VIBS lecture 2 Microscopy Notes

What is Microscopy

  • Using a microscope to view details of cells and tissues that are otherwise undetectable with just our eyes.

  • Magnification: increase in size.

  • Resolution: smallest distance between two points that can be distinguished.

Magnification

  • Magnification is the increase in size of the specimen being viewed.

Resolution

  • Resolution is the smallest distance between two points that can be distinguished.

What is Histology?

  • Microanatomy: the study of cells and tissues.

  • Correlates structure with function.

  • Used in diagnosing diseases and in research.

Tissue: How to Prepare Tissue for Microscopy

  • Collection and trimming

  • Fixation

  • Processing

  • Embedding

  • Sectioning

  • Staining

SPECIMEN

  • (Note: The slide lists SPECIMEN but provides no further detail in the transcript.)

STAINING IN LIGHT MICROSCOPY

  • A. Hematoxylin and Eosin (H&E)

  • B. Periodic Acid/Schiff (PAS)

  • C. Toluidine Blue

  • Color provides clues about:

    • Shape

    • Size

    • Intensity of staining

Hematoxylin

  • Stains BLUE.

  • Opposites attract in histology: acidic structures love bases – stain blue.

  • Basophilic structures: DNA and RNA present.

  • Nucleolus basophilic; chromatin (heterochromatin vs euchromatin) basophilic; condensed DNA is basophilic.

  • Nuclear envelope stains blue (note: not the membrane seen with light microscopy).

  • Proteins (and chromatin) accumulated at the membrane can be stained.

  • Ribosomes stain blue in the cytoplasm.

  • Reference: Fyodorov D V.; et al. 2017

Eosin

  • Eosin stains RED/PINK.

  • Basic structures are acidophilic and stain with eosin.

  • Cytoplasmic staining highlights fibrous proteins and mitochondria in the cytoplasm.

  • Eosin is the most common counterstain to hematoxylin.

Light Microscopy vs Electron Microscopy

  • Light microscopy is great to view tissues and cells.

  • What about ultrastructure (mitochondria, rough ER, etc.)?

  • Electron Microscopy (EM) provides ultrastructural detail.

EM: Types and Magnifications

  • Conventional Transmission Electron Microscopy (TEM)

  • Scanning Electron Microscopy (SEM; surface features)

  • Carbon Replica TEM

  • EMs at different magnifications

Scanning vs Transmission Electron Microscopy

  • Scanning Electron Microscopy (SEM): 3D image, great for surface features of cells.

  • Transmission Electron Microscopy (TEM): 2D image, great for cellular/organelle detail.

Typical TEM Carbon Replica

  • Ref code # 1, 19

  • Tsuji, T. and Fujimoto, T. (2017). Freeze-fracture-etching Electron Microscopy for Facile Analysis of Yeast Ultrastructure. Bio-protocol 7(18): e2556.

  • Tsuji, T., Fujimoto, M., Tatematsu, T., et al. (2017).

ORGAN, TISSUE, CELL, SYSTEM, PROTOPLASM

  • ORGAN – Two or more types of tissues; larger functional unit (e.g., skin, kidney, intestine, blood vessels).

  • TISSUE – Groups of cells with the same general function and texture.

  • CELL – Smallest unit of protoplasm; simplest animals consist of a single cell.

  • ORGAN SYSTEM – Several organs (e.g., respiratory, digestive, reproductive systems).

  • PROTOPLASM – Living substance.

Cells Contain Organelles

1) MEMBRANOUS ORGANELLES – common structures and metabolic functions: cell membrane, RER, SER, Golgi, mitochondria, lysosomes.
2) NON-MEMBRANOUS ORGANELLES – cytoskeletal components: microtubules, microfilaments, intermediate filaments; free ribosomes.
3) INCLUSIONS – expendables: nutrients, pigments, secretory granules.

Organelles and Inclusions (Functional Map)

  • The cell contains:

    • Cell membrane: gatekeeper; double layer of phospholipids; controls water flow; outer limit; separates cell from environment.

    • Nucleus: archive of cell’s DNA; double membrane; holds DNA; involved in cell division; directs protein production by ribosomes.

    • Mitochondria: power plant; produces ATP (cell energy);

    • Has a double membrane; can vary in shape.

    • Endoplasmic Reticulum (ER):

    • Rough ER (rER): synthesizes proteins to be exported or to be part of plasma membrane.

    • Smooth ER (sER): no ribosomes; synthesizes phospholipids, steroid hormones; detoxification; glycogen metabolism.

    • Golgi Apparatus: modifies (adds sugar to) and packages proteins; “packing department.”

    • Lysosomes: intracellular digestion; turnover of cellular components; lysosomes are difficult to see with light microscopy on H&E sections; EM is needed to visualize them.

    • Cytoskeleton (Non-membranous organelles): microtubules, microfilaments, intermediate filaments; provide structure; direct intracellular/extracellular movement; involved in mitosis.

    • Free ribosomes: ribosomes not attached to ER; synthesize proteins used within the cytoplasm; under LM, cells rich in ribosomes (polyribosomes + rER) show increased basophilia in cytoplasm.

Inclusions

  • Cytoplasmic deposits:

    • Nutrients: glycogen, lipid

    • Pigments: melanin granules

    • Secretory granules: e.g., zymogen granules of pancreas

  • The cell can live without these inclusions; lipid may be present in cells.

Function-Driven Organelle Abundance (Example Exercise)

  • Depending on a cell’s function, certain organelles may be abundant.

  • Example: A neuronal cell body has abundant protein synthesis; infer which organelle is likely abundant (rough ER and ribosomes).

  • Lab tip: Before lab, understand organelle functions and be able to identify organelles in EM and LM images.

The Cell Cycle

  • The cell cycle is the series of events in a cell’s life.

  • Interphase:

    • G₁ (First gap phase)

    • S (Synthesis phase)

    • G₂ (Second gap phase)

  • M phase: Mitosis and cytokinesis

  • Mitosis stages: Prophase, Prometaphase, Metaphase, Anaphase, Telophase; followed by Cytokinesis.

  • Key structures:

    • Sister chromatids

    • Centrosome

    • Microtubule

    • Spindle microtubules

    • Nuclear envelope (re-forms in Telophase)

Checkpoints in the Cell Cycle

  • G₁/S checkpoint

  • G₂/S checkpoint

The Cell in Context

  • Nucleolus, Nucleus, Cytoplasm are major cellular compartments.

  • Nucleus houses DNA and coordinates production via ribosomes; nucleolus is part of ribosome synthesis.

When Do Cells Divide? Turnover and Mitotic Figures

  • Cellular turnover varies by tissue type.

  • Epithelium: higher turnover rate than other tissues.

  • Neurons: long-lived cells; low turnover.

  • In normal tissues, mitotic figures are not commonly seen on light microscopy.

  • Mitosis lasts textmitosis=3.4 hourst_{ ext{mitosis}} = 3.4\ \text{hours}.

  • Mitotic figures can be found in rapidly renewing tissues such as the intestinal epithelium; turnover there is 5−6 days5-6\ \text{days}.

Variation in Cell Shape and Staining

  • Cells vary in shape and size; shape correlates with function.

  • Organelles can influence H&E staining.

  • The Cell Nucleus varies across cell types (examples shown include cells from lung, cardiac muscle, neuronal cells, and fibroblasts).

Objectives (Recap)

  • Describe the difference in magnification and resolution.

  • Compare and contrast different staining types for light microscopy (LM).

  • Identify cell/organelles in electron micrographs (EMs) and describe organelle function.

  • Given a cell and its function, predict which organelles would be abundant.

  • Recognize the different types of EMs and why EMs are helpful.

  • Identify cells in mitosis and discuss the clinical importance of identifying cells in mitosis.


Microscopy is essential for viewing cellular and tissue details, relying on magnification and resolution. Histology, the study of microanatomy, correlates structure with function and involves precise tissue preparation steps: collection, fixation, processing, embedding, sectioning, and staining. Common light microscopy stains include Hematoxylin (stains acidic/basophilic structures blue, like DNA and ribosomes) and Eosin (stains basic/acidophilic structures red/pink, like cytoplasm and mitochondria).

Beyond light microscopy, Electron Microscopy (EM) provides ultrastructural detail not visible with conventional methods. Transmission Electron Microscopy (TEM) offers 2D cellular and organelle views, while Scanning Electron Microscopy (SEM) provides 3D surface features. Biological organization progresses from protoplasm to cells, tissues, organs, and organ systems.

Cells contain various organelles: membranous (e.g., cell membrane, nucleus, mitochondria, ER, Golgi, lysosomes) and non-membranous (e.g., cytoskeleton, free ribosomes), each with specialized functions like energy production (mitochondria), protein synthesis and modification (ER, Golgi, ribosomes), and digestion (lysosomes). Cells also store inclusions like nutrients and pigments. The abundance of specific organelles reflects a cell's primary function.

The cell cycle, vital for growth and repair, includes Interphase (G₁, S, G₂) and M phase (Mitosis and Cytokinesis), regulated by checkpoints. Cellular turnover rates vary by tissue type, with mitotic figures indicating active division, important for both normal physiological processes and disease diagnosis.