A.

Darkfield

Principle:

A dark field microscope uses an opaque disk that is placed underneath the condenser lens, this
is done so that the only light that is scattered by objects on the slide can reach the eye. The light
is then reflected by particles on the slide instead of coming up through the specimen; in this
microscope, everything can be seen regardless of color. The usual image is bright white against
a dark background. Objects that are pigmented usually seen in “false colors”, that is, the reflected
light is of a color different than the color of the object.

Special Feature:

This microscope is ideal for unstained, transparent and absorb little or no light organisms such
as algae, plankton, diatoms, insects, fibers, hairs, yeast and protozoa as well as some minerals
and crystals, thin polymer and some ceramics; examining external details, such as outlines,
edges, grain boundaries and surface defects than internal structures is more useful in this
microscope.

Function:

There are other microscopes that are capable of dark field illumination such as stereo and
standard compound microscopes. In bright field illumination, the object is lit from the stage,
resulting in a larger, contrasted image that can be studied while in a dark field microscope
blocks this central light with a condenser so that oblique rays hit the object.

Application:

Dark field microscopy is also used in the research of live bacterium, as well as mounted cells
and tissues

Sources:

Caprette, D. R. (2012, August 10). Dark Field Viewing. Retrieved January 28, 2019, from
https://www.ruf.rice.edu/~bioslabs/methods/microscopy/dfield.html

Anderson, H. (2018). Using a Dark Field Microscope - Buyer's Guide, Uses and Advantages.
Retrieved January 28, 2019, from https://www.microscopemaster.com/dark-field-
microscope.html
B. UV
Principle: Ultraviolet (UV) microscopy is a type of light microscopy that utilizes UV light to
generate a magnified image of the sample being analyzed. As a result of the shorter
wavelength of UV light than visible light, it is possible to view samples with greater
magnification and resolution.

Special Feature: Improved image resolution and increased contrast enhancement are
the primary strength of UV microscopy. The resolution of an optical microscope depends
on the wavelength of the light source. The short wavelength of UV light helps to improve
the image resolution beyond the diffraction limit of optical microscopes using normal
white light. The response of the sample to UV light is greater than that achieved by use
of white light, in respect to the surroundings. As a result, there is an increased contrast
in the image created by the microscope so that it is easier to view the samples.

Function: UV microscopy is a type of light microscopy that uses UV light to view samples
at a greater resolution than is possible with visible light. The light source typically ranges
from deep blue to UV light wavelengths (180-400 nm) to give a magnification
approximately double that which can be achieved with white light.

Application:

UV microscopy uses UV light as the source for the microscope, rather than visible light.
UV light has a wavelength between 180 and 400 nm, which is less than the wavelength
of visible light (400-700 nm). The resolution power of a microscope is directly
proportional to the wavelength of the light. This means that it is possible to observe
smaller objects with a light source that has a smaller wavelength. In fact, UV microscopy
is able to produce magnification of images to a high resolution, approximately double
that of visible light.

Sources:
Smith, Y. (2019, January 25). What is Ultraviolet Microscopy? Retrieved January 28, 2019, from
https://www.news-medical.net/life-sciences/What-is-Ultraviolet-Microscopy.aspx

C. Confocal
Principle:

The confocal microscope uses fluorescence optics. Instead of illuminating the whole
sample at once, laser light is focused onto a defined spot at a specific depth within the
sample. This leads to the emission of fluorescent light at exactly this point. A pinhole
inside the optical pathway cuts off signals that are out of focus, thus allowing only the
fluorescence signals from the illuminated spot to enter the light detector.

Special Feature:
As a distinctive feature, confocal microscopy enables the creation of sharp images of the
exact plane of focus, without any disturbing fluorescent light from the background or
other regions of the specimen. Therefore, structures within thicker objects can be
conveniently visualized using confocal microscopy. Furthermore, by stacking several
images from different optical planes, 3D structures can be analyzed. The sample
penetration depth is limited, however, when using confocal microscopy. Thicker objects,
like large spheroids, organoids, tissue, and small animals, should instead be optimally
imaged using two-photon microscopy or LSFM.

Function:

By scanning the specimen in a raster pattern, images of one single optical plane are
created. 3D objects can be visualized by scanning several optical planes and stacking
them using a suitable microscopy deconvolution software (z-stack). It is also possible to
analyze multicolor immunofluorescence staining using state-of-the-art confocal
microscopes that include several lasers and emission/excitation filters.

Application:

Confocal microscopy is broadly used to resolve the detailed structure of specific objects
within the cell. Similar to widefield fluorescence microscopy, various components of
living and fixed cells or tissue sections can be specifically labeled using
immunofluorescence, for example, and then visualized in high resolution.

Sources:

Microscopy Techniques and Culture Surfaces: Find the Perfect Match. (n.d.). Retrieved
January 28, 2019, from https://ibidi.com/content/216-confocal-microscopy