In the previous tutorial we outlined some details regarding the history of microscopy,
and the main types that were innovated in the 20th century, which we commonly use today.
Out of all the different and fascinating-looking microscopes that scientists use to study microorganisms,
the most common one that anyone will use in their early microbiologist ventures is the
light or optic microscope.
This is also the type of microscope that just about everyone is familiar with, and would
recognize instantly from high school biology class, but there is a lot to talk about in
terms of its operation, so let’s get a closer look now.
The light microscope, as the name implies, uses light to visualize something.
The light passes through a lens which focuses the light onto the specimen.
After passing through the specimen, the light passes through the objective lens which magnifies
the image from 4 to 100 times its original size.
Next, the light travels through the tube of the microscope and the eyepiece which further
magnifies the image by a factor of 10 or 15, before it finally enters your eye.
Some light microscopes can magnify an image up to 1,200 times its original size.
In order to use a microscope properly, one must know the names of all its components,
as well as their functions.
So let’s take a look at a typical light microscope.
Any light microscope has a stage that the specimen sits on, and stage clips to hold
the specimen in place, which is usually nestled between a slide and a cover slip.
Attached to the stage is the diaphragm, which controls the contrast of the image by controlling
the amount of light from the light source that you expose the specimen to.
On the sides of the stage are the slide adjustment knobs, which are used to move the specimen
so that what you want to see is in your field of view.
Above the stage are the objectives that are held by the nosepiece, which can be rotated
so that different objective magnifications may be employed to view the specimen.
The tube of the microscope connects the nosepiece to the eyepiece, which further magnifies the
image and is the part one looks through to view the specimen.
There are also focus knobs on the side of the microscope, the larger one being the coarse
focus knob, used for larger changes in focus, and the smaller one being the fine focus knob,
used for smaller adjustments in focus.
To put these parts to use, one can start by plugging the light microscope in and turning
on the light source.
Then secure the specimen on the stage using the stage clips, before rotating the nosepiece
so that the smallest objective is in place over the specimen.
First, use the coarse focus knob to clear up the image then use the slide control knobs
to center the specimen in your field of view.
A tip for this is that the image in the microscope is inverted, so when you move the slide to
the right, it will move to the left in your field of view and vice versa.
Next, use the fine adjustment knob to refine the image.
This can also involve adjusting the eyepiece or eyepieces to further focus the image of
the specimen.
A tip for adjusting eyepieces is that if there are two eyepieces, the focus for either eye
might be different given that your eyes are not identical.
Additionally, the focus setup differs slightly between individuals with different prescriptions.
Each person may have to readjust the focus when using the microscope.
After the focus is set, rotate the nosepiece to the objective appropriate for your purposes.
Usually, between switching objects, it is necessary to use the fine focus knob to clear
up the image, but avoid using the coarse focus.
Make sure to note what objective you are on when you make observations.
Also note that the total magnification will be 10 times the objective as most eyepieces
magnify the image further by a power of 10.
It’s important that when using the 100x objective, it is set up for oil immersion
to improve the resolution of the image.
To do this, rotate the nosepiece so that the specimen is between the 40 and 100x objectives,
and add a drop of oil on top of the coverslip which sits over the specimen.
Then rotate the nosepiece so that the 100x objective is submerged in the oil and over
the specimen, without scraping the lens.
Finally, after making all of your observations with the light microscope you will need to
turn off the light source, unplug the microscope, dispose or save the specimen properly, use
lens paper to wipe off any oil on the lenses, and replace the microscope cover.
Light microscopes are commonly used to study bacteria, slices of tissue, liquid samples,
and some mitochondria.
Inverted light microscopes, which have the light source coming from the top and the objective
under the stage, are often used to study tissue cultures like cells covered by liquid media,
as cells settle to the bottom of the flask where the objective can pick up the image.
This is in contrast with a standard light microscope, with the light source below and
objective above the specimen, which would have trouble visualizing the cells through
the liquid medium.
Light microscopes are easy to use, and also relatively inexpensive, making them common
in high school and undergraduate labs.
Also, with a light microscope, one can visualize living specimens over time with a low likelihood
of damaging the specimen.
However, this microscopy technique is limited in resolution and therefore magnification,
making it impossible to see structures in as much detail as with other microscopes that
we will talk about in a moment, so let’s move forward and check out something much
more powerful.