Ocean Optics High-sensitivity Spectrometer Assists Signal Transduction Research at the Netherlands Cancer Institute
Application material and images provided by dr K. Jalink, the Netherlands Cancer Institute, Amsterdam
Microscopic and micro-spectroscopic techniques have become powerful tools to aid fundamental cancer research. We study the basic unit of life: the cell. Until a few decades ago, cells were primarily studied using biochemical and genetic tools, and indeed these studies have taught us much about the fundamental chemical reactions that make up the living cell. However, these techniques tell us virtually nothing about the localization of biomolecules in the cell, about their mutual interactions or their activation state. Furthermore, very fast reactions usually escape detection.
Microscopical techniques, of course, do report on localization, which is pivotal to our understanding of the cell. We have learned that the cell is compartmentalized: many biomolecules have their own little corner in a cell (in “cellular organelles” like vesicles, in particular membranes, or associated with the cytoskeleton) and only in that context can we apprehend their function in the cell. What was still lacking is a detailed dynamic view of the functioning biomolecules. The resolution of the microscope is limited (Abbes law) to about half the wavelength of light, or ~250 nm. Molecules are about 2 orders of magnitude smaller so we cannot see molecules, their interactions, their conformation or activity state, with even the best transmitted light microscopes.
The GFP (Green Fluorescent Protein) revolution has changed that. GFP is an auto-fluorescent protein isolated from the jellyfish Aequorea victoria (the discovery of GFP earned the Nobel Prize in Chemistry in 2008). The gene that encodes GFP was cloned (i.e., the complete DNA sequence for GFP was determined) and when we put a synthetic version of that gene into mammalian cells, the cells read the gene, translate it into proteins, and start to show green fluorescence. That means, when excited with blue light, they glow green.
Fig. 1. Green Fluorescent Protein
Funny, but that’s not why we do it. The real power of this technique lies in that it brings molecular contrast to microscopy. We can make a molecular fusion of the DNA encoding GFP with the DNA that encodes any protein we wish to study. When we put that back in a cell, it is translated into proteins, but now it is our protein of interest fused with a fluorescent label. Thus, we have equipped each copy of our protein with its own little green lamp. And under the fluorescence microscope they stand out like light bulbs far away in the night. Anything that glows must be our protein, and thus we have molecular contrast.
That was 20 years ago. In the meantime, things have evolved rapidly. Many variants of GFP have been found in nature (mainly at tropical coral reefs! Not a bad job to hunt for them) or were created artificially by mutagenesis. Variants are now available with basically any color of the rainbow. That means that we can now image many different proteins, each fused to a lamp in their own specific color, within the same cell. We can make time-lapse movies of that, and see how they behave. Some may enter the nucleus when the cell gets activated, while others may be rapidly degraded: the light is then turned off in that color. But of course we need instrumentation then, to visualize many different colors at the same time. The microscopes have rapidly evolved from simple snap-shot machines into very advanced, very expensive multi-wavelength and quantitative recording instruments, with excitation light sources at all colors and many different detection filters to separate the different fluorescent proteins according to the color of their emission.
With that comes the need to characterize all spectra of different fluorescent proteins, and to characterize and select the optimal filters for each combination of fluorescent proteins expressed in a cell. Our Ocean Optics spectrometers are invaluable. We obtained originally a USB2000, which, after many years of good service, we upgraded to the QE65000. Attached to a cuvette, the spectrometer rapidly and quantitatively takes spectra of purified fluorescent proteins in solution. We also use the flexible SpectraSuite software to quickly characterize the hundreds of light filters that are around in the lab. Just take a baseline spectrum of a white lamp, then put the filter over the spectrometer and take a second spectrum. Simply divide the two, and the filter is fully characterized in seconds. Very basic characterizations, but really essential to get the best out of your microscope … and cells!
The USB2000 already did that well, but for a second, quite advanced application we need the ultimate sensitivity of the QE65000. As said, fluorescence microscopy brought molecular contrast. But it still did not bring the high resolution needed to study molecular interactions and protein activation states. Here, another “trick” comes into play: FRET, or Fluorescence Resonance Energy Transfer. Imagine that we have two proteins that might interact, but we’ll never know because they are too small to see (even the fluorescence microscope yields blurry images of them). Now, let’s label one of them with GFP, so when excited blue, it shines green. The other one we label with a Red FP; this one we have to excite green for it to shine bright red.
Fig. 2. Spectral FRET Detection of CFP and YFP made easy
Now, when the two proteins do interact, that is, they are literally touching each other, a physical phenomenon called “resonance” takes place. When we shine only blue light onto GFP, the molecule gets in an excited state, but that excited state may hop over (by electron resonance) to the RFP. Thus, the GFP loses its energy without ever shining green, and the RFP gets excited and emits red light, without ever being hit by green light! The important thing is that this happens only at nanometer range, so only when the proteins are within molecular distance. The beauty of FRET is that it converts the fundamentally unknown (nanometer distance) into something we can easily detect: loss of green light, and gain of red signal. A simple spectrum suffices to deduce molecular interactions!
That sounds easy, but it really isn’t. FRET signals are usually very small, noisy, and the spectra of the FPs are far from ideal. The GFP and RFP spectra overlap significantly, and it thus becomes necessary to disentangle the compound spectrum mathematically. Again, this is a straightforward task with the QE65000. Any emission from the cells (whether the proteins interact or not) should be a linear combination of the GFP and RFP spectra, and of cellular background fluorescence. Each of those can be measured with precision, and the compound spectrum of the cell can be simply decomposed into S = a.GFP + b.RFP + c.AutoFluo. From that, the degree of FRET follows.
FRET has become an extremely powerful technique that we use routinely, often in collaborations with cell-biological groups here at the Netherlands Cancer Institute or from abroad. They are interested in whether two proteins interact in the cell, and if so, where. We ourselves also use FRET in so-called bio-sensors. These are genetic constructs with a fluorescent donor (e.g., GFP) and acceptor (RFP) that are made to report on some kind of cellular messenger molecule. For example, Ca2+ ions are universal and important messenger ions in the cell. A protein called Calmodulin binds to Ca2+, and it then changes shape (it folds over). When GFP and RFP are attached to either end of Calmodulin, they get in closer proximity when Ca2+ binds, and the degree of FRET increases. By simply reading that out with a spectrometer device, we get a dynamic and detailed impression of the changing Ca2+ levels in the cell. Many other messengers can be read out by this technique!
Editor’s note: The two Ocean Optics spectrometers mentioned here are legacy models that performed well but since have been enhanced in newer models. The USB2000+ Spectrometer and the QE65 Pro Spectrometer have improved electronics, optical designs and other features.
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