[Rice Catalyst Issue 14]

seeing THE unseen:
OPTICAL
MICROSCOPY
By Carlson Nguyen
eneath our fingertips, in the air we
breathe, and on virtually every
surface imaginable, there are countless
microbes living just like you and me.
The study of such microbes, invisible to the
human eye, can aid scientists in unraveling
mysteries regarding biological mechanisms.
This, in turn, facilitates our understanding
of many physiological conditions, such as
precancerous lesions and malignant
tumors, and the potential pathways for
treating them. In order to study these
hidden microbial worlds, we need to
develop specific tools for their imaging and
observation. For many scientists, visualization
often comes in the form of optical
microscopy techniques. However, as with
any instrument, using microscopy
techniques requires an understanding of
their purpose, capabilities, and limitations.
Here, we’ll take a peek into the world of
these microscopy techniques and how they
enable the progression of science.
Microscopes are not new inventions by any
means; they have existed since the early
17th century when the first simple (one
lens) and compound (two or more lenses)
microscopes were created—allowing for up
to a hundredfold magnification of objects. 1
These early compound models functioned
by using a lens, close to the sample, to
collect sunlight and magnify images. These
images could be further magnified by
adding another lens near the eyepiece. As
one might imagine, these images contained
many imperfections, such as color distortions,
and were nowhere near the level of
8 | C A T A L Y S T 2022-2023
quality achievable through modern
microscopes. Today, optical microscopy
techniques have been refined to achieve
greater magnification, serve specialized
functions and minimize image aberrations—allowing
for much higher resolution
Today, optical microscopy
techniques have been
refined to achieve greater
magnification, serve
specialized functions and
minimize image aberrations—allowing
for much
higher resolution and
detail in a variety of
applications.
and detail in a variety of applications.
Optical microscopy techniques can
generally be divided into two categories:
transmitted light and epi-fluorescence light.
Both are techniques that allow for the
visualization of cells and their components
but differ in their capabilities and uses.
Although they are separate categories,
these techniques are often used in
conjunction with one another to provide a
better interpretation of a biological sample.
In the first category, transmitted light
microscopy is the general term used for any
type of microscopy where the light is
transmitted from a source on the opposite
side of the specimen to the objective
lens—including the stereotypical microscope
you might find in a high school
classroom. 2 This group of techniques is
based on the differences in a sample’s
refractive index, which determines how
much a path of light is bent when it enters
the sample. Within this category, Brightfield
microscopy is the most basic form of
transmitted light microscopy where
incident light, or light that falls onto the
sample, transmits through a sample and
creates contrast through denser areas. 2,3
However, this contrast is lower than that of
other techniques; therefore, brightfield
requires thick samples of high refractive
indices or chemical staining for the best
results. This is important because staining
of biological samples will often kill cells and
prevent live-cell imaging, thus making other
visualization techniques necessary. On the
other hand, phase contrast microscopy
makes up for the brightfield limited
contrast by converting ‘phase shifts’ to a
change in the image’s light intensity. In this
case, light passes through a phase condenser,
which concentrates and defocuses light
before it reaches the sample. The diffracted
and background light passes through a
phase plate/objective lens, which slows