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Microscopes are found in almost all fields of science, and are used to make small objects more easily visible to the naked eye. Depending on the type, they are used in various areas, from general biology to quantum physics.
Microscopes are organized and grouped based on what method of magnification they use. Optical microscopes, for example, use light and a series of mirrors to produce a magnified image of a sample. The two main categories of optical microscopes are simple and compound.
The difference between simple and compound microscopes is that simple microscopes only use one mirror, while compound microscopes use multiple. The most common optical microscopes use visible light as the light source. However, microscopy utilizes other light spectrums as well.
Fluorescence refers to the emission of light due to the absorption of light. When a material with GFP or other fluorescent dye is exposed to electromagnetic radiation, it absorbs that radiation and re-emits some of it. The time difference, less than a millisecond, between when the organic or inorganic object absorbs the radiation and emits it is short due to the photons’ inherent absorption and emission time.
The object will stop emitting fluorescent light once it stops being hit by radiation. When the materials emit light after the radiation source has gone, it is called phosphorescence. The fluorescent objects’ re-emitted light from the fluorochromes has lower energy and thus a lower wavelength signature, making it exploitable in different analytical techniques.
An example of fluorescence can be seen when one uses a blacklight, and previously unseen colors suddenly are illuminated. Other common examples of autofluorescence include minerals and various bioluminescent animals and plants like algae, fish, and insects.
A fluorescence microscope is a type of light microscope that exploits fluorescence and phosphorescence to identify and observe specific microscopic objects. The area of the material to be observed is dyed with fluorophore tags that will illuminate only the intended parts of the sample.
The specimen is struck with electromagnetic radiation of a specific excitation wavelength that is subsequently absorbed by the fluorophores, which then fluoresce radiation with longer wavelengths than the incident light. This difference in wavelengths can then be used to filter out the light from the light source and only analyze light from the fluorescent sample by using a spectral emission filter.
Detailed imaging of live cells, antibodies, and more can be easily achieved using the fluorescence microscope with proper microscopy techniques. The main components that make up these imaging systems include:
Different filter cubes and beam splitters are chosen depending on the specific fluorescent protein used to label the specimen. Single fluorophores are imaged at one time, meaning that a narrow wavelength or one “color” that is emitted from the sample is observed at one time.
High-resolution multi-colored images can be composed by sewing multiple one-color images together. Understanding how to properly perform your fluorescence imaging is critical to fluorescence microscopy since running a sample too many times can cause photobleaching, causing the fluorescent molecules to stop responding.
The most widely used type of fluorescent microscope is epifluorescence or epi-illumination microscopes.
Most microscopes have light travel from the light source, through the sample, and into the objective lens where the image magnification occurs, deriving the refractive index. This means the light source shines from beneath the sample. In epifluorescence microscopy, the specimen is illuminated from above. This means that the emitted fluorescence and the illuminating light both travel through the same objective lens.
Due to the excitation light traveling through the objective lens to get to the sample and not the other way around, less diffraction occurs overall, meaning less light needs to be filtered out to isolate the fluorescent light.
A problem that can occur in fluorescence microscopes is that in thicker objects, the light fluorescing from the background matter will drown out the light fluorescing from the object’s surface.
TIRF microscopes are used to focus on just these surface-bound fluorophores’ emitted light. This is done by using evanescent waves for excitation of the fluorophores rather than direct light from a lamp. Evanescent fields or waves are spatially concentrated in the area where they occur.
This means that when these waves occur, they only excite the fluorophores on the sample’s surface. TIRF’s sub-micron surface selectivity makes it the primary method for single-molecule detection due to its specificity.
For a long time, most of the scientific community accepted the fact that optical microscopy would never be used to observe nano-dimensional objects. The conventional wisdom was that the limitations on the resolution of wavelengths that can be obtained would limit the usefulness of optical microscopy.
The 2014 Nobel Laureates in Chemistry, however, has completely changed this. Eric Betzig, William Moerner, and Stefan Hell were all awarded the Nobel Prize in Chemistry “for the development of super-resolved fluorescence microscopy.” Their discovery of super-resolution microscopy has brought optical microscopy into the nano dimension, making visualization at the 250-nanometer range simple.
In 2000, Stefan Hell developed stimulated emission depletion (STED) microscopy, which utilizes two lasers to achieve nano-level microscopy. One laser is used to fluoresce specific molecules, while the second is used to cancel out undesired wavelengths.
Eric Betzig and William Moerner developed single-molecule microscopy, which allows them to turn molecules’ fluorescent qualities on and off again. Using multiple images with different molecules, either on or off, they can also achieve nano-level microscopy. These are revolutionary techniques that allow scientists to take a better look at the world around us.
Nanoscopy techniques are now being used to look at synaptic behavior between nerve cells in the brain to better understand diseases like Alzheimer’s, Parkinson’s, and Huntington’s disease.
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