GROUP IV January 25, 2017

De Los Santos, Mike

Fernandez, Christeline Mae
Gaerlan, Jeisela
Genilla, Mary Rose

EXERCISE 1
THE MICROSCOPE: PRINCIPLE, PARTS, USE AND CARE

I. Introduction

Microscope comes from the Greek words, mikros which means small and
skopein which means to look. It is an instrument used to see objects that are too small
for the naked eye. In the late 17th century, Anton van Leeuwenhoek of Holland created
a simple single-lens microscope. Similar to a modern magnifier in structure, this novel
invention differed in that its magnification reached more than 200 times. This new
microscope allowed Leeuwenhoek to discover microorganisms and spermatozoa.
Around the same time, Robert Hooke of England created a compound microscope that
had two lenses, objective and ocular lenses. Hooke observed cork tissue and called it
cells because it looked like the small cells of honeycomb. Thus he coined the biological
term cell. There are many kinds of microscope but there are two most used in
laboratories, one of which is the optical microscope which uses light to capture an
image, hence its name. Another one is the electron microscope which emits electron
beam towards objects to magnify them. Magnification is the enlargement of an image
while resolution is the ability to see fine detail in an image; the latter can be quantified
by separation of two distinguishable points. The level of resolution an optical instrument
has, is dependent on the size of its aperture. The human eye is an optical instrument
that has a small aperture, called pupil. Because of its size, humans have to use
microscope when viewing objects smaller than 0.1 mm, the resolving power of human
eye.
Nowadays, a general microscope mainly consists of an objective lens, ocular
lens, lens tube, stage, and reflector. An object placed on the stage is magnified through
the objective lens. When the target is focused, a magnified image can be observed
through the ocular lens. At the end of this exercise it is expected to recognize the
different kinds of microscope, their parts, function and proper case. It is also assumed to
know how to compute the microscopic parameters like Total Magnification and
Resolving Power of each objective using Abbes equation and to know their practical
application.
II. Methodology

The Limit of Resolution was computed using the Abbes Equation:

Wherein the constant 0.61 is Abbes constant derived from coherence and
visibility. And lambda is the wavelength of light illuminating the microscope, the
wavelength of the visible white light is 550 nm. Eta is the refractive index of the medium
between objective lens and glass slide. Air has an index of 1.0, water has 1.33, linseed
oil has 1.48, cedar wood oil has 1.50 and Canada balsam has 1.52. While theta is half
the value of angular aperture also known as cone angle; it is the cone of light that enters
the objective lens.
For proper use and care, the microscope was kept in an upright position, held by
the right hand and supported by the left hand. For cleaning of lenses and other parts,
the microscope lenses were wiped and its mechanical lenses were cleaned with dry
lens paper. Xylene is the solvent used to clean hardened oil sticking from the Oil
Immersion Objective. The working distance of the objectives were measured. The
working distance usually decreases with increasing objective magnification. The
microscope was also checked if it is parfocal to avoid the breakage of cover slips, and
approximately 0.17 mm thickness cover slip was used. The mirror of the microscope
was slowly adjusted to use light that is most comfortable to the eyes. Both eyes were
kept open while viewing under the microscope, one eye into the oculars and the other
into the illustrations that are simultaneously done.

III. Results & Discussion

Table 1
Microscopic Parameters LPO HPO OIO
Numerical Aperture 0.25 0.65 1.25
Half of Angular Aperture 14.48 40.54 56.44
Angular Aperture 28.96 81.08 112.89
Resolving Power 1.34 0.32 0.27

The table was filled with the data engraved on each objectives. The angular
aperture and the resolving power were computed using the Abbes equation. Based on
the table above, the resolving power increases as the lambda increases, thus both are
directly proportional. While the resolving power decreases as the numerical aperture
increases, thus both are inversely proportional.
Table 2
Place a if you can see the object under each objectives, if not
Dimension of object LPO HPO OIO
0.08
0.01
0.29
0.15

To be able to see the object using the microscope, the resolving power should be
equal or less than the dimension of the object being observed. On the first column
under LPO, the resolving power is 1.34 micrometers while the dimension is 0.08
millimeters, therefore the object is visible through the microscope. Conversely, the
object which has a dimension of 0.29 micrometers is smaller than the resolving power,
therefore it is not visible.

IV. Conclusion

While useful, magnification is much less important than resolution. Resolution is

the ability to see fine detail in an image. Therefore the smaller the resolution, the better
we see details of the image. More simply, good resolution of two nearby points provides
a clear view of two distinct points, while poor resolution would result in a single blurry
image. Since magnifying blurry image would not serve much purpose, resolution is
clearly more important than magnification. Concave mirrors are used in microscope
rather than plain mirror because it gathers more light on the specimen, plain mirror is
not advisable because it doesnt form a real image, thus if it were to be used, theres
nothing to be visible in the eyepiece.
Viruses are not visible in the light compound microscope because the diameter of
viruses are lesser than the limit of resolution of the microscope which is 200 nm. To be
able to see much smaller objects, decrease the lambda, increase the numerical
aperture or use filters. These are some adaptions in the electron microscope making
viruses visible.

V. References

Tille, P. (2013). Diagnostics Microbiology. In P. Tille, Diagnostics Microbiology (p. 68).

Singapore: Elsevier
Parichha, A. (2016, March 31). Limit of resolution and resolving power of microscope.
Retrieved January 30, 2017, from https://www.youtube.com/watch?v=xPVG