My Research on Single-Molecule Localization Microscopy for Five Levels of Understanding
By Josh Prindle
UVA ChemSciComm
Primary School:
There are things much smaller than you and me. So small, in fact, that you cannot even see them with your own two eyes! How do we know this? Microscopes! Microscopes allow us to “zoom in” on the natural world. For example, if we were to use a microscope to look at a leaf you picked from a tree, we would see a bunch of oddly shaped circles called cells. It takes millions of plant cells to create just one leaf! Now, if we were to “zoom in” even further on an individual plant cell, we would begin to see even smaller things called molecules that are inside of the cell. Historically, scientists have only been able to use microscopes to see objects of a certain size, such as the size of a plant cell. Recently, a group of clever scientists figured out how to “zoom in” even further to see the tiny molecules within the plant cell, a technique known as “super-resolution” microscopy.
High School:
In school, you have probably used a microscope to visualize things that you are not able to see with the naked eye. We have had the ability to “zoom in” on the natural world for hundreds of years since the invention of the microscope in 1590! The conventional microscope allows us to “zoom in” on things that are as small as 200 nanometers, or 0.00000006 feet. Now, that may seem small, but the natural world is made up of things much, much smaller. For example, you may be aware that humans are made up things called cells. Inside of these cells are thousands upon thousands of small molecules known as proteins. As scientists, we want to study proteins and understand how they help a cell function. However, conventional microscopy approaches are unable to detect such small molecules. This is where super-resolution microscopy becomes useful, as it allows us to visualize individual proteins inside of the cell! Super-resolution microscopy is still a new scientific technique, but it has given scientists the ability to see things much smaller than we could have ever imagined.
College:
Conventional light microscopy approaches allow us to visualize cells, such as bacteria, human cells, and plant cells. However, for hundreds of years, we were unable to visualize anything smaller than the bacterial cell, which is approximately ten times smaller than a human cell. This is due to the physical nature of visible light, also known as the diffraction-limit. To put this into perspective, imagine visualizing two small objects with the conventional light microscope. If those two objects happen to be within 200 nanometers of one another, we would be unable to determine whether one or two objects are present. Therefore, the resolution of conventional light microscopes is 200 nanometers. Approximately thirty years ago, scientists discovered that the resolution limit could be broken through complex physical chemistry and optical engineering. The current state of the art single-molecule localization microscopes can achieve resolutions of up to 1 nanometer, allowing for the visualization of molecular structures with incredible detail.
Graduate Student in the Discipline:
Conventional light microscopy techniques do not necessarily need labeling strategies for visualization, yet fluorescence labeling allows the microscope user to visualize specific components of their sample. For example, in a conventional fluorescence microscopy experiment, one can label the cell membrane with an intercalating dye, allowing the microscope user to visualize the resulting fluorescence signal upon activating the fluorescent molecule. This fluorescence signal, known as the point-spread-function (PSF), is directly related to the resolution of the conventional light microscope, as it is approximately 200 nanometers (at best) in diameter. The PSF diameter cannot be made any smaller with conventional approaches due to the diffraction limit of light, so any objects within 200 nanometers of one another are unresolvable. Additionally, when exposed to the appropriate excitation wavelength, every fluorescent molecule is in a fluorescence-ON state at the same time, making it impossible for the user to distinguish one fluorophore from another. Single-molecule localization microscopy overcomes the diffraction-limit by utilizing light- or chemically-controlled fluorophores that turn on and off at different points in time. This allows the microscope user to localize individual fluorophores by capturing movies of these blinking events. Once localized, the PSF can be further mathematically manipulated through Gaussian fitting, bringing the PSF diameter and, subsequently, the resolution of the microscope to ~5-40 nanometers. Therefore, by reconstructing the individual localizations in a fluorescence microscopy movie to create a static image, scientists are now able to achieve high-resolution images of incredibly intricate molecular structures.
Expert in the Field:
The performance of fluorescence microscopy has been dramatically improved for two reasons: recently developed fluorescent probes with unique photophysics allow for precise localization of emitters in space and time, and mathematical fitting of the point-spread-function (PSF) allows for a much more accurate estimation of the emitters’ position. Historically, super-resolution fluorescence microscopy experiments have relied on imaging large, static biomolecular structures, such as actin networks, to showcase the power of the technique. While these 2-dimensional images provide incredible structural detail, imaging of static structures doesn’t allow the microscope user to capture dynamic information. In order for this to be achieved, scientists have optically engineered the PSF of the conventional light microscope so that it’s diffraction pattern above and below the focal plane is asymmetric. This asymmetry therefore allows one to localize, with super-resolution quality, PSFs in the z-dimension. Given this and the temporal control over our fluorescence signal, we can now capture dynamic information of our fluorescent molecules with super-resolution quality, such as a protein traversing the inside of a cell or a quantum dot diffusing through water. The future of single-molecule imaging is bright, as this new capability of tracking fluorescent molecules over time has opened up numerous different analytical approaches to uncover complex physical phenomena.