NAME: Daniela Velez Jimenez

DATE: 09/28/2023
Laboratory Report 4 (Light microscopy)
Procedure 5.12 The dissection Microscope
 Image1. Collection of images for the dissection microscope experiment

- Range of magnification: 7-30

-Distance from stage to lenses in lowest magnification: 9.6cm
-Diameter of the field in lowest magnification: 2.62cm
-Diameter of the field in highest magnification: 0.55cm
The lowest power field of view was 4.76 times greater than the highest power field of view

Procedure 5.5 Initial use of the Compound Light Microscope

Image 2. Red blood cells using 4x magnification (left) and 400x magnification (right)
 Image 3. “e” sheet slide using 4x, 100x, and 400x magnification, respectively.

Procedure 5.6 Adjusting diaphragm for contrast

 Image 4. Flower seen using 400x magnification, before and after variation of contrast.

After moving the Aperture Diaphragm Control Lever (ADCL) to the right, the visibility and the definition
of the image increases, therefore, the resolution is higher, and the image becomes clearer. When the
ADCL is moved to the left, the illumination is less concentrated, the contrast is very low, and the
resolution decreases.

Procedure 5.7 Working distance

Below are the results from the measurement of the working distance of each objective:

- Scanning objective (4x): 1.0 cm = 10 mm

- Low power objective (100x): 0.1 cm = 1.0 mm
- High power objective (400x): 0.0050 cm = 0.50 mm

To know these distances is important to prevent accidents such as the collision of the lenses
with the slide; it is also important when selecting a microscope for a specific application to
ensure it is suitable for the desired use.
Procedure 5.8 Depth of field

Image 5. Slide with three colored threads, seen using 4x, 100x, and 400x magnification, respectively.

- Using the scanning objective (4x) it is possible to determine the order of the threads.
First is located the blue one, then the yellow one and finally the red one.
- Using the high-power objective (400x) it is not possible to determine this order, when
moving the fine adjustment knob the order of the threads seem to change.

Procedure 5.9 Wet mount

 Image 6. Hay infusion seen using 400x magnification.

Procedure 5.11A Diameter and Area of the Field of View (FOV)

Using a metric ruler, the Diameter of the FOV (𝑆 𝑓𝑜𝑣𝐷 ) was measured:

𝑆 𝑓𝑜𝑣𝐷 =2.35𝑚𝑚
Then, using the following formula,

( 𝑆 𝑓𝑜𝑣𝐷 ) ( 𝑆𝑚𝑎𝑔 )
=𝑁𝐻 𝑃 𝑓𝑜𝑣𝐷
𝑁𝐻 𝑃 𝑚𝑎𝑔
𝑆=𝑠𝑐𝑎𝑛𝑛𝑖𝑛𝑔 𝑜𝑏𝑗𝑒𝑐𝑡𝑖𝑣𝑒 ; 𝑚𝑎𝑔=𝑜𝑏𝑗𝑒𝑐𝑡𝑖𝑣𝑒 𝑚𝑎𝑔𝑛𝑖𝑓𝑖𝑐𝑎𝑡𝑖𝑜𝑛
𝑁𝐻𝑃=𝑁𝑒𝑥𝑡 𝐻𝑖𝑔h𝑒𝑟 𝑃𝑜𝑤𝑒𝑟 𝑜𝑏𝑗𝑒𝑐𝑡𝑖𝑣𝑒 ; 𝑓𝑜𝑣𝐷=𝐹𝑖𝑒𝑙𝑑 𝑂𝑓 𝑉𝑖𝑒𝑤 𝐷𝑖𝑎𝑚𝑒𝑡𝑒𝑟
The NHP objective (for magnification 100x) was calculated:

( 2.35 𝑚𝑚 )( 4 )
𝑁𝐻 𝑃 𝑓𝑜𝑣𝐷 = =0.0940 𝑚𝑚
( 100 )
Finally, using the following formulas,
𝑆 𝑓𝑜𝑣𝐷
=𝑆 𝑓𝑜𝑣𝑟 =𝐹𝑂𝑉 𝑟𝑎𝑑𝑖𝑢𝑠
2

2
𝜋 ( 𝑆 𝑓𝑜𝑣𝑟 ) =𝑆 𝑓𝑜𝑣𝐴=𝐹𝑂𝑉 𝑎𝑟𝑒𝑎

The FOV radius and area were calculated:

2.35𝑚𝑚
=1.18 𝑚𝑚=𝐹𝑂𝑉 𝑟𝑎𝑑𝑖𝑢𝑠
2.00

2 2
𝜋 ( 1.18 𝑚𝑚 ) =4.37 𝑚 𝑚 =𝐹𝑂𝑉 𝑎𝑟𝑒𝑎
QUESTIONS:
1. What is the definition of resolution? Why is this ability important to a biologist or
anyone else using a microscope?
Resolution is the ability to see detail by separating characteristics of an object into individual
entities or points. It is important to biology and microscopy because it allows microscopes to
magnify very small objects (impossible to be detailed by human eye), so that the scientist can
observe specimens as a clear image and identify their components.
2. What is the resolution of the human eye? Compound light microscope? Electron
Microscope? What is the difference in magnitude between the resolution of the
human eye and the resolution of the compound light microscope? How would you
overcome this difference to view small objects using the compound light
microscope?

 The minimum distance between objects that the human eye can resolve is about 0.1mm
at 25cm from the eye.
 The theoretical limit of resolution for a light microscope is 0.20𝜇 𝑚
 The limit of resolution of the most powerful electron microscopes is 0.21nm

1 mm is 1000𝜇 𝑚, then the resolution of the light microscope in mm is 0.0002mm, which means
that is approximately 1000 times smaller than 0.1mm.
The compound microscopes use a set of two lenses (ocular and objective). Using this type of
microscope, it is possible to magnify the resolution of it, allowing the human eye to observe
organisms from different sizes. The total magnification (Mt) is the product of the magnifications
of the objective lenses (Mo) and the ocular or eyepiece lenses (Me):
𝑀𝑡=𝑀𝑜 × 𝑀𝑒
3. What is the difference between a transmission electron microscope (TEM) and a
scanning electron microscope (SEM)? When would you use one instead of the
other?
Electron microscopes use a beam of electrons as their source of illumination, instead of visible
light energy. However, there are two types of this microscopes; the TEM, where the electrons
pass through a thin section of the object producing a 2-dimensional image, and the SEM, where
the electrons scan the surface of the object producing a 3-dimensional image. For that reason, I
would use the TEM for thin specimens, for example thin films; while SEM would be very useful
for the observation of surface structures.
4. What is the major drawback with an electron microscope (EM) that doesn’t exist
with a compound light microscope?
The major drawback with Electron microscopes is that the organisms must be stained or
covered in metal atoms, to produce a clear image. Therefore, the organisms cannot be living at
the time of observation.
 5. How is scanning probe microscope (SPM) different from an EM?
A SPM is different from EM, because what scans the surface is not a beam of electrons, but
instead is a group of tips or probes. Although the resolution is very similar to the other EM, in
this case the organisms can be alive in an aqueous environment, because there is no need of
stains.
6. List the two general types of light microscopes. What is the difference between
them?
 Simple light microscope: they have only a single lens; they are small and have limited
resolution
 Compound light microscope: they have two sets of lenses; they are bigger and have
higher resolution

7. What physical phenomenon is responsible for the production of magnification?

What scholar was thought to be the first to use this phenomenon to magnify
images?
The physical phenomenon is the Refraction of light, in other words, when the light passes
through a lens, it is refracted in a specific angle, which depends on the refractive index of the
lens material and the angle of incidence of the light. Additionally, the amount of refraction
depends on the shape of the lens and the distance between the lens and the organism. It is also
used in applications such as eyeglasses, telescopes, and cameras.
The Arabian scholar Ibn al-Haytham is known as the first person to explain how lens work as
magnifiers and to deduce that the curvature of the glass, or lens is the cause of producing the
magnification.