1.3 Microscopy

Introduction

The discovery and study of cells progressed with the invention of microscopes. Without microscopes, cells could not have been discovered or studied, because most cells are too small to be seen with the naked eye.

Definition

Microscopy is the technique used to view objects that cannot be seen by the naked eye.

In simple words: Microscopy is the use of microscopes to see very small things.

Core Concept

The microscopes that you use in the laboratory are light microscopes.

• Light microscopes use visible light to illuminate specimens.
• They use a two-lens system.
• The two lenses present in a light microscope are:

As the light microscope uses two lenses, it is also known as a compound microscope.

Detailed Explanation

Parts of a Light Microscope

The main components of a light microscope are:

Working Principle of a Light Microscope

The working principle of a light microscope is as follows:

1. The light source illuminates the object. - Light passes through the specimen from below.

2. The first image of the object is formed by the objective lens. - This image is real, inverted, and magnified.

3. The image formed by the objective functions as the object for the eyepiece. - The eyepiece takes the image from the objective lens and magnifies it further.

4. The eyepiece produces the final, virtual, and magnified image. - This is the image you see when you look through the eyepiece.

5. The final image produced is inverted with respect to the object. - This means if the specimen is placed right-side up, it will appear upside down.

• Light passes through the specimen.
• The objective lens makes a magnified, inverted image.
• The eyepiece magnifies that image again.
• You see a final image that is magnified and inverted.

Magnification

Definition: Magnification is the capacity of an optical instrument to increase the size of an object than its original size.

In simple words: Magnification is how much bigger a microscope makes an object look.

• Objects which cannot be seen by the naked eye can be observed by increasing magnification.
• Different lenses have different magnification powers.
• Magnification is represented by the letter 'X', which means "the number of times than original size."

Example: - A lens of 10X magnification can increase the size of an object of 1 μm to 10 μm.

Total Magnification: The total magnification can be calculated by multiplying the objective lens value by the ocular lens value.

Total Magnification = Objective Lens Magnification × Ocular Lens Magnification

Example: - If the objective lens is 40X and the ocular lens is 10X: Total Magnification = 40 × 10 = 400X

Maximum Magnification of a Light Microscope: - Maximum magnification of a light microscope is 1500X using a 100X objective and 15X ocular lens.

Magnification of an Electron Microscope: - An electron microscope can magnify specimens between 1 and 50 million times depending on which type is used.

Resolution

Definition: Resolution is a measure of the microscope's ability to distinguish between two points which are close together on an object.

In simple words: Resolution is how clearly a microscope can show two nearby points as separate points.

• If a microscope can clearly distinguish between the two points, it has a good or high resolution.
• If it cannot distinguish them, it has low resolution.

• The resolution of the naked eye is 0.1 mm.
• This resolution cannot be increased by increasing magnification.

Resolving Power of a Light Microscope: - The resolving power of a light microscope is 250 nm.

Resolving Power of an Electron Microscope: - The resolving power of an electron microscope is 0.2 nm.

Difference Between Magnification and Resolution

• Magnification = How big the object looks.
• Resolution = How clear the object looks.

Important: Increasing magnification without increasing resolution will only make the image bigger, not clearer.

Intermediate Level

The light microscope is also called a compound microscope because it uses two lenses.

How the two lenses work together:

• Forms a real, inverted, magnified image of the specimen.
• This image is formed inside the microscope.

• Acts like a magnifying glass on the image formed by the objective lens.
• Produces a final virtual, magnified image.
• The final image is inverted with respect to the original object.

Calculating Total Magnification:

M_total = M_objective × M_ocular

• Objective = 10X, Ocular = 10X
• Total magnification = 10 × 10 = 100X

• Resolution depends on the wavelength of light used.
• Shorter wavelengths give better resolution.
• Light microscopes use visible light (400–700 nm), so their resolution is limited to about 250 nm.
• Electron microscopes use electron beams (about 1 nm), so their resolution is much better (0.2 nm).

• Because magnification without resolution only makes the image bigger, not clearer.
• If two points are closer than the resolution limit, they will appear as one blurred point, no matter how much you magnify.

Important Points

• 1. Microscopy is the technique used to view objects that cannot be seen by the naked eye.
• 2. Light microscopes use visible light to illuminate specimens.
• 3. Light microscopes use a two-lens system: ocular lens and objective lens.
• 4. Because they use two lenses, light microscopes are also called compound microscopes.
• 5. Working principle:
• - Light source illuminates the object.
• - Objective lens forms a real, inverted, magnified image.
• - Eyepiece produces the final virtual, magnified image.
• - Final image is inverted with respect to the object.
• 6. Magnification is the capacity to increase the size of an object.
• 7. Total magnification = objective lens value × ocular lens value.
• 8. Maximum magnification of a light microscope = 1500X (100X objective + 15X ocular).
• 9. Resolution is the ability to distinguish between two close points.
• 10. Resolution of naked eye = **0.1 mm**.
• 11. Resolving power of light microscope = 250 nm.
• 12. Resolving power of electron microscope = 0.2 nm.
• 13. Magnification = how big; Resolution = how clear.
• 14. Increasing magnification does not increase resolution.

Remember

• - Microscopy = technique to view small objects
• - Light microscope = compound microscope (two lenses)
• - Two lenses: ocular (eyepiece) + objective
• - Total magnification = objective × ocular
• - Maximum magnification of light microscope = 1500X
• - **Resolution of naked eye** = 0.1 mm
• - Resolving power of light microscope = 250 nm
• - Resolving power of electron microscope = 0.2 nm
• - Magnification = size; Resolution = clarity
• - Increasing magnification does not increase resolution

Common Mistakes

• 1. Confusing magnification with resolution – Magnification makes things bigger; resolution makes things clearer. They are not the same.
• 2. Thinking that increasing magnification increases resolution – This is incorrect. Resolution is limited by the wavelength of light.
• 3. Forgetting that the final image is inverted – The final image in a light microscope is inverted with respect to the object.
• 4. Mixing up objective and ocular lens – Objective lens is near the specimen; ocular lens is in the eyepiece.
• 5. Forgetting the formula for total magnification – Total magnification = objective × ocular.
• 6. Using wrong units – Resolution of naked eye is in mm; resolving power of microscopes is in nm.
• 7. Thinking light microscopes can see viruses – Light microscopes have a resolution of 250 nm, so they cannot see viruses (which are much smaller).
• 8. Thinking electron microscopes can see living specimens – Electron microscopes can only see dead, dried specimens (this will be covered in 1.3.1).

Quick Revision

• Microscopy is the technique used to view objects that cannot be seen by the naked eye.
• Light microscopes use visible light and a two-lens system (ocular + objective).
• They are also called compound microscopes.
• Working principle:
• Magnification is the capacity to increase the size of an object.
• Total magnification = objective lens value × ocular lens value.
• Maximum magnification of a light microscope = 1500X.
• Resolution is the ability to distinguish between two close points.
• Resolution of naked eye = **0.1 mm**.
• Resolving power of light microscope = 250 nm.
• Resolving power of electron microscope = 0.2 nm.
• Magnification vs Resolution:
• Magnification = how big
• Resolution = how clear
• Increasing magnification does not increase resolution.

60-Second Revision

• Microscopy = technique to view small objects
• Light microscope = compound microscope (two lenses)
• Two lenses: ocular (eyepiece) + objective
• Total magnification = objective × ocular
• **Max magnification (light)** = 1500X
• **Resolution (naked eye)** = 0.1 mm
• Resolving power (light) = 250 nm
• Resolving power (electron) = 0.2 nm
• Magnification = size; Resolution = clarity
• Warning: Increasing magnification does not increase resolution. The final image is inverted.