MICROSCOPY AND SLIDE PREPARATION

NAME- MADDI SAI KRUPALI

SEC- 2A
ID- 211000547
1. Light microscopy

1.What is the difference between magnification and resolution .

A. Magnification is the ability to make small objects seem larger, such as
making a microscopic organism visible. Resolution is the ability to
distinguish two objects from each other. Light microscopy has limits to both
its resolution and its magnification.
2.What does the term parfocal mean ?
A. being or having lenses or lens sets (such as eyepieces) with the
corresponding focal points all in the same plane.
3.what is your observation on the size of each objective relative to its
magnifying power ?
A. The magnifying power of a telescope is determined by the focal length of
the objective. The longer the focal length, the more the magnification is.
The intermediate image produced by the objective lens has a size , where
is the focal length of the objective lens and is the angular size of the
source.
4. what is the effect of opening and closing the iris diaphragm ? how does it
helps in focusing ?
A. In light microscopy the iris diaphragm controls the size of the opening
between the specimen and condenser, through which light passes. Closing
the iris diaphragm will reduce the amount of illumination of the specimen
but increases the amount of contrast.
5. what is the effect of raising and lowering the condenser ? how does it
helps in focusing ?
A. lowering the condenser causes less light to get to the specimen. they
suggest to keep it fully raised because of the disadvantages it has. ... A
drop of immersion oil is used to help concentrate the light, otherwise the
image will be too dark and blurred.
6. what is the effect of immersion of oil in microscopy?
A. In light microscopy, oil immersion is a technique used to increase the
resolving power of a microscope. This is achieved by immersing both the
objective lens and the specimen in a transparent oil of high refractive index,
thereby increasing the numerical aperture of the objective lens.
7.why is the lpo placed in the position ( in other cases no objective is
placed in the position ) ,when the microscope is stored or carried .
A. The low-power objective is farther away from the stage than the other
objectives so the lens is less likely to get scrapped during handling.
8. Note the horizontal and vertical scales on the mechanical stage . What is
the function of these scale.
A. Horizontal and Vertical Scale Graticules, Electron Microscopy Sciences.
These scales are used for the measuring of lengths of specimen or
distances between points on a variety of different shaped objects.
9. What is the optimal position of the substage condenser in routine
microscopic procedure.
A. The substage condenser gathers light from the microscope light source
and concentrates it into a cone of light that illuminates the specimen with
uniform intensity over the entire viewfield. It is critical that the condenser
light cone be properly adjusted to optimize the intensity and angle of light
entering the objective front lens.
10. What is most commonly used objective in routine microbiology explain.
A. A compound microscope is the most common type of microscope used
today, which mechanism is explained earlier. It is basically a microscope
that has a lens or a camera on it that has a compound medium in between.
This compound medium allows for magnifications in a very fine scale.
12.
a. Inability to bring the specimen into sharp focus
A. Inability to bring the specimen into sharp focus. • Take the slide out,
wipe off the oil and start all over again. Start with the lower power 40X,
increase to 100X and then 400X. Finally, 1000X with oil immersion.
b. Insufficient light while viewing the specimen.
A. Insufficient light while viewing the specimen. • Adjust the field diaphragm
level to increase the amount of light through the specimen.
C. Artifacts in the microscopic field
A. Not unlike the case with photobleaching, you can minimize this artifact
by using lower illumination and shortening the exposure. Another option is
that you can also use fluorophores with longer wavelengths. Longer
wavelengths of light have less energy, and are therefore less likely to
induce ROS formation.

2.Dark field microscopy .

1.what is the best instance to use the dark field microscopy .
A. Dark-field microscopy is ideally used to illuminate unstained samples
causing them to appear brightly lit against a dark background. This type of
microscope contains a special condenser that scatters light and causes it to
reflect off the specimen at an angle.
2. Why is the dark field and the sample light when a dark field microscope
is used to examine a sample ? What is the practical benefit of a microscope
? Does it have any setbacks?
A. A dark field microscope is arranged so that the light source is blocked
off, causing light to scatter as it hits the specimen. This is ideal for making
objects with refractive values similar to the background appear bright
against a dark background.
3. What is the difference in light path of the dark field microscope against
bright field microscope ?
A. In general the dark-field image lacks the low spatial frequencies
associated with the bright-field image, making the image a high-passed
version of the underlying structure. While the dark-field image may first
appear to be a negative of the bright-field image, different effects are visible
in each..
4. What is the function of the dark field stop in the dark field microscope ?
A. Light Source: enters the microscope and hits the dark field patch stop,
which is a disc used to block light from entering the condenser and leaves
a circular ring of illumination. Condenser Lens: collects outer ring of
illumination and focuses it on the sample..

3.Phase contrast microscope.

1. What is the function of the annular diaphragm in the phase contrast
microscope.?
A. The light rays are allowed to pass through the annular groove. Through
the annular groove of the annular diaphragm, the light rays fall on the
specimen or object to be studied. At the back focal plane of the objective
develops an image.
2. What are the best practical applications of the phase contrast
microscope? Explain briefly.
A. Phase contrast is by far the most frequently used method in biological
light microscopy. It is an established microscopy technique in cell culture
and live cell imaging. When using this inexpensive technique, living cells
can be observed in their natural state without previous fixation or labeling.
3. What happens to the phase of diffracted light in comparison to
undiffracted light in a phase contrast microscope.?
A. Transparent specimens imaged by phase contrast techniques diffract
light that is retarded by one-quarter wavelength (90 degrees) with respect
to undiffracted (surround) incident illumination, whereas opaque
specimens, such as diffraction gratings, diffract light that is 180-degrees
(one-half wavelength) out of phase ...