A sharp look into the nitty gritty
At first glance, creative chaos seems to reign in biological cells. Thousands of different proteins whirl around inside and strangely find their destination. A new microscopy technique with almost unprecedented resolution reveals which protein is to be found where. Small and full of diversity - that's exactly the combination that scientists have their problems with. "Small" means not much to pick out with the naked eye. In order to be able to see something, appropriate tricks are necessary. Which wouldn't be so bad, because physicists and chemists know the structure of their crystals and molecules down to the tiniest detail. If it weren't for this unpleasant, typically biological diversity.
Because compared to the relatively monotonous tests of the physicists and chemists, the biologists are incredibly colorful. Nobody knows exactly how many different proteins are found in an ordinary cell, but the number is certainly in the thousands. And each of them is in thousands of copies. And that was just the proteins, but then there are the lipids, carbohydrates, peptides and so on. With the best will in the world, this hustle and bustle cannot be de alt with using the sophisticated methods of chemistry and physics.
The microscope remains. However, anyone who thinks of the good old lens system with mirror and slide from school days when hearing this word will have a little difficulty recognizing it when they see modern research microscopes. What's attached to the left and right these days, hissing through the path of rays and counting photons, is almost reminiscent of the apparatus in physical laboratories. What a miracle – physics has long joined forces with biology to bring diversity and precision in the microscope under a cover glass, despite all the difficulties.
US scientists led by Eric Betzig from the Howard Hughes Medical Institute are now reporting the latest success in this constant effort. Their recipe operates under the name PhotoActivated Localization Microscopy, or PALM for short, and is a kind of super-precise photographic localization system for individual protein types in the apparent chaos of the cell interior. Which only works because lasers provide the light and instead of people, computers look through the optics with sensitive sensors.
In order to detect a specific protein with PALM, however, some preliminary molecular biological work is necessary. Strictly speaking, the microscope does not see this target protein, but another one that fluoresces in the right light. However, in order for this fluorescent molecule to be able to reveal the location of the target protein, the two must be fused together. That would be difficult to do at the protein level, so it is better to manipulate the genes and attach the blueprint for the fluorescent marker directly to those of the target object. When the cell needs its protein, it uses this buggy guide to make a larger version that can glow at one end. And already PALM can warm up for his spy services.
The cell quickly undergoes the usual procedures for high-resolution microscopy beforehand: it is shock-frozen to prevent it from moving or deforming and, if necessary, cut into thin slices. As a finished specimen, it then comes into the beam path and can take a short breather here, because PALM first needs a dark reference. In a way, the calm before the lightning storm.
It really starts as soon as the laser goes into action. He stimulates the fluorescent substances on the proteins with constant light pulses. These absorb the energy and shortly thereafter emit it again in a slightly depleted form as longer-wave fluorescent light. A highly sensitive sensor, which is already sufficient for single photons, registers the light and forwards the information to a connected computer with special software.
So far the procedure would be nothing new. But PALM is busy. The system collects up to several hundred thousand individual images, in which this and that protein light up. The software uses the smeared light dots to predict exactly where the protein is likely to be and what its image would look like if it were subject to random fluctuations caused by the physical properties of the optics and the light. This creates a map of the probability density map that includes more proteins the more individual images are included in the calculations. And as the smeared fluorescent spots become accurate dots, resolution increases in rarely reached regions. The proteins are allowed to sit together up to a few nanometers before PALM can no longer distinguish them as separate objects.
Betig and his colleagues have used several examples to demonstrate that this method is in fact practicable. They examined the distribution of proteins in the membranes of lysosomes, mitochondria and the cytoplasm. Membrane-bound proteins lend themselves particularly well to such studies because they are already largely fixed and are practically nailed in place after freezing. If they were illuminated and measured from different perspectives, PALM could even create a three-dimensional map of their distribution. Admittedly, this is a dream of the future, but the scientists are already working on such plans.
Despite all the joy in the beautifully resolved images, PALM has to contend with a number of weak points. In this way, only those proteins can be localized that can readily be coupled with a fluorescent protein and neither lose their shape nor their function or simply migrate to a different location. Also, PALM is not for living cells. It takes between two and twelve hours to extract a complete set of individual images – no cell stands still for that long. But the researchers are already working on faster variants.
With PALM, the view into the apparent chaos becomes a lot sharper again. Even if - or precisely because - the new microscopy technology still needs improvement in many areas, we can eagerly await the insights it will bring us. And which tricks the biologists will learn from the physicists next. Cells always offer us enough variety to observe.
