Redacted Lecture 1.2 Light Microscopy - Slides
Page 3: Relative Cell Sizes
Human Cell Dimensions
Red Blood Cells (RBCs): 7.2 µm
Most human cells: 10–20 µm
Keratinocytes: 20-30 µm
Oocytes: ~100 µm
Neutrophils: 10-12 µm
Page 4: Measurement Relationships
Scale Conversion
Relationship among:
Millimetres (mm)
Micrometres (µm)
Nanometres (nm)
Page 5: Limit of Resolution
Defining Resolution
Unresolved vs. Resolved:
The minimum distance at which two objects can be distinguished as separate entities.
Page 6: Types of Microscopy
Comparison of Microscopy Techniques
Transmission Electron Microscopy (TEM):
Electron source and detection mechanism.
Scanning Electron Microscopy (SEM):
Produces 3D images from electron beams.
Light Microscopy:
Uses visible light and lenses to magnify specimens.
Page 7: Wavelength and Resolution
Understanding Wavelength Impact
Visible Light Spectrum:
Violet: = 380 nm
Deep Red: = 700 nm
Practical Limits of Resolution:
Light Microscope: ~200 nm
Electron Microscope: ~0.1 nm
Resolution is inversely proportional to wavelength.
Page 8: Resolution Limits
Comparing Limits in Different Structures
Mammalian cells: 10-100 µm in diameter
Nucleus: ~6 µm
Mitochondrion: ~0.5-2 µm
Ribosome: ~25 nm
Page 9: Intestinal Epithelium
Microscopic Structures
Brush border (microvilli):
Microvilli: ~90 nm in diameter, ~1 µm in length
Columnar epithelial cells
Page 10: Histology in Diagnosis
Importance of Histology
Quote:
"Chemistry brings the clarification of living processes nearer than does anatomy. Each anatomical change must have been preceded by a chemical one." – Rudolf Virchow (1821-1902)
Page 11: Histological Changes in Malignancies
Cancer Diagnosis
Characteristic histological changes in malignancies.
Importance of cancer 'staging' for treatment and prognosis.
Page 12: Clinical Case Scenario
Differential Diagnoses for Skin Lesions
Considerations for an unusual mole:
Atypical benign mole?
Seborrheic wart?
Malignant melanoma?
Page 13: Histology of Melanomas
Superficial Spreading Melanoma
Characteristics of dividing melanocytes in the epidermis.
Overview of granular layer in epidermis.
Page 14: Breslow Thickness
Prognostic Indicator in Melanoma
Survival rates associated with Breslow thickness measures:
<1 mm: 95-100%
1.0-2.0 mm: 80-96%
2.0-4.0 mm: 60-75%
4.0 mm: 37-50%
Page 15: Breslow and Clark's Thickness
Tumor Invasion Levels
Breakdown of tumor invasion according to Clark's levels and Breslow thickness
Page 16: Histological Techniques
Slide Preparation Process
Fixation: Chemical preservation of tissue.
Embedding/Processing: Dehydration and solidification of the tissue.
Sectioning: Cutting of solidified tissue.
Staining: Coloring of tissue sections.
Collection: Tissue sample collection (Biopsy).
Viewing/Analysis: Microscopic viewing of stained tissue.
Page 17: Common Biopsy Techniques
Overview of Biopsy Methods
Needle Biopsies:
Endoscopic Biopsies:
Transvascular Biopsies:
Curettage:
Direct Incision:
Page 18: Examples of Biopsy Procedures
Specific Techniques
Smear: Cervix or buccal cavity
Curettage: Endometrial lining of uterus
Direct Incision: Skin, mouth, larynx
Page 19: Additional Biopsy Procedures
Technique Applications
Needle: Brain, breast, liver
Endoscopic: Lung, intestine
Transvascular: Heart, liver
Page 20: Tissue Processing Overview
The Fixation Process
Fresh biopsy (wet and bloody) undergoes fixation (e.g. glutaraldehyde, formaldehyde) to preserve structure and prevent decomposition.
Page 21: Tissue Embedding
Preparation Steps
Dehydration: Using ethanol (70-100%)
Embedding: Biopsy impregnated & embedded in wax (at 56°C)
Page 22: Sectioning Technique
Microtome Operation
Utilizing microtome to cut thin sections (1-10 µm, usually 3-5 µm) for microscopic analysis.
Page 23: Artefacts in Tissue Processing
Potential Issues
Artefacts can arise such as air bubbles or tears due to microtome issues.
Page 24: Histological Staining Techniques
Overview of H&E Staining
Haematoxylin: Basic dye; stains acidic components (e.g., nucleus, chromatin).
Eosin: Acidic dye; stains basic components (e.g., cytoplasmic proteins).
Page 25: H&E Staining in Action
Gastric Pit Analysis
Schematic detailing smooth muscle and epithelial staining patterns.
Page 26: PAS Staining
Special Stains for Carbohydrates
Periodic Acid-Schiff (PAS) reaction: Stains carbohydrates and glycoproteins magenta.
Page 27: Histological Analysis of Villi
Intestinal Villi Examination
Identification of cell types and structures using PAS stain.
Page 28: Quality Control in Histology
Ensuring Accuracy
Quality control checks for proper sectioning and staining processes.
Page 29: Digital Scanning in Histology
Modern Analysis Techniques
Use of high-throughput digital slide scanners for analysis.
Page 30: Pancreatic Histology
Examination of Pancreatic Tissues
Overview of exocrine (Acini), endocrine (Islet of Langerhans) structures via H&E stain.
Page 31: Chromatin Structure in Nuclei
Nuclear Morphology
Differentiation of heterochromatin and euchromatin based on staining and transcriptional activity.
Page 32: Nissl Bodies and Neuronal Health
Importance in Protein Synthesis
Nissl bodies as sites of synthesis in large motor neurons, affected by neuronal injury.
Page 33: Histological Considerations
Important Factors in Analysis
Multiple factors influencing histological evaluation must be considered.
Page 34: Plane of Sectioning
Analyzing Skin Glands
Discussion on sectional views affecting gland examination.
Page 35: Regions of Interest
Importance of Sectioning
Identifying key areas in various planes.
Page 36: Section Planes
Types of Sectioning
Explanation of oblique, transverse, and longitudinal sections.
Page 37: Microscopy Dynamics
Understanding Wave Phases
Differences between waves in-phase and out-of-phase affecting microscopy images.
Page 38: Red Blood Cell Analysis
Epithelial Observations
Examination of cell types using microscopy.
Page 39: Phase Contrast Microscopy
Light Dynamics
Two waves from phase-contrast microscopy create bright and dim contrasts for analysis.
Page 40: Phase Contrast Microscopy Details
Techniques and Applications
Enables visualization of living cells without staining, enhancing contrast.
Page 41: Confocal Microscopy
Advanced Imaging Techniques
High-resolution imaging and 3D reconstruction via confocal microscopy.
Page 42: Darkfield Microscopy
Detection Applications
Useful for identifying specific pathogens in specimen samples.