3.1: Introduction to Light Microscopy

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Learning Objectives

Understand the importance of the microscope in microbiology.
Learn and be able to identify the parts of the microscope and their purpose.
- Learn how to calculate total magnification.
- Learn the difference between dry and oil objectives.
Define magnification, resolution, and contrast.
- Understand the difference between the virtual and real image generated by the microscope.
Gain experience using a compound light microscope: successfully turning the microscope on, focusing a specimen using oil immersion, and cleaning/caring for the microscope.

Introduction

As early as 460BC, Greek-age philosophers and physicians, like Hippocrates, hypothesized that infection and disease had natural causes found within people and their environments. Their suspicions wouldn't be proven true, however, until the 1600s with the development of the microscope. Antone van Leeuwenhoek, a Dutch merchant, is credited for being the first person to develop a microscope powerful enough to view microscopic organisms in 1675. His observation of pond water and subsequent discovery of microscopic single-celled organisms, which he called animalcules, helped launch the field of microbiology. With the development of more sophisticated and powerful microscopes, microscopy is now a key tool in the microbiology field. Microscopes are used for clinical diagnoses, observation and determination of pathogenic effects on human cells, identification of microbes in environmental samples, university research, and more. (Hartline, 2023) Thus, learning how to use and apply the microscope is a significant portion of this course.

There are three main principles of microscopy: Magnification, Resolution, and Contrast. Magnification is the ability of a lens to enlarge specimen images in comparison to the real, physical object. Objective lenses with different magnification powers are used to observe specimens. For example, an objective lens with a power of 40x will make the specimen appear 40 times larger than its original size. Magnification is a powerful tool for observing microbes, but the use of objective lenses alone is not sufficient enough to produce a clear, usable image. Resolution is the ability to identify two distinct points or regions in a microscopy image, ie the ability to identify separate bacterial cells clearly. High-resolution images will appear clear and sharp, while low-resolution images will be fuzzy. Resolution can be increased by altering the pathway of light through the microscope. The more light centered on a specimen, the clearer the image will be. We can increase the ability of the objective lens to gather light through the use of immersion oil.

Immersion Oil is a unique oil used solely on high power, wet or oil objective lenses (for this course it will be the 100x objective lens). The oil physically touches the microscope slide and objective lens, altering light refraction, and decreasing light scattering. Thus, the oil increases light exposure of the specimen and increases the resolution of the final image. With magnification and resolution perfected, microscopy still encounters the problem of contrast. Contrast is the ability to see the specimen in relation to the color of the background. Most microbes are transparent and difficult to impossible to see on a microscope. To increase the contrast of the microbial cell body versus slide background, special dyes (called stains) are used. These stains will target the external cell body, internal cell structures, or even slide background, adding color to the sample and increasing contrast.

File:Opthalmology AMD Super Resolution Cremer.png

Magnification: Through the use of lenses and light, microscopic species, like yeast (pictured above), can be easily observed.

Resolution: Sections of eye tissue were observed by microscopy. The image on the left has poor resolution and is fuzzy. The image on the right has high resolution and individual regions of eye tissue can be observed and labeled.

Contrast: By using stains, like the Gram Stain above, transparent bacterial cells can be easily observed against their background.

Table 1. These images help demonstrate the three main principles of microbiology. Magnification: increasing the size of microbes, like yeast, using lenses and light. Resolution: the ability to see cells or specimen regions as distinct, separate images. Contrast: the ability to see a specimen clearly against the color of the background.

$Photograph a shows a close-up of a lens from a brightfield microscope. The lens is nearly touching the slide below it and there is oil spanning the space between the lens and the slide. Diagram b light traveling from the light source through the microscope slide. Without oil, the light refracts as it passes through the glass. Many of these refracted light beams do not meet the lens of the microscope. With immersion oil, the light beams travel from the slide, through the immersion oil, to the microscope lens with minimal refraction.$

Figure 1. A) Immersion oil will physically touch the slide/specimen and oil-safe objective. B) Resolution can be increased through the use of oil immersion. Oil helps decrease light scattering/refraction and increase illumination of the specimen, which will make cells and specimen regions clearer.

Parts of a Microscope

In this course, you will use a Compound Light Microscope. This type of microscope uses visible light to observe specimens and is also called a brightfield microscope. Compound light microscopes are made with 2 lenses that magnify or compound their strength. Advantages of this microscope include: the ability to view living and heat prepped specimens, straightforward operation, and the ability to easily update equipment with technological advancements. Understanding the parts of the microscope and how to use them are key to success in this course.

Table 2: Compound Light Microscope Parts and Functions

1. Eyepiece and Ocular Lens: The eye piece is a structural component that contains the ocular lens and is the part of the microscope an individual looks into. The ocular lens is a magnifying lens with a power of 10x.

2. Nose Piece: A structural component that contains and rotates the objective lenses.

3. Objective Lenses: Magnifying lenses with different magnification power that observe the specimen.

4. Coarse Adjustment Knob: Adjusts the focus of the microscope by moving the stage in large increments, creating a focused "big picture" where cell details are not yet observable but the overall cell body is clearly focused. Typically used with lower power objectives like the 4x objective lense.

5. Fine Adjustment Knob: Adjusts the focus of the microscope by moving the stage in small increments, creating a sharp image of individual cells and cell details. Typically used with higher power objectives like the 10x, 40x, and 100x objective lenses.

6./9. Mechanical Stage and Stage Clips: Movable metal clips are used to hold a microscope slide in place on a platform called the stage.

7. Illuminator: Light source, typically a high-intensity light bulb located below the stage in the base of the microscope.

8a. Diaphragm: Adjusts the amount of light that passes between the condenser and specimen, altering specimen brightness.

8b. Condenser: Light passes from the illuminator to the condenser which will focus the light on the specimen.

Compound Light Microscope Handling and Care Instructions:

Improper handling and care of a microscope can damage the equipment. Make sure you read and understand the following steps to use a microscope safely and correctly.

To take the microscope out of the microscope cabinet:

Use your dominant hand to hold the arm of the microscope, and your non-dominant hand to hold the base of the microscope. (See Figure 2)
Carry the microscope upright, flush with your body. That way the ocular lenses located in the eyepiece do not fall out and prevent other microscope structures from shifting.
Make sure the electrical cord is wrapped or removed from the microscope, avoid tripping on the cord.

To return the microscope to the microscope cabinet:

Turn off the microscope.
Remove your slide. Pre-made slides go back in the Bio 225 slide-holder box. Student prepared slides are disposed of in the hard plastic sharps bin.
Turn the revolving nosepiece to the lowest power objective, the 4x.
Lower the stage all the way down.
Use lens cleaner and lens paper to wipe down the 100x oil objective.
1. The 4x, 10x, and 40x objectives can be wiped down with dry lens paper. Lens cleaner should not be used on these "dry" objectives.
Disconnect from power source.
Wrap cord around the microscope and cover with the protective plastic sheet.
Place the microscope back in the cabinet and close the cabinet door.

Figure 2. All movable microscopes should be properly supported during transport to prevent equipment damage or personal injury. When moving the microscope, one hand should always support the base and one hand should hold/support the arm.

Total Magnification

The compound light microscope is made with a pair of ocular lenses and multiple objective lenses that have different magnification powers. The Ocular Lenses are located in the eye piece of a microscope, the part of the microscope an individual looks into. The Objective Lenses are connected to a rotating nose piece and magnify the specimen at variable strengths. Since the objective lenses are the magnifying lens that physically observes the specimen, these lenses create the real image. The ocular lens observes the magnified, mirrored image created by the objectives, meaning the ocular lens looks at a virtual image. The virtual image is produced by combining the magnification power of the ocular lens and objective lens, a value called the total magnification power. Total magnification is important as it tells us how many times the original size of the specimen has been magnified. Total magnification should always be included in microscopy photos and even in the observation illustrations students will draw in this course. Total magnification is calculated with the following formula:

Ocular Lens Magnification x Objective Lens Magnification = Total Magnification

The ocular lens of the compound light microscope always has a magnification power of 10x. In this course we will use objective lenses with magnification powers of: 4x, 10x, 40x, and 100x. Thus, total magnification for this course will be:

Ocular Lens Magnification	Objective Lens Magnification	Total Magnification
10x	4x	40x
10x	10x	100x
10x	40x	400x
10x	100x	1,000x

Table 3. Total magnification is determined by multiplying the power of the ocular lens by the objective lens. In this course, we will use the 4x, 10x, 40x, and 100x objective lens. The power of the ocular lens is always 10x for the compound light microscope.

Focusing A Compound Light Microscope

Read the following steps on how to focus a compound light microscope before completing the microscope activity.

Follow instructions from "Compound Light Microscope Handling and Care Instructions:" to collect a microscope from the cabinet.
Place the microscope on a level surface, the lab bench, and plug it in such that the cord does not create a tripping hazard.
Turn the light on using the light switch located in the microscope base.
Adjust the eye piece and ocular lenses to best fit your face and viewing comfort.
Place a slide on the stage of the microscope and properly position the stage clips such that the clips "hug" the slide on either side and are not on top of the slide.
Move the stage control knobs to position the specimen directly over the path of the light.
Using the 4x objective, turn the course focus knob to raise the stage.
Look through the microscope and use the course focus knob to focus on the sample.
1. You should be able to focus on the "big picture" at this step. Cell structures and details will not be visible, but groups of cell bodies should be in focus.
2. Use the stage control knobs to slowly move the slide left, right, up, or down to find your specimen.
Switch to the 10x objective lens without changing any other settings on the microscope.
Using the 10x objective, turn the fine focus knob only to sharpen the image and focus on cell shape and structure.
1. Try not to use the coarse knob, you risk losing your specimen and focus.
When the 10x image is sharp and focused, move to the 40x objective without changing any other settings on the microscope.
Using the 40x objective, turn the fine focus knob only to modify the focus.
1. The specimen should mostly be in focus and only minor adjustments should be necessary.
When the 40x image is sharp and focused, move the nose piece such that the 40x dry and 100x oil objective straddle each end of the slide, meaning no objective lens should be touching the slide. (See Figure 3)
Add a small amount of immersion oil directly to your specimen/slide then slowly move the 100x oil immersion objective lense into place.
Using the 100x oil immersion lens, turn the fine focus knob only to modify the focus.
1. The specimen should mostly be in focus and only minor adjustments should be necessary.
Record observations then follow instructions from "Compound Light Microscope Handling and Care Instructions" to clean the microscope and return it the the cabinet.

NOTE: If your sample goes out of focus and is "lost", clean your objective lenses, wipe the oil off, go back to the 4x objective (Step 7), and re-start the focusing process.

**You CAN NOT switch between the 40x dry and 100x oil lens after oil has been added to the slide. The 40x objective is a "dry" lens meaning no liquids should touch it or it will be damaged. The 100x objective is a "wet" oil lens meaning it is specially crafted to handle liquids. Switching between the 40x and 100x lens after applying oil is going to damage the 40x objective. If you lose your sample when using the 100x objective, you must clean the lens, slide, and re-start the focusing process.

**You can change the light exposure (brightness) by using the the rheostat (a dial) located on the microscope base. This may help you focus the image when switching between objectives.

Figure 3. Oil should never touch the dry 40x objective. To place oil on a slide, move the 40x objective away from the slide, but do not bring the 100x oil objective into place yet. Instead, the 40x and 100x objective should straddle each side of the slide, leaving the specimen uncovered. Add oil to the uncovered specimen then slowly move the 100x oil objective into place.

Prokaryotic Microorganisms

Prokaryotes are ubiquitous, found everywhere, and play a key role in human welfare and disease. By using the microscope, we can visualize these potentially beneficial or pathogenic microbes to better understand their cell structure and function. In this course, we will specifically be using the microscope to observe different bacterial species. All bacterial cells have significant differences in their cell shape, arrangement, and size. These differences make up the bacteria's cell morphology. Bacterial cell morphology contributes to their pathogenicity and can be used to assist with clinical diagnosis and research. Common cell shapes we can see with a microscope are shown in Figure 4.

Figure 4. Magnification of bacteria reveals their cell shape and arrangement, their cell morphology. In this course, students will observe coccus (round cells) and bacillus (rod-shaped cells).

Real-World Connections

Malaria Diagnostic Tests

Malaria is a blood parasite caused by protozoans in the Plasmodium genus. The parasite is transmitted from mosquitoes to humans and most commonly found in regions of Asia and Africa. The disease is characterized by red blood cell lysis leading to extreme fever, chills, and organ failure or death. The “gold standard” for diagnosing malaria infections is using a microscope to identify the Plasmodium parasite.

Patients suspected to have malaria will need to submit a blood sample for microscopic evaluation. The blood samples will be spread on a microscope in a thin or thick blood smear and stained with dyes like Giemsa. Giemsa stains use multiple dyes, like eosin and methylene blue, to stain human red blood cells and the protozoan parasite. This stain allows us to see if the parasite is present inside the red blood cells.

Microscopic evaluation of malaria has many advantages. The equipment and stains needed to observe malaria are widely available. After staining, determining if the parasite is present in a patient’s blood can be completed within a few hours as the staining procedure is simple and quick to complete. False negatives and false positives are very unlikely to occur in this method as the stain is highly specific for the parasite.

Attributions

"Microbiology Laboratory Manual: Labs, 1.4 Microscopy" by Dr. Rosanna Hartline, LibreTexts: Biology, West Hills College Lemoore is licensed under CC BY-NC-SA 4.0

"Microbiology Laboratory Manual: Labs, 1.5 Get to Know the Microscope and Microbes" by Dr. Rosanna Hartline, LibreTexts: Biology, West Hills College Lemoore is licensed under CC BY-NC-SA 4.0

"Microbiology Textbook: Chapter 1, An Invisible World " by OpenStax, Digital ISBN 13: 978-1-947172-2 is licensed under CC BY 4.0

"Microbiology Textbook: Chapter 2, How We See the Invisible World" by Openstax, Digital ISBN 13: 978-1-947172-23-4 is licensed under CC BY 4

"Microbiology Textbook: Chapter 3, The Cell" by OpenStax, Digital ISBN 13: 978-1-947172-23-4 is licensed under CC BY 4.0

"Microbiology Textbook: Chapter 4, Prokaryotic Diversity" by OpenStax, Digital ISBN 13: 978-1-947172-23-4 is licensed under CC BY 4.0

"Microbiology Textbook: Chapter 25, Circulatory and Lymphatic System Infections" by Openstax, Use of CDC Materials Statement is licensed under CC BY 4.0

"Malaria: Malaria Diagnostic Tests" by Centers for Disease Control and Prevention, Use of CDC Materials Statement is in the Public Domain

"DPDx-Laboratory Identification of Parasite of Public Health Concern" by Centers for Disease Control and Prevention, Use of CDC Materials Statement is in the Public Domain

Image Citations

Figure 1, "Microbiology Textbook: Chapter 2, How We See the Invisible World" by Openstax, Digital ISBN 13: 978-1-947172-23-4 is licensed under CC BY 4

Figure 2, Modified From: "Microscope - school laboratory (1).jpg" by Kvr.lohith, WikiMedia Commons is licensed under CC BY-SA 4.0

Figure 3, Modified From: "µBrews Episode 3 – Focusing & Changing Lenses on a Microscope" by Sui Generis Brewing, Creative Commons Attribution license (reuse allowed), YouTube is licensed under CC BY

Figure 4, "Microbiology Textbook: Chapter 1, An Invisible World " by OpenStax, Digital ISBN 13: 978-1-947172-2 is licensed under CC BY 4.0

Table 1, Modified From:

"Microbiology Textbook: Chapter 1, An Invisible World " by OpenStax, Digital ISBN 13: 978-1-947172-2 is licensed under CC BY 4.0
"Opthalmology AMD Super Resolution Cremer.png" by Andy Nestl, Wikimedia Commons is licensed under CC BY-SA 3.0
"Gram stain 01.jpg" by Y tambe, WikiMedia Commons is licensed under CC BY-SA 3.0

Table 2, "Microbiology Textbook: Chapter 2, How We See the Invisible World" by Openstax, Digital ISBN 13: 978-1-947172-23-4 is licensed under CC BY 4

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