MICROSCOPY

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Book:	MICROSCOPY

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Date:	Tuesday, 9 December 2025, 5:09 AM

1. INRODUCTION TO MICROSCOPY
- 1.1. PARTS OF A MICROSCOPE
- 1.2. HOW TO OPERATE A MICROSCOPE
2. CONCEPTS IN MICROSCOPY
- 2.1. APPLICATIONS ON THE CONCEPTS IN MICROSCOPY
3. TYPES OF MICROSCOPE
- 3.1. LIGHT MICROSCOPE WORKING PRINCIPALS
- 3.2. FLUORESCENCE MICROSCOPE WORKING PRINCIPLES

1. INRODUCTION TO MICROSCOPY

Microscopy is the technique of using a microscope to view objects and specimens that are too small to be seen with the naked eye. It provides crucial information by using magnification (making objects appear larger) and resolution (the ability to distinguish between two close objects). Key types include optical (using light and lenses), scanning probe, and electron microscopy, each with its own uses in fields like biology, medicine, and materials science.

1.1. PARTS OF A MICROSCOPE

Structural parts

Arm: Connects the head to the base and is used to carry the microscope.
Base: The bottom support of the microscope, which may also house the illuminator.

Optical parts

Eyepiece (Ocular Lens):

The lens at the top that the user looks through; it magnifies the image from the objective lens.

Body Tube (Eyepiece Tube):

Connects the eyepiece to the objective lenses.

Objective Lenses:

Lenses with different magnifications (e.g., 4x, 10x, 40x) that are mounted on the revolving nosepiece.

Revolving Nosepiece (Turret):

Holds the objective lenses and allows you to rotate them into place to change magnification.

Illuminator:

The light source, typically a built-in lamp, that shines light through the specimen.

Condenser:

A lens system located under the stage that focuses the light from the illuminator onto the specimen.

Diaphragm (Iris Diaphragm):

Controls the amount of light that passes through the specimen, affecting brightness and contrast.

Mechanical and focusing parts

Stage: The platform where the specimen slide is placed.

Stage Clips: Hold the slide securely on the stage.

Focus Knobs (Coarse and Fine):

Coarse Adjustment: Moves the stage up and down for large changes in focus.

Fine Adjustment: Moves the stage in small increments for sharp, precise focusing.

1.2. HOW TO OPERATE A MICROSCOPE

Start with the lowest power:

Rotate the nosepiece to click the lowest power objective lens into position.
Place the slide:

Secure the slide on the stage, making sure the specimen is over the hole in the stage.
Position the objective lens:

Use the coarse focus knob to move the stage up until the objective lens is very close to the slide, but not touching it.
Look through the eyepiece:

Slowly turn the coarse focus knob to move the stage away from the lens until the specimen comes into view.
Sharpen the focus:

Use the fine adjustment knob to get a clear, sharp image.
Adjust the light:

Use the diaphragm to control the amount of light. You may need to increase the light for higher magnifications.

2. CONCEPTS IN MICROSCOPY

Key concepts

Magnification:

This is the process of making an object appear larger than its actual size. A compound microscope achieves high magnification by using two lens systems: the objective lens, which is close to the specimen, and the ocular lens, which is in the eyepiece. The total magnification is the product of the magnifications of both lenses.
Resolution:

This is the ability to distinguish between two closely spaced points as separate entities. Higher resolution means finer detail can be seen. It is measured in linear units, like micrometers (

$mu m$

).
Illumination:

A light source is used to shine light through the specimen, allowing the lenses to create a magnified image. Some advanced techniques, like phase contrast, use light variations to create contrast in transparent specimens.
Contrast:

This is the difference in intensity between different parts of an image, which is crucial for visibility. Contrast-enhancing techniques can be used to make features more distinct.

2.1. APPLICATIONS ON THE CONCEPTS IN MICROSCOPY

Biology and medicine

Disease diagnosis:

Microscopes are used to identify and analyze pathogens like bacteria and viruses to diagnose diseases.
Cell biology:

They are essential for studying the detailed structure and function of cells, and for researching biological processes at a molecular level.
Medical research:

They enable the visualization of molecular-scale biological processes by using techniques like fluorescence microscopy.

Materials science and nanotechnology

Materials analysis:

Microscopes are used to examine the structure and composition of materials like metals, polymers, and ceramics.
Nanotechnology:

They are critical for studying phenomena at the nanometer scale to develop new materials and devices.
Quality control:

They are used to analyze the internal structures of electronic components, like microchips, to ensure quality and identify defects.

Forensic science

Evidence analysis:

Microscopes help analyze physical evidence such as fibers, hair, and residues found at crime scenes.
Ballistics:

They can be used to match bullets to a gun by examining the microscopic markings left on the bullet's surface.

Other industrial applications

Manufacturing:

They are used in industrial settings for tasks like inspecting parts, ensuring technical cleanliness, and analyzing material failure.
Research and development:

Microscopy is a key tool in R&D across various fields for observing and analyzing new materials and technologies.

3. TYPES OF MICROSCOPE

Light microscopes

Principle: Use light and a system of lenses to magnify images.
Examples:
- Compound microscope: Uses multiple lenses for higher magnification, often used in labs and schools.
- Stereo (or dissecting) microscope: Provides a 3D view of larger, opaque objects, used for tasks like dissection or inspecting circuit boards.
- Fluorescence microscope: Uses fluorescent light to highlight specific parts of a specimen.
- Confocal microscope: Uses lasers to create high-resolution 3D images of fluorescently labeled specimens.
Brightfield microscope: A common type that produces a dark image on a bright background.

Electron microscopes

Principle:

Use a beam of electrons instead of light to achieve much higher magnification and resolution. Cannot be used to view living cells.

Examples:

Transmission Electron Microscope (TEM): Transmits electrons through a thin specimen to create an image.

Scanning Electron Microscope (SEM): Scans a beam of electrons over a specimen's surface to create a detailed 3D image.

Scanning probe microscopes

Principle:

Use a physical probe that moves across the surface of a specimen to map its topography.

Examples:

Atomic Force Microscope (AFM): A type of scanning probe microscope that uses a physical probe to "feel" the surface of a specimen at an atomic level.

3.1. LIGHT MICROSCOPE WORKING PRINCIPALS

A light microscope works by passing light through a specimen, which is then magnified by two sets of lenses: the objective lens and the eyepiece lens. The objective lens creates an initial magnified, real image, and the eyepiece lens further magnifies this intermediate image, creating a larger, virtual image that the observer sees. A light source, condenser, and diaphragm control and focus the light for illumination.

Detailed breakdown:

Illumination:

A light source, such as an LED or bulb, shines light up through the stage where the specimen is placed.
Light control:

The diaphragm and condenser regulate and focus the amount of light passing through the specimen to create contrast and clear illumination.
Magnification:
Objective lens: The first lens, positioned close to the specimen, gathers light and produces an initial, magnified "real image".
Eyepiece lens: The second lens, or eyepiece, magnifies the real image from the objective lens, creating a final, much larger "virtual image" that is visible to the viewer's eye.

Image formation:

The final image is a magnified, inverted version of the original specimen because of the way the light is bent and focused by the lenses.

Observation:

The viewer looks through the eyepiece to see this final, highly magnified image

3.2. FLUORESCENCE MICROSCOPE WORKING PRINCIPLES

A fluorescence microscope works by exciting a fluorescent dye (fluorophore) in a specimen with high-energy, short-wavelength light, causing it to emit lower-energy, longer-wavelength light. An excitation filter selects the specific wavelength from the light source, which is then reflected by a dichroic mirror onto the specimen. The emitted fluorescent light is then transmitted back through the same dichroic mirror and an emission filter, which blocks any remaining excitation light, before being detected by a camera or the observer's eye.

Step-by-step working principle

Excitation:

Light from a specialized light source (like a mercury vapor lamp or laser) passes through an excitation filter that selects a specific wavelength.
Reflection:

The filtered excitation light is reflected by a dichroic mirror (or "dichroic beamsplitter") at a 45° angle towards the objective lens.
Illumination:

The objective lens focuses the excitation light onto the fluorophore-labeled specimen.
Emission:

The fluorophore absorbs the excitation light and emits light at a longer wavelength (a different color).
Transmission:

This emitted light travels back through the objective lens and the same dichroic mirror, which is now designed to transmit the longer wavelength of the emitted light.
Filtering:

The emitted light then passes through an emission (or "barrier") filter that blocks any residual excitation light, allowing only the desired fluorescent light to reach the detector.
Detection:

The filtered fluorescent light is then captured by a detector, such as a camera or the observer's eye, to form a bright, colored image of the specimen against a dark background