluorescent properties, this protein was able to absorb ultraviolet light and, in return, release its own light as a green glow. It wasn’t long before researchers discovered that this protein could be attached to cellular components, such as proteins and DNA, and used to observe cellular processes at a molecular level.
This marked the discovery of the infamous Green Fluorescent Protein (GFP) - a protein that has become a key asset in the study of biological systems, even to this day.
Figure 1: Structure and activation of the Green Fluorescent Protein.
This was only the beginning of “harnessing nature’s glow” to help further biological and medicinal research.
The power of medicinal imaging:
In order to begin looking at the use of bioluminescence in medicinal imaging, we first need to understand what medicinal imaging is. Medicinal imaging is the use of non-invasive technology to produce images of anatomical structures that are usually hidden from our sight. Imaging can be used for observing internal systems and tissues in real time, and to detect abnormalities within these systems, thus allowing for fast and easy diagnosis of diseases.
Over the years there have been many different methods for observing internal processes. Some of the more well-known methods of imaging include X-ray radiography, CAT scans, Magnetic resonance imaging (MRI), and ultrasound - and these are just to name a few. Although these imaging methods are non-invasive procedures, many of them are way too expensive for practical use and can also be potentially harmful to patients (for example x-ray radiation).
Fluorescent vs bioluminescent imaging: what’s the difference?
Another highly used imaging method is fluorescent imaging. Fluorescent imaging is an ideal examination technique that uses compounds called fluorophores. These compounds are capable of emitting light after being excited by an external light source. When these compounds enter an excited state they become unstable, and as the excited material stabilizes within the excited state, energy is released as heat. When the material relaxes back down to a ground state, photons of energy are re-released from the material in the form of light.
Figure 2: Fluorescence at its finest. This figure shows the energy levels involved in light release of a fluorophore. A is the energy from the external light source, B is the energy lost through heat, and C is the light energy released by the fluorophore. The E’s represent the different excitation states, with E0 being the ground state.
Different fluorescent stains and markers can be used to tag molecules within cells for observational studies. One of the more commonly used fluorescent markers is the infamous GFP that I mentioned earlier.
Unfortunately fluorescent imaging has many limitations, the first being that fluorophores lose their ability to give off light over time. This is due to an event called photobleaching - which refers to damage that accumulates within the fluorophore from the constant excitement of its electrons –this causes the fluorophore to slowly break down and also reduces the time allotted for observation.
Although the use of fluorescent imaging allows us to observe living cells in their natural habitat, it also leaves our cells open to phototoxicity. This is a toxic response that occurs when light travelling from an external light source (needed to excite the fluorophore) comes in contact with our cells. Phototoxicity can be compared to UV damage from sun rays. (And we definitely know that UV damage is not good for our skin!) Fluorophores are also known to generate reactive chemical species which can enhance this phototoxic effect.
To bypass the downfalls of fluorescent imaging, researchers have been looking into the possibilities of using bioluminescent imaging instead.
The Bioluminescent difference:
Bioluminescent imaging (BLI) is a sensitive, and relatively new, tool that is used to detect light energy given off from bioluminescently tagged cells and tissues to study in-vivo pocesses. Unlike fluorescent imaging, BLI does not need an external light source, since it produces its own light.
BLI works by adding bioluminescent reporter proteins to the tissues, cells, or molecules are being studied, and these natural reporter molecules are derived from bioluminescent organisms such as fireflies (D-luciferin), cnidarians (Coelenterazine and Renilla luciferin), and bacteria (the LUX operon). These bioluminescent markers can be added to cells in different ways. A couple of examples include: using modified DNA (ex: transgenic mice) or using antigens and antibodies specific to the target cells to attach the reporters molecules.
The light produced through BLI is too small to be seen with the naked eye. Imaging technology like charge coupled cameras are used to detect the light through the tissue. This camera device works by converting the incoming photons into electrical charges. These electrical charges can then be converted into an image.
Figure 3: An example of how BLI works. 1. Bioluminescent reporter tags are added to the cell to be studied, in this case cancer cells. 2. Luciferin is then added via injection to activate the luminescent enzymes and produce light. 3. The light from the reporter molecules is then visualized using CCD cameras allowing for observation of the cancer cells.
Although BLI has only been used in small in-vivo animal studies so far, it still shows a lot of promise in someday becoming a mainstream imaging technique.
The main problem with BLI is that it depends on the presence of oxygen as well as the addition of luciferin in order to work. It is also not yet powerful enough for use in larger animal testing, mostly due to the fact that BLI signals do not travel very far through tissue, so visualization is restricted to only a few centimeters deep.
But beside these few issues, there are plenty of reasons to use bioluminescent imaging!
The benefits to using BLI include the fact that it does not need an external light source, so cells involved in the screening are not exposed to any phototoxicity and remain damage free. BLI is also much cheaper compared to other imaging methods (for example x-ray machinery), it is non-invasive, and it is an easy way to visualize a variety of in vivo cellular events as they are happening in real time. BLI can be used to study and diagnose a very wide range of molecular functions and diseases, including: gene function, cell trafficking, tumor development in cancers, disease progression, protein-protein interactions, bacterial and viral infection progression. It can also be used to test the effectiveness of newly developed drugs and antibiotics.
Future Prospects for this technology:
So what can we do with this technology?
The medicinal potential of bioluminescent imaging is enormous. With this technology we might someday be able to map brain activity in a new level of detail, or track electrical impulses translating into muscular actions. It could be used as an alternate process for diagnosis of diseases, replacing radioactive or other harmful techniques that had been previously used, or it could be merged with other imaging techniques to look into deeper tissues. BLI could also be used as an aid in drug discovery in pharmaceuticals by acting as a visual aid to determine the effectiveness of drugs.
And with millions of blood tests and scans and operations being performed every day, using bioluminescent imaging could revolutionize the way we see things both medicine and in science.
Since this technology is still developing, there is a ways to go before it’s ready for human use, but who knows where this technology could take us next? Bioluminescent imaging might someday even become as famous as the GFP.
-Lauren
Additional readings and references:
http://www.the-scientist.com/?articles.view/articleNo/41699/title/Picturing-Infection/
http://www.biolume.net/tumorlight.htm
Papers on bioluminescent imaging:
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2713342/pdf/PROCATS26537.pdf
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3274065/pdf/sensors-11-00180.pdf