LECTURE 6:

1. Introduction to Optical Microscope:

- Uses magnifying lenses to produce magnified images in the eyepiece.

- Concepts covered include magnification, useful magnification, resolution, depth of field, and depth of
focus.

2. Magnification:

- Useful Magnification: There’s a limit to magnification; too much magnification reduces image clarity.
Magnification must be meaningful and related to the resolution.

- The thin lens equation is used to describe the relationship between object distance (u), image
distance (v), and focal length (f).

- Magnification is defined by the ratio of the image distance to the object distance.

3. Thin Lens Equation:

- Describes how light interacts with a lens to form an image.

- Image formation involves rays passing through the focal point and the center of the lens, leading to
an inverted image.

v
- Magnification: M = , where v = image distance and u = object distance.
u

4. Compound Microscope:

- Consists of multiple lenses to magnify the image.

- Components include light source, apertures, condenser lens, half-silvered mirror, objective lens, and
eyepiece/projector lens.

- Metallurgical vs Biological Microscopes:

- Metallurgical microscopes use reflected light to view the surface.

- Biological microscopes use transmitted light through the sample.

5. Image Formation in Compound Microscope:

- Light from the source is focused by the condenser lens and passes through the objective lens,
reflecting off the sample.
 - Reflected rays return to the objective lens, creating an intermediate magnified image.

- Further magnification can be achieved, but adding lenses decreases resolution.

6. Limitations of Optical Microscopes:

- Beyond a certain magnification (around 1400X), optical microscopes lose resolution, causing blurred
images.

- Comparison with Scanning Electron Microscope (SEM):

- At 300X, optical microscopes and SEM show similar sharpness.

- At 1400X, SEM provides much sharper images than optical microscopes due to better resolution.

7. Resolution:

- Resolution is the ability to distinguish two close points as separate entities.

- It is related to the smallest distance between two points that can still be seen as distinct.

8. Airy Rings:

- Occurs when light passes through a small aperture and gets diffracted, forming a central bright spot
surrounded by rings (Airy discs).

- Intensity decreases with each ring, with a central maximum and minima between maxima.

- These diffraction patterns limit the resolution of microscopes, as closely spaced objects may appear
as one.

Key Takeaways:

- Optical microscopes have a practical limit for magnification due to resolution constraints.

- Resolution is more important than mere magnification for clarity in microscopy.

- The principles of lens behavior, such as magnification and the thin lens equation, are fundamental to
understanding microscopes.

- Advanced microscopes like SEM offer higher resolution than optical microscopes at high
magnifications.

**Notes on Airy Rings, Resolution, and Depth of Field in Microscopy**

### Airy Rings and Beam Diffraction:

- A **parallel beam of light** passing through an aperture forms **Airy rings**, appearing as a central
bright spot surrounded by rings.

- As light passes through multiple apertures and lenses, Airy rings form at each stage, causing the beam
to divide into multiple beams.

- This **diffraction pattern** impacts the resolution, as Airy rings from different sources interact,
reducing image clarity.

### Resolution and Rayleigh Criterion:

- **Resolution** refers to the ability to distinguish two close features as separate entities.

- The **Rayleigh criterion** defines the minimum distance between two sources where they can still be
seen as separate. This occurs when the maximum of one Airy disc coincides with the minimum of
another.

- The formula for resolution is:

\[

r_1 = \frac{1.22 \lambda}{\mu \sin(\alpha)}

\]

where:

- \( \lambda \) = wavelength of light

- \( \mu \) = refractive index of the lens

- \( \alpha \) = half-aperture angle

- Reducing \( \lambda \), increasing \( \alpha \), or using lenses with a higher refractive index improves
resolution.

- **Microscope resolution** can be improved by:

- Using shorter wavelengths (e.g., green light with 400 nm wavelength).

- Increasing the aperture angle to increase the numerical aperture.

- Using materials with a high refractive index (e.g., \( \mu = 1.7 \) in optical microscopes).

### Airy Ring Interference and Image Clarity:

- As light passes through apertures, Airy rings from different features interact, reducing the **image
resolution**.

- Increasing aperture size improves resolution but can reduce the **depth of field**.

### Depth of Field (DOF):

- **Depth of field** is the range within which an object remains in focus.

- If an object lies within the range determined by the resolution, it remains in focus.

- The depth of field can be calculated by:

\[

h = \frac{1.22 \lambda}{\mu \sin(\alpha) \tan(\alpha)}

\]

- **Larger aperture** angles reduce depth of field but improve resolution.

- A **smaller aperture** increases depth of field but reduces resolution, a trade-off often managed in
**SLR cameras**.

### Depth of Focus:

- **Depth of focus** refers to the eyepiece's tolerance for varying focal distances.

- It is proportional to the square of the magnification times the depth of field.

- Unlike the depth of field, depth of focus is typically much larger, giving flexibility in maintaining focus
over different viewing positions.

### Application in Electron and Optical Microscopy:

- **Electron microscopes** offer high resolution due to the shorter wavelength of electrons.

- **Optical microscopes** are limited by the visible light spectrum, with green light often used for its
favorable wavelength.

### Practical Considerations:

- In microscopy, increasing the aperture or using a higher refractive index medium can improve image
resolution.

- When photographing, reducing aperture size increases the depth of field, allowing more subjects in the
scene to remain in focus.
- However, this reduces resolution. In contrast, larger apertures improve resolution but limit depth of
field.

### Issues with Optical Systems

1. **Resolution Problem:**

- Resolution determines how close two objects can be before they are seen as distinct entities.

2. **Optical Aberrations:**

- Optical aberrations cause imperfections in the image formation by lenses.

- High-quality optical systems are expensive due to the effort required to minimize aberrations.

- The cost of microscopes and cameras varies widely based on how well the optical system manages
aberrations.

3. **Ideal Optical System:**

- In an ideal system, all light rays from an object should converge at the same point to create a clear
and sharp image.

- In real systems, light may not focus on a single point due to aberrations.

### Types of Optical Aberrations

1. **Spherical Aberration:**

- Caused when light rays far from the optical axis (marginal rays) focus at a point closer to the lens,
while rays closer to the optical axis (paraxial rays) focus further from the lens.

- This results in two different focal points, leading to blurred images.

- Example: Images appear sharp in the center but out of focus on the periphery, as seen in common
magnifying lenses.

- High-quality lenses and microscopes are designed to correct spherical aberrations.

2. **Chromatic Aberration:**

- Occurs when light with a range of wavelengths (e.g., white light) passes through a lens.

- Different wavelengths of light (colors) refract by varying amounts, causing them to focus at different
points.

- Blue light focuses closer to the lens, while red light focuses further away.

- This creates color fringing or "halos" around objects, such as a blue or red halo around edges in
images.

- Example: A pigeon image with a red halo around its edges due to chromatic aberration.

### Correction of Aberrations

- Advanced and expensive optical systems (like high-end cameras and microscopes) are designed to
minimize spherical and chromatic aberrations for clearer, sharper images.

### Key Points from Lecture on Optical Systems and Aberrations

#### 1. **Microscope Cost and Optical Quality**

- Price variations in cameras and microscopes (from cheap to very expensive) are largely due to the
quality of lenses and how well they correct optical aberrations.

- High-quality optical systems are expensive because they are designed to be aberration-free, ensuring
better image clarity.

#### 2. **Optical Aberrations**

- **Aberrations** are issues in optical systems where light rays do not converge at a single focal point,
leading to blurred or distorted images. Key types include:

#### 3. **Spherical Aberration**

- Occurs when light rays at different distances from the optical axis focus at different points.

- **Marginal rays** (rays farther from the center of the lens) focus closer to the lens, while **paraxial
rays** (rays near the center) focus farther away.

- Example: In images, the center may be in focus while the edges appear blurry, common in basic
magnifying lenses.
#### 4. **Chromatic Aberration**

- Happens when light of different wavelengths (colors) focuses at different points.

- White light, composed of multiple wavelengths, is split into different colors. Blue light focuses closer to
the lens, while red light focuses farther away.

- Leads to colored halos around objects (e.g., a red halo around a pigeon’s body in an image), reducing
image sharpness.

- **Correction**: Use of achromatic lenses or monochromatic light (like green filters) to reduce
chromatic aberrations.

#### 5. **Astigmatism**

- Rays passing through different planes (e.g., vertical vs. horizontal) of a lens focus at different points.

- Leads to two separate focal points depending on the direction of the light.

- Correction: Use of cylindrical lenses along with spherical lenses (common in eyeglass prescriptions for
people with astigmatism).

#### 6. **Corrective Lenses in Microscopes**

- **Achromatic Objectives**:

- Combination of convex and concave lenses made from different materials.

- Correct chromatic aberrations for red and blue light and spherical aberrations for green light.

- Using a **green filter** improves image quality by eliminating chromatic aberrations and minimizing
spherical aberrations.

- **Semi-Apochromat (Fluorite) Lenses**:

- Correct chromatic aberrations for red and blue light.

- Correct spherical aberrations for blue and green light.

- **Apochromatic Lenses**:

- Most complex and expensive.

- Correct chromatic aberrations for four colors: deep blue, blue, green, and red.

- Correct spherical aberrations for two or three colors: deep blue, blue, and green.

- These lenses are used for high-precision, high-quality imaging.

#### 7. **Lens Complexity and Manufacturing**

- Complex objectives like apochromatic lenses are composed of many individual lens elements (convex
and concave), all with carefully controlled curvatures and materials.

- **Astigmatism** can be caused by manufacturing defects, such as inconsistencies in the curvature of

lenses, but can be corrected with better manufacturing processes.

- The more complex and precise the lens, the higher the cost, which reflects in the quality of the
microscope's performance.

#### 8. **Impact on Cost**

- The more advanced the objective (achromat, fluorite, apochromat), the higher the cost due to the
complexity of lens design and manufacturing precision.

- High-end microscopes provide significantly better image clarity but at a much higher price point.

#### 9. **Use of Green Filters**

- In microscopy, **green filters** are often used in metallography labs to enhance image clarity.

- Green filters eliminate other wavelengths, thus minimizing chromatic aberration and improving the
focus of images, particularly with black-and-white imaging.

### Microscope Use and Sample Preparation (Metallography Practice)

- **Introduction:**

- Focus on the practical use of microscopes and sample preparation for viewing microstructures, known
as metallography.

- The process includes several steps to prepare a flat, polished, and optically flat sample for microscopic
examination.

### Steps for Sample Preparation:

1. **Grinding:**

- Used to flatten the sample by removing surface irregularities (undulations or curvatures).

- A coarse abrasive is used for fast material removal.

- Requires careful handling to avoid introducing additional surface problems such as curvature.

2. **Polishing Using Abrasive Papers:**

- Begins with coarse abrasive particles and progresses to finer ones.

- Each step involves rotating the sample 90 degrees and polishing in a new direction to remove the
scratches left by the previous step.

- **Emery Paper:**

- Traditionally used, made from a natural mix of abrasive particles like aluminum oxide.

- Designations: 1/0 (coarse) to 4/0 (fine).

- **Silicon Carbide Paper:**

- Modern abrasive paper made of uniform particles for consistent polishing.

- Wet polishing can be done to reduce heat and avoid sample damage.

- Designations: P100 to P2000, with particle sizes ranging from 162 µm to 10 µm.

3. **Final Polishing Using Cloth:**

- Polishing is done with finer abrasive particles, often suspended in water.

- **Materials Used:**

- For steel: Alumina particles in suspension.

- For aluminum: Magnesia powder dissolved in distilled water.

- **Diamond Paste:** Popular for various samples with precise particle sizes (9 µm, 3 µm, 1 µm, etc.).

- Different cloths should be used for different particle sizes to avoid contamination and maintain
polishing quality.

### Purpose of Polishing:

- **Objective:** Achieve a mirror-like or optically flat finish on the sample.

- Important for keeping the entire sample in focus under a microscope, given the small depth of field
(micron range) of optical microscopes.

- Polishing progressively reduces surface roughness and prepares the sample for microstructural
examination.

### Final Steps:

4. **Etching:**

- After polishing, the sample is etched to reveal the microstructure.

- Introduces new features or surface roughness for clearer observation of material phases and
structures.

### Key Points:

- Each polishing step must be done meticulously to remove the damaged layer introduced by the
previous step.

- A finer abrasive particle size progressively leads to better surface quality, essential for microscopic
analysis.

- **Cloth Polishing:**

- Slower but results in better surface finish with less damage to the sample.

- Diamond paste can be used for fine polishing.

- Ensure a clean environment and separate cloths for different particle sizes to avoid contamination.

### Polishing and Microstructure Observation

1. **Polishing Techniques:**

- Disc polisher: A semiautomatic tool where the disc rotates and a cloth is used for polishing.

- Once polished, the surface must be inspected for microstructure.

2. **Etching Process:**

- Etching reveals microstructure by reacting with the material's constituents differently.

 - Reveals features like grain boundaries and phases by causing surface undulations, which microscopes
capture.

- Etchants react with different materials uniquely, revealing phases or grain boundaries.

3. **Common Etchants:**

- **Steel:** 2% Nital (2% HNO₃, 98% methanol; ethanol is typically preferred but not easily available in
India).

- **Aluminum Alloys:** Keller's reagent.

- Numerous etchants exist, and many materials may require different types depending on the desired
observation.

4. **Polishing as an Art:**

- Mastering polishing and etching takes time.

- Different methods (e.g., swabbing instead of immersing) may yield better results.

- Personal experimentation is encouraged to achieve optimal microstructures.

- Example: Swabbing aluminum lithium alloy with Keller's reagent gave better results than immersing
it.

5. **Microscopic Imaging:**

- A polished surface with no etching reflects light back evenly, resulting in a uniform bright image.

- With etching:

- Phases that react with the etchant develop surface roughness, scattering light and appearing dark.

- Phases that don’t react appear bright, as they reflect light directly back into the objective lens.

- For a single-phase material, grain boundaries react more vigorously with etchants, causing light to
scatter, making the grain boundaries appear darker than the grains.

6. **Microscope Overview:**

- **Parts:**

- Light source.

- Filters.
 - Half-silvered mirror (directs light towards the sample).

- Objective lens.

- Eye piece for viewing.

- The sample is placed on a movable stage, with X and Y adjustments for precise measurement.

- Microscopes have a coarse adjustment for rough focus and fine adjustment for precise focus.

7. **Microscopy Procedure:**

- Always start with the lowest magnification (10x) for coarse focusing.

- Switch to higher magnifications (20x, 50x) using only fine adjustment to avoid damaging lenses.

- Higher magnifications have shorter working distances, so coarse adjustment can risk the sample
hitting the lens.

- After focusing, view features of the microstructure clearly at each magnification level.

8. **Sample Preparation for Microscopy:**

- After etching, thoroughly wash the sample in running water to remove acid residues.

- Dry the sample using an air blower (not a hair dryer) to avoid damaging it.

- Ensure the sample is completely dry before using it on the microscope.

- Clean and dry hands before handling the sample or microscope.

9. **Microscope Maintenance:**

- Always cover the microscope when not in use to prevent dust collection and fungal growth on lenses.

- Optics are the most critical part of the microscope and should be carefully maintained.