1 Lab 2 Microscopy

LAB 2 

2401 AP I

Name: __________________________ Date: ______________

Learning How to Use A Compound Microscope

A compound microscope functions by utilizing a series of lenses to magnify small specimens, making it an essential tool in advanced anatomy and physiology labs. The process begins when light from a source illuminates the specimen, which is usually placed on a glass slide on the stage. The condenser focuses the light onto the specimen, enhancing visibility and contrast. As light passes through the specimen, it is refracted by the objective lenses, which come in varying magnifications—typically ranging from 4x to 100x. The eyepiece lens further magnifies the image, allowing the observer to view the specimen in detail. By adjusting the focus knobs, users can achieve a clear image. This multi-lens system enables the observation of cellular structures, tissues, and microorganisms, making it invaluable for health science students studying the intricacies of human anatomy and physiology.

Caring for a compound microscope is crucial to ensure its longevity and proper functioning. Regular maintenance includes cleaning the lenses with lens paper to remove dust and smudges, as dirt can significantly impair image quality. After each use, the microscope should be covered to prevent dust accumulation. Additionally, it is essential to handle the instrument with care—always carry it by the arm and base to avoid damage. However, despite its advantages, the compound microscope has limitations. It may struggle to resolve larger specimens or those that are opaque, as it relies on transmitted light. Furthermore, the depth of field can be limited, making it challenging to focus on thicker specimens. Understanding these constraints is vital for students, as it highlights the importance of selecting the appropriate microscopy technique or equipment for different types of biological samples in health science applications.

Figure 1. Compound Microscope (Top figure). A. SEM B. TEM C. Light Microscope

 

Table 1.1 Parts of a Microscope

Compound Microscope Part	Function
1. Eyepiece (Ocular Lens)	The lens through which the viewer looks; typically has a magnification of 10x.
2. Objective Lenses	Set of lenses with varying magnifications (e.g., 4x, 10x, 40x, 100x) used to focus on the specimen.
3. Stage	The flat platform where the slide is placed for observation; often equipped with clips.
4. Stage Adjustment Knobs	Controls the movement of the stage vertically and horizontally for precise positioning of the slide.
5. Condenser	Focuses light onto the specimen; can be adjusted to enhance illumination.
6. Iris Diaphragm	Regulates the amount of light entering the condenser; controls contrast and resolution.
7. Substage lamp (Light Source)	Provides illumination for viewing the specimen; can be a mirror or an electric lamp.
8. Base	The bottom support structure of the microscope; houses the light source in some models.
9. Arm	The part that connects the base to the eyepiece; provides stability and support.
10. Coarse Focus Knob	Adjusts the distance between the objective lens and the specimen for initial focusing.
11. Fine Focus Knob	Allows for precise adjustments to focus on the specimen after coarse focusing.
12. Mechanical Stage	A stage with knobs that allow for precise movement of the slide in the X and Y directions.
13. Revolving Nosepiece	Holds multiple objective lenses and allows for quick switching between them.
15. Optical (Body) Tube	The part that connects the eyepiece to the objective lenses; ensures proper alignment of light.
16. Glass Slide	A thin flat piece of glass that holds the specimen; essential for sample observation.
17. Cover Slip	A thin piece of glass placed over the specimen to protect it and improve imaging clarity.
18. Parfocal Adjustment	A feature that allows the microscope to maintain focus when switching between objective lenses.
19. Field Diaphragm	Controls the diameter of the area illuminated by the light source, enhancing contrast.
20. Stage Clips	Prevents the slide from moving off the stage during observation, ensuring stability.

Activity 1.1

Instructions: Using the table above, label the microscope pictures below by write the number of the structure from the table on the lines in the microscope pics below.

Figure 2. Parts on a Nikon E200 Compound Microscope

Activity 1.2 Finding Calculations: Total Magnification, Depth of field, Field of View, and Working Distance

There are several important calculations that you need to become familiar with when using a microscope. These include the total magnification (M), depth of field (DOF), field of view (FOV), working distance (WD), and the ability to estimate the size of a specimen.

 

Table 1.2  Terms Used When Working With  a Microscope

Variable	Definition	Analogy
DO	Depth of Field: The range of distance within which the specimen remains in focus.	Think of DOF like the window of clarity in a camera lens; the larger the window, the more of the scene stays in focus.
N.A.	Numerical Aperture: A dimensionless number that characterizes the range of angles over which the system can accept or emit light.	Imagine N.A. as the size of a funnel; a wider funnel can catch more light (or liquid) from different angles.
Lambda λ	Wavelength of Light: The distance between successive peaks of a wave, typically measured in nanometers (nm) or micrometers (μm).	Consider wavelength as the length of waves in the ocean; shorter waves are like higher frequencies, while longer waves are like lower frequencies.
M	Magnification: The ratio of the size of the image of the specimen to its actual size.	Think of magnification like a magnifying glass; it enlarges the view of small objects, making details clearer to see.
FOV	Field of View: The diameter of the area visible through the microscope at a given magnification.	Imagine FOV as the view from a window; the bigger the window (or the lower the magnification), the more you can see outside.
Size of Specimen	The estimated size of the specimen based on how many can fit in the field of view.	Think of this as measuring how many people can stand side by side in a hallway; knowing how many fit helps determine individual sizes.
WD	Working Distance: The distance between the objective lens and the slide when the specimen is in focus.	Consider WD like the distance between a photographer and a subject; too close might result in blurry images, while the right distance ensures clarity.

Part A.

Finding Total Magnification

Calculating the total magnification is the first skill that you should familiarize yourself with. To calculate this value, you must multiply the magnification on the ocular times the objective lens in place. For example, if you have an ocular with a magnification of 20, and use it with the 100x objective, the total magnification would be 20 times 100x, which is 2000x.

Fill in Table 1.2 below. Show your work on how you calculated total magnification for each in the white space provided.

Table 1.2 Total Magnification Calculations

Power	Ocular Magnification	Objective Magnification	Total Magnification
Scanning
Low
High

Show your work:

Scanning

Low

High

Part B

Finding Depth of Field

Calculating the depth of field (DOF) is crucial for understanding how much of a specimen remains in focus at a given magnification level. The DOF is the thickness of the specimen where it is sharp, at a given focus level. The depth of field can be calculated using the Rayleigh criterion optics formula, and derive DOF = λ/ (N.A)² , where N.A. is the numerical aperture of the objective lens, λ is the wavelength of light (typically around 550 nm for visible light). This formula indicates that as the magnification increases, the depth of field decreases, meaning less of the specimen will be in focus simultaneously. Understanding DOF is particularly important in anatomy and physiology labs, as it helps students visualize and differentiate between structures that may be layered or closely spaced.

Calculate the DOF values using the information below by showing how the values were obtained. Use the white space provided. Use the formula, DOF = λ/ (N.A)².

Example 1 (Rayleigh Criterion)

Given:
N.A. = 0.14, λ = 550 nm (which is 0.55 μm)

DOF = 0.02806 mm = 28.06 micrometers (microns)

Using the formula for the Nikon E200 however, we get .879 if we neglect e, which is the smallest distance that can be resolved when a detector is placed in image plane of the objective. For Nikon microscopes, e can vary between 4 – 24 microns. The formulas can be found at Nikon DOF Calculator.

Example 2 (Rayleigh Criterion)

Given:
N.A. = 0.3, λ = 550 nm (0.55 μm)

DOF = 0.00611 mm = 6.11 micrometers (microns)

The depth of field values varies according to the type of microscope use. For example, the formula below is the DOF specific to a Nikon E200 Eclipse, we can select the objective ‘CFI Plan Achromat DL 10x 0.25NA’ , we can use the formula below, and get the results in Example 3, which is a DOF of 10.4 microns.

Figure 1.3 (Depth of Field formula for a Nikon microscope)

Practice Adding Numbers Where They Belong:

dtot=λ⋅nNA2+nM⋅NAe

Example 3 (Nikon vs. Rayleigh Criterion)

Given:
N.A. = 0.25, λ = 550 nm (which is 0.55 μm)

Using the formula, DOF = λ/ (N.A)²

DOF = 0.0088 mm} = 8.8, micrometers (microns)

Recalculate with Nikon DOF Formula: If we re-calculated using the Nikon E200 and an e value of 4um for a Nikon 10x objective, ‘CFI Plan Achromat DL 10x 0.25NA’, we will get a very different DOF.

DOF = 0.00104 mm = 10.4 micrometers (microns)

Notice that without the e value included, the two DOF values, 8.8 and 10.4 are not the same.

Part C.

Finding Field of View

Calculating the field of view (FOV) is essential for determining the observable area of the specimen through the microscope. The field of view can be estimated using the formula: FOV = (Field Number) / (Objective Magnification). The field number is typically provided by the manufacturer of the eyepiece and represents the diameter of the area visible through the eyepiece at a specific magnification. For example, if an eyepiece has a field number of 20 mm and the objective lens is set to 10x, the field of view would be calculated as FOV = 20 mm / 10 = 2 mm. Knowing the field of view allows students to gauge the scale of the specimen they are examining, making it easier to identify structures and their relationships within a biological sample. We can use the calculation of the field of view to estimate the size of a specimen.

Figure 1.3 Objective lens, field of view, and working distance illustration

Calculate the FOV values using the information below by showing how the values were obtained. Use the white space provided.

Example 1

Given:
Field Number = 25 mm, Objective Magnification = 4

FOV = 6.25

Example 2

Given:
Field Number = 30 mm, Objective Magnification = 10

FOV = 3 mm

Part D.

Working Distance

Calculating the working distance (WD) is vital for ensuring that the microscope can focus on the specimen without causing damage to either the lens or the slide. Working distance is defined as the distance between the objective lens and the slide when the specimen is in focus. It can typically be found in the specifications provided by the microscope manufacturer, but it can also be measured directly with a ruler or caliper. For instance, if an objective lens has a working distance of 0.2 mm, this means that the lens must be positioned 0.2 mm away from the slide to achieve optimal focus. Understanding working distance is particularly important in anatomy and physiology labs, as it aids in selecting the appropriate objective lens for various specimens while preventing accidental contact that could damage the lens or the sample.

Using a ruler, measure the working distance of your 4X, 10X, and 40X. in centimeters (cm), then fill in Table 1.3. Note that your answers must be in mm.

Table 1.3 Working Distance

Objective	Working Distance (mm)
4x
10x
40x

Describe the procedure for measuring the WD of an objective:

 

Part E.

Estimating the Size of a Specimen

Figure 1.4. Overview of how to estimate the size of a specimen

Estimation of the size of a specimen can be found a couple of ways:

Method 1 Using ocular units from a graticule

Some microscopes have an eyepiece micrometer. An eyepiece micrometer (also called a graticule), is a small glass disc with an etched measurement scale that can be placed inside the eyepiece. The micrometer needs to be calibrated by placing a stage micrometer on the microscope stage, then aligning the two micrometers, then counting the number of divisions that correspond to a known distance on the stage micrometers.

Figure 1.5. Calibration of an eyepiece micrometer

In the example above, there are 100 ocular units for the eyepiece micrometer. The stage micrometer measures 1.0 mm across, therefore, each ocular on eyepiece is 1/100, which is .01 mm. If we apply this same method Figure 2, 70 ocular units measures .46mm, therefore, each ocular unit in the eyepiece is .46/70, which is .00675 mm each.

Here are data from the Nikon E200 that has been calibrated, where ou= ocular units:

At 4x the 10ou is 1.2 mm, at 10X the 10ou is 0.5 mm, and at 40X the 10ou is 0.125 mm.

To estimate the size of a specimen, count the number of ocular units it occupies, then convert the number of ocular units to the equivalent number of mm. For example, if a specimen took up 1 ou of the Nikon microscope when viewed at 4x, this works out to be 1.2mm/10 = .12 mm for 1 ocular unit, which is what we need. Thus, the width of that specimen would be .12 mm or 120 microns.

Method 2

Another method is to use a stage ruler or small ruler and measure the FOV. Then estimate how many of the specimen you are viewing can fit in the FOV at that magnification. This is best done at the scanning objective.

Activity 1.3

Practice Problems

Use these methods below for the calculations for practice problems. Show your work.

Depth of Field: DOF = λ / NA^2
Where:

N.A. = Numerical Aperture of the objective lens

λ = Wavelength of light (typically 550 nm for visible light)

Field of View (FOV):

Field of View = Field Number /Objective Magnification
Where:

Field Number is typically provided by the manufacturer of the eyepiece

Working Distance (WD):
Working distance is typically provided in the specifications of the microscope, but can also be measured directly.

Estimating Size of a Specimen:
To estimate the size of a specimen under a microscope, one can use the formula:
Size of Specimen =Diameter of FOV/Number of Specimens that Fit Across FOV.
This method allows for an estimation of size based on how many specimens can fit within the observable field of view.

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Table 1.4 Depth of Field Practice Problems

Show your work for DOF practice problems:

Table 1.5 Field of View Practice Problems

Field Number (mm)	Objective Magnification	FOV (mm)
20	4
20	10
25	40
25	100
30	20

Show your work for FOV practice problems:

Table 1.6 Working Distance Practice Problems

Objective Magnification	Working Distance (mm)
4
10
40
100
20

Show your work for WD practice problems:

Table 1.7 Size of Specimen Practice Problems

Diameter of FOV (mm)	Number of Specimens Across FOV	Size of Specimen (mm)
5	2
10	5
4	4
6	3
8	2

Show your work for estimating the size of the specimen:

Activity 1.4 Cheek Cell Stain and Size Estimation

Pre-Prepared Slides

Materials:

3 Pre-prepared slides

Light Microscope

Warm-up: First examine 3 pre-prepared slides. Sketch them, then fill in the information below:

______________ _________________ ________________

Total magnifications

Isolate Human Cheek Cells

Materials:

Toothpick or Sterile Cotton Swab

Blank microscope slide

Cover slips

Gloves

Methylene blue

Light Microscope

Make a smear on a blank slide by gently rubbing the inside of the buccal area of your mouth with a toothpick or sterile swab and smearing it in the center of the slide. Your smear should not be larger than a dime. Place a small drop of methylene blue on the smear, then cover with a cover slip

View your smear at 4x, sketch, and calculate the total magnification and working distance at each objective:

 

 

 

___________________________ _________________

Total Magnification Working distance

 

 

View your smear at 10x, sketch in the space below, and calculate total magnification:

___________________________ _________________

Total Magnification Working distance

 

View your smear with the 40X objective:

 

 

 

___________________________ _________________

Total Magnification Working distance

 

Now estimate the size of one of your cheek cells using both Method 1 and 2. Write a description of your procedure, step by step. Describe which method you think is more accurate and why. Be sure to include the magnification you used. Take a snapshot of each method. For Method 2, be sure the micrometer is visible in your snapshot!

Method 1 Estimated size of cheek cell = _____________

Method 2 Estimated size of cheek cell = _____________