3. Understanding and operating an optical microscope
Blood Academy August 19, 2024
Microscopes can vary in shape and size, but they generally consist of three main parts: the head, the arm, and the base.
Head
The head is the top part of the microscope, housing the eyepieces and the prisms. The prisms split the light, allowing the image to be viewed through both eyes, providing a stereoscopic view. This component is essential for directing light to the eyepieces and creating a three-dimensional effect in the viewed image.
Base
The base, located at the bottom, usually contains the light source and is designed to be heavy for stability. This stability is crucial because any movement can disrupt the viewing experience, making it difficult to observe fine details in the specimen.
Arm
The arm connects the head to the base and serves as a handle for carrying the microscope. When transporting a microscope, it is important to support it by holding both the arm and the base to ensure safety and prevent damage to the delicate components.
Additional Microscope Components
Several additional components are important in delivering an optical microscope’s functionality. Proper handling and adjustment of these components ensure accurate visualisation and analysis of microscopic specimens. The location and function of these additional components are shown below in Figure 5.4.
Eyepiece (Ocular Lens)
The eyepiece is the lens through which the viewer looks, further magnifying the image produced by the objective lenses. The total magnification is the product of the magnification of the eyepiece and the objective lens. Typically, the eyepiece magnification ranges from 5x to 30x.
Interpupillary Adjustment
This adjustment allows the distance between the eyepieces to be changed to match the user’s interpupillary distance, ensuring a comfortable and clear viewing experience for users with different eye distances.
Nosepiece
The nosepiece holds the objective lenses and can be rotated to switch between different magnifications quickly, allowing for varied levels of detail in the observed specimen. This feature is essential for examining different aspects of the sample at varying magnifications.
Objective Lenses
Objective lenses are located in the nosepiece and provide various magnification levels, typically ranging from 4x to 100x. High-power lenses, such as the 100x, require closer proximity to the specimen and often require immersion oil. The choice of objective lens depends on the level of detail required for the examination.
Stage
The stage is the platform where the microscope slide is placed, often equipped with clips to hold the slide securely. It can be adjusted with knobs to move the slide smoothly and precisely, a feature known as a mechanical stage. These controls, usually located next to the stage, allow precise movement of the slide in the X and Y directions. This enables the user to systematically scan the entire slide area and locate specific regions of interest.
Diaphragm (Iris)
The diaphragm, or iris, regulates the amount and intensity of light passing through the specimen. It is typically a rotating disc located below the stage, allowing precise light control to enhance image quality.
Condenser
Positioned between the stage and diaphragm, the condenser consists of lenses that focus light onto the specimen, improving illumination, clarity, and contrast.
Focus Knobs
Focus knobs allow the user to adjust the focus of the microscope. The coarse focus knob makes larger adjustments for initial focusing, while the fine focus knob provides precise adjustments for detailed observation. Proper use of these knobs is crucial for achieving a clear and sharp image.
Light Source
The light source, often built into the microscope’s base, provides the necessary illumination for viewing specimens. It can be a mirror reflecting external light or an internal light bulb, with adjustable intensity to suit different specimens and magnifications.
Filters
Filters can be used to enhance contrast or specific details of the specimen. They are placed in the light path between the light source and the condenser. Common filters include blue filters to increase contrast and green filters for reducing chromatic aberration.
Oil Immersion
High-power objective lenses, typically 50x and 100x, often require immersion oil. This oil bridges the gap between the lens and the slide, reducing light refraction and enhancing resolution. Without immersion oil, images at high magnification can appear blurry due to light scattering.
A key property of immersion oil is its index of refraction, which is similar to that of glass. This similarity helps minimise light refraction, enhancing the image’s clarity and brightness.
The resolving power of a microscope is significantly improved with the use of immersion oil. By reducing light scattering and maintaining a consistent light path, immersion oil enables higher resolution and sharper images at high magnifications.
Various types of oils are used in microscopy, with the most common being non-drying synthetic oils. Some oils are formulated to be less viscous, making them easier to apply and remove, while others are designed for specific optical properties required for certain types of microscopy.
When using immersion oil, apply only a small amount to the slide. Ensure it does not contact lower power lenses, which can obscure the image. Clean any oil residue from lenses with a solvent like methanol if necessary.
Troubleshooting Common Microscope Issues
When using a microscope, encountering problems such as no light or difficulty focusing the slide can disrupt your observations. Here, we’ll address these two common issues and provide solutions to resolve them effectively.
No Light
First, ensure the microscope is securely plugged into a working power supply. Verify that the outlet is providing electricity, as an interruption in the power supply could cause the issue.
Next, inspect the condenser. If it is positioned too low, it can obstruct light from reaching the slide. Adjust the condenser to an appropriate height to facilitate proper illumination.
Examine the diaphragm to ensure it is not fully closed. The diaphragm controls the amount of light passing through the specimen. Open it to allow sufficient light to enhance visibility.
Lastly, check the light bulb for any signs of malfunction. Inspect the bulb to determine if it is burnt out or damaged. If the bulb is not functioning correctly, replace it with a new one to restore proper lighting to the microscope.
Difficulty Focusing the Slide
First, check the slide orientation to ensure it is correctly placed on the stage. If the slide is positioned upside down, the image will appear blurry. Correcting the orientation should enhance clarity.
Next, adjust the stage height. Verify that the stage is not set too high, as this can affect focus. Gradually lower the stage and attempt to refocus the image. Proper stage height is crucial for achieving a sharp image.
Ensure that the slide is securely clipped onto the stage. If the slide is not fastened properly, it can move during examination, causing blurriness. Confirm that the clips hold the slide firmly in place to maintain stability.
Also, make sure the slide is lying completely flat on the stage. If the slide is tilted or uneven, the image will appear blurry. Ensure that the slide’s entire surface is in contact with the stage for optimal focus.
Next, find the specimen in the field of view. Ensure that the specimen is within the viewing area by gently moving the slide to locate the area of interest. Proper alignment is essential for accurate observation.
If you are using an oil immersion lens, check for oil on the objective lens. Ensure there is sufficient oil between the slide and the lens, as insufficient oil can cause poor resolution. Additionally, if oil has accidentally gotten onto a dry objective lens, clean the lens carefully with lens paper or an appropriate solvent to restore clarity.
Additional Considerations
It is essential to consider the overall maintenance and care of the microscope to ensure its optimal performance and longevity. Regular cleaning and calibration of the optical components are crucial for preventing light and image clarity issues. Additionally, storing the microscope in a dust-free environment helps protect the delicate parts and ensures long-term functionality. Regular maintenance checks and timely replacement of parts can prevent many common issues and extend the life of the equipment. By adhering to these practices, the reliability and precision of the microscope can be maintained consistently.