Class 2 Microscopy Transcript DS

Class 2 Topic: Microscopy

The second class in Cell Biology 2026, focusing on microscopy as a core experimental pillar of cell biology, alongside biochemistry and genetics. The lecture was recorded due to a winter storm canceling in-person class on Monday; in-class work on microscopy is planned for the following Wednesday.

Three Pillars of Cell Biology Experimentation

1. Biochemistry, 2. Genetics, 3. Microscopy. These fields are combined to answer questions and discover new information about cells.

Two Main Types of Microscopy

1. Light Microscopy: Uses light as the beam source. 2. Electron Microscopy: Uses electrons as the beam source.

Light Microscopy Subtypes

A) White Light Microscopy: Uses all visible wavelengths. Types: Brightfield, Phase Contrast, Nomarski/Differential Interference Contrast (DIC). B) Fluorescence Microscopy: Key identifier is a very dark background with a stark, bright (white or colored) signal.

Electron Microscopy Subtypes

1. Scanning Electron Microscopy (SEM). 2. Transmission Electron Microscopy (TEM).

Microscopy Comparison Table (on Canvas)

A table organizing information by: Beam Type, Signal Detected, and specific techniques (including three types of fluorescence microscopy). Students are encouraged to expand it by listing advantages and disadvantages for each technique to aid comparison and understanding.

Key Characteristics for Evaluating Microscopy

Sample preparation, Image generation process, Cellular features detectable, Resolution, Contrast.

Process for Selecting a Microscopy Technique

Start with a biological question, then consider the characteristics of different microscopy types to select one or more suitable techniques to address that question.

Detailed Considerations for Choosing Microscopy

1. Detectability of the object (protein, cell part, whole cell). 2. Required resolution to distinguish parts. 3. Sufficient contrast. 4. Sample thickness (critical for tissues/whole organisms). 5. Need for 3D information. 6. Requirement for live cells. 7. Imaging speed/duration (snapshot vs. time-lapse). 8. Image analysis/quantification needs. 9. Availability of molecular tools (antibodies, fusion proteins). 10. Availability of the microscope. 11. Cost and time. General Principle: Choose the simplest sufficient approach (lowest time, cost, and resources).

Common Microscopy Techniques Covered

1. H&E Staining, 2. Immunohistochemistry (IHC), 3. Immunofluorescence Microscopy / Fluorescent Fusion Protein Microscopy, 4. Immunogold Electron Microscopy.

H&E Staining (Hematoxylin and Eosin)

A histology technique to visualize cells and tissues, often used with patient samples. Hematoxylin: Blue/purple dye that binds nucleic acids (e.g., DNA in nucleus). Eosin: Red/pink dye that binds proteins (can be intracellular or extracellular). Visualized using white light microscopy, typically brightfield. Blue/purple regions indicate nuclei; pink regions indicate protein-rich areas.

Immunohistochemistry (IHC)

Technique to detect specific proteins in tissue samples. "Histo" refers to tissue; "immuno" refers to antibody use. Uses antibodies linked to an enzyme or fluorescent dye to detect antigens (typically proteins). Example: Breast cancer tissue with nuclei stained blue/purple and a brown enzymatic product indicating antibody binding and thus presence of a specific protein.

Detecting Proteins by Fluorescence (Two Approaches)

1. Immunofluorescence Microscopy: Uses an antibody (primary) to bind the protein; visualization via attached fluorescent molecule or secondary antibody. 2. Fluorescent Fusion Protein Microscopy: Protein of interest is genetically fused to a fluorescent protein (e.g., GFP). Class Focus: Understanding that a primary antibody detects an antigen; details of secondary antibodies are not required. Note: All approaches (H&E, IHC, fluorescence) are used commonly and interchangeably; sometimes multiple are used to confirm results.

Immunogold Electron Microscopy

Similar concept to immunofluorescence, but antibodies are linked to gold nanoparticles. The electron-dense gold appears as dense black dots in EM images, allowing precise protein localization at high resolution. Example: Used to show gold-labeled proteins located centrally in nuclear pores during transport between cytoplasm and nucleus.

Course Goals for Microscopy Unit

1. Describe important characteristics of general microscopy types. 2. Justify the selection of a specific microscopy type for a given biological question. 3. Identify the type of microscopy used based on an image (a skill developed with practice). 4. Interpret microscopy images to deduce information about cells or proteins.

Figure Analysis Assignment

Goal: Analyze a published microscopy figure from a peer-reviewed paper that visualizes a protein related to the endomembrane system (the next course topic). Purpose: Practice applying microscopy knowledge, learn about the endomembrane system, and communicate effectively. Logistics: Can be done individually or in groups up to 3; due February 11th; start early; read instructions and rubric on Canvas; an example assignment is provided. Optional Pre-approval: Submit just the figure by February 4th via a separate Canvas link for feedback on its appropriateness. Support is available via office hours.

Upcoming Schedule (Weather-Dependent)

Planned in-person class on Wednesday: Topic "Cell Architecture" (Class 3). The in-class work for the microscopy unit will also be completed then. Pre-class videos and assignment for Wednesday's class must be completed by noon Wednesday. Office hours may be virtual on Monday, resuming in-person late