Hey everyone, QuickBike Chemistry Basics here. Let's talk about super resolution microscopy. Super resolution microscopy is a microscopy technique that gives highly resolved images of the specimen at the molecular level. Technically, super resolution microscope is a modified confocal microscope.
First, let's try to understand the function of a confocal microscope. Confocal microscope uses a laser as a source of excitation light. The laser is focused in a small region of the specimen and it scans the specimen in x and y direction. As the laser scans the specimen, the images of each region of the specimen are taken and combined into one single image using a software. Now here comes an interesting thing.
What if we zoom the confocal image and try to see the fluorescence of the single excitation spot. When we do this the image gets blurred. This is because a single excitation spot of laser can have many closely spaced fluorescence molecule that cannot be resolved. So the question is how can we achieve a super resolution?
The idea of getting super resolution is simple. If we can decrease the excitation spot of laser then we can have individual fluorescent molecules to give fluorescence. However, according to laws of physics, it is practically impossible to have such a small excitation spot. This phenomenon is known as the point spread function. To understand point spread function, consider an imaginary example where the water waves try to meet at a point.
You will notice that the diameter of a central bulge region where the water waves meet cannot be less than the water waves. A similar situation arises with light. When light is focused in a small region using a convex lens, the smallest spot that the lens can focus cannot be less than the wavelength of light. This phenomenon is known as the point spread function. For example, if the light uses 500 nanometers, the smallest spot that a convex lens can focus cannot be less than 500 nanometers.
Same thing happens with the confocal microscope. The smallest spot of a laser that can be focused cannot be less than the wavelength of excitation light. To get super resolution, we must try to break the point spread function and get a very small excitation spot.
So the next question that arises is how can we break the point spread function? According to laws of physics, it is practically impossible to break the point spread function. To solve this problem, scientists use a brilliant idea.
Look carefully. In a confocal microscope, what we ultimately detect is the fluorescence in the specimen. Now, what happens in fluorescence?
The electron absorbs the radiation of specific wavelength and gets excited. When this electron comes back to ground state, the fluorescence is emitted. Now here comes a brilliant idea.
This excited electron can be re-excited to higher energy level such that when it comes back to the ground state, the fluorescence emitted is of a different wavelength. This phenomenon is known as stimulant emission. Now the microscope has an emission filter that allows only one wavelength to pass while the other wavelengths are blocked.
In this case the filter allows only green light to pass while it blocks the other wavelength. As the other wavelengths are blocked, this fluorescence is no longer detected and hence it is called fluorescence depletion. Let's see what happens in fluorescence depletion.
The excitation laser is focused on the specimen as a result of which the fluorescence molecule within the range of the excitation spot gives fluorescence. In the next step, depletion lasers focus around the excitation spot. This results in the depletion of fluorescence.
Only the fluorescent molecule in the center of the excitation spot gives fluorescence, while the surrounding molecules of the fluorescence is depleted. When the objective focuses this fluorescent molecule, the image formed is blurred. This happens because of the point spread function.
Even though the molecule may be few nanometers in diameter, the smallest spot that an objective can focus cannot be less than the fluorescence wavelength emitted by the molecule. Because we are using depletion laser, we already know there is a single molecule giving fluorescence. Hence, using a computer software, we tell the computer to consider only the fluorescence of the central pixels of the point spread function.
The rest of the fluorescence is ignored and this is how a single molecule is located. Further, if we notice the image carefully, the image of the molecule is simply a spot. This is because of the fact the dimension of the molecule is so less, the resolution of the molecule is diffraction limited.
Hence, whatever the shape of molecule is, it is only seen as a single spot.