We just learned about the innovations in the
middle of the 20th century that resulted in the development of electron microscopy, which
allowed us to visualize things much smaller than what had previously been seen with light
microscopes.
As we said, with electron microscopes, scientists could study biological systems in more detail,
but it was still difficult to identify or distinguish between different structures with
accuracy.
Another innovation in microscopy, called fluorescence microscopy, answered this problem, providing
the scientific community with another tool to visualize and study the microscopic world.
Fluorescence microscopy functions, as one might imagine, on the principle of fluorescence.
So let’s first understand what this word means.
Fluorescence occurs when a fluorescent molecule or protein absorbs a high-energy photon which
excites an electron within the molecule.
When the electron returns to its lower energy state, this emits a photon of light that is
at a lower energy than the photon that originally excited the electron.
An example of this is certain kinds of fluorescent objects that absorb light in the UV range,
which we can’t see, and then emit visible light, a phenomenon responsible for a variety
of glow-in-the-dark commercial products.
As we recall from our study of classical physics, the energies of photons are inversely proportional
to their wavelength.
If a photon has a long wavelength, it is lower in energy, compared to a photon with a shorter
wavelength, which will be higher in energy.
By filtering, exciting, and detecting these differences in photon wavelengths, fluorescence
microscopes are able to visualize and differentiate between parts of the specimen that are labeled
with different fluorescent molecules or proteins.
To get more specific, in fluorescence microscopy the wavelengths fall within the visual spectrum,
which means the different wavelengths correspond with different colors.
For example, a photon with a wavelength of 650-780 nanometers is seen by the human eye
as red.
Different parts of the specimen are labeled with fluorescent molecules called fluorophores.
There are variations of fluorophores that are excited at different wavelengths and therefore
emit photons of different wavelengths.
This allows scientists to distinguish between different structures, as they can label structures
with different fluorophores and excite the fluorophores with specific wavelengths of
light to visualize different structures at a particular time.
When using any form of fluorescence microscopy it is important to avoid something called
photobleaching, which means exciting the fluorophore too many times, which causes the photons the
molecule emits to be too weak to be detected by the microscope.
Photobleaching can be avoided by limiting the amount of images taken of the specimen,
as well as limiting the exposure of the specimen to light after the fluorophores are added.
The initial form of fluorescence microscopy that was developed, which is still used today,
is called Widefield Fluorescence Microscopy.
For Widefield Microscopy, specimens are either fixed in place, or left alive and functioning.
The fluorophores are introduced to the specimen and can be modified in multiple ways so that
they bind to certain structures.
Most commonly, fluorophores are linked to antibodies that are made to detect and attach
to certain cellular structures, like parts of the nucleus or cytoskeletal filaments.
After the different fluorophores for the different structures in the specimen are added, the
specimen is set up between a slide and cover slip, and secured onto the stage of the microscope.
Next, the lazer emits a photon of light which passes through an excitation filter that only
allows photons of a certain wavelength to pass through.
Dichroic mirrors then angle the photon beam through the objective lens that focuses the
photon onto the sample.
The photon reaches the sample, which in turn emits a photon of lesser energy.
The emitted photons create an image that is magnified by the objective lens as it passes
through.
The emitted photons then pass through the same dichroic mirror that reflects the excitation
photon, which is possible because of its longer wavelength.
The emission photons then pass through the emission filter that separates them from the
surrounding radiation or background emissions.
The detector then detects the wavelengths of the emission photons and tells the computer
to pseudocolor, or fake color, the image with the color that was preassigned by the researcher
to that wavelength.
The image is then projected onto a computer screen.
After images are taken with each excitation filter relevant to the labeled sample, one
can save each image separately or overlay images on the computer to view the relative
locations of the labeled structures.
This technique is commonly used to explore the interactions of proteins with other proteins
or structures in a cell.
For example, a researcher looking at cell death could look at the positioning of two
proteins, first in a cell going through cell death, and then a healthy cell.
If in the dying cell the proteins colocalize, or their fluorescent images overlap, but in
the healthy cell they do not colocalize, the data suggests that these proteins interacting
have the potential to play a role in cell death.
Although, it is important to note that these images cannot prove the interaction of proteins,
but simply serve as a starting point for further research.
Another type of Fluorescence Microscopy is Confocal Fluorescent Microscopy, which is
very similar to Widefield.
The specimens are prepared the same way, and the functionality of the microscope is the
same as Widefield, except that before the emitted photon reaches the detector, it passes
through a pinhole which only lets a very small part of the image through.
This allows for better filtering of the photon emitted by the labeled structure from any
background fluorescence.
Because of the function of the pinhole, the excitation photon scans across the sample,
meaning that you only get a small part of the image at a time through the pinhole.
This allows for better resolution compared to the Widefield Microscope, which allows
scientists to look at thicker samples because of the low levels of background fluorescence.
The precision of Confocal Microscopes are so advanced that scientists can even scan
different depths of a sample and compile the images into a 3D image.
Although, this precision does have its trade offs, as the creation of the image with the
Confocal Microscope does take longer and is more likely to photobleach or destroy samples
in the imaging process.
Confocal microscopy can be used to visualize genetic material, structural components of
cells, assess the colocalization of structures, and study the viability of cells.
Although Confocal Microscopes do not have as high a resolution as electron microscopes,
unlike electron microscopes, they are able to identify and distinguish between structures
within a specimen.
Again we’ve covered quite a bit in this one tutorial, so let’s quickly summarize.
Fluorescence Microscopy uses the principle of fluorescence to visualize a specimen.
In short, the principle of fluorescence is that when a fluorescent molecule is excited
by a high energy photon it will then emit a lower energy photon.
These emitted photons are detected by the microscope to create an image of the fluorescently
labeled structure.
In Fluorescence Microscopy, specific structures are labeled with certain fluorescent molecules
called fluorophores so that they can be differentiated in the imaging process.
Widefield Fluorescence Microscopy is a standard form of Fluorescence Microscopy that is used
to visualize thin samples and structures that do not need to be viewed with high levels
of resolution.
Confocal Fluorescence Microscopy uses a pinhole to better filter the emitted photons from
the target fluorophores in the specimen, so that it can construct 3D images at a high
resolution and with little background fluorescence.
And with that, over the past few tutorials, we have gained a reasonable understanding
of the history of microscopy, the various forms that are in use today, how they work,
and what they are useful for.