Lecture 2: Cells and Microscopy

Scale of Cells

  • Cell biology studies objects of different sizes.
  • It's important to develop a sense for the relative sizes of cells, organelles, and molecules.
  • To see objects smaller than approximately 0.2 mm, a microscope is needed.
  • Successive 10x magnifications are used to visualize objects from 20 mm down to 0.2 nm.

History of Microscopy and the Discovery of Cells

  • Early microscopists aimed to enlarge small living objects.
  • Robert Hooke (1665)
    • First to describe "cells" in cork.
    • Used a compound microscope with separate objective and eyepiece.
  • Antonius van Leeuwenhoek
    • First to describe protozoa (1674), sperm (1677), and bacteria (1683).
    • Used a "simplex" microscope with a single lens.
    • Achieved 275x magnification with a single lens.

The Cell Theory (1838-1839)

  • Theodor Schwann & Matthias Schleiden
    1. All life forms are made from one or more cells.
    2. The cell is the smallest form of life.
  • Rudolf Virchow (~10 years later)
    1. Every cell originates from a pre-existing cell.
  • Walther Flemming (1879)
    • Described chromosomes and mitosis.

Microscopes

Light Microscope

  • Light is transmitted through the specimen.
  • Resolution: 0.2μm0.2 \mu m (200 nm), about 1000x better than the unaided eye.
  • Used to view live cells.
  • Methods to achieve higher contrast:
    • Phase contrast
    • Differential interference contrast (DIC or Nomarski optics)
    • Tissue fixing and staining

Electron Microscope

Transmission Electron Microscope (TEM)
  • Resolution: 0.2 nm, about 1,000,000x better than the unaided eye, and 1,000x better than the light microscope.
Scanning Electron Microscope (SEM)
  • Resolution: 3-20 nm, approximately 10x worse than TEM.
  • Major strength: large depth of focus.
  • Limitation: can only observe the surface.

Fluorescence Microscopy

  • A special type of light microscopy that utilizes the properties of fluorescent molecules.
    1. Fluorescent molecules preferentially absorb light of a specific wavelength (excitation light).
    2. Fluorescent molecules emit light at a longer wavelength than the one they are excited with (emission light).
  • Fluorescent molecules can highlight specific locations or molecules inside the cell (e.g., actin filaments, microtubules, intermediate filaments).
  • Super-resolution fluorescence microscopy
    • Allows researchers to overcome the 200 nm resolution limit.
    • Achieves a resolution limit of 20 nm (for microtubules).

Confocal Microscopy

  • An advanced form of fluorescence microscopy.
  • Features:
    • Sharp images due to selective collection of light from a narrow focal plane.
    • Enables optical sectioning and 3-D reconstruction.
    • Excitation by a laser (single wavelength light) improves co-localization capability using different fluorescent markers.
  • Used to determine if proteins are present in the same or different compartments within the cell.

Summary: Different Kinds of Microscopy

  • Light Transmission
    • Bright-field
    • Phase contrast
    • DIC (Nomarski)
    • Fluorescence
      • Wide-field
      • Confocal
  • Electron
    • TEM (transmission electron microscopy)
    • SEM (scanning electron microscopy)

When to Use Different Kinds of Microscopy (Pros and Cons)

  • Conventional light (bright-field, phase, and DIC) microscopy
    • Pros:
      • Easiest to do.
      • Can look at live cells.
    • Cons:
      • Limited ability to visualize structures.
      • Sample must be transparent, otherwise requires sectioning.
  • Electron microscopy
    • Pros:
      • Greatest resolution, can see smallest objects
    • TEM:
      • Cons: Requires thin sectioning and contrasting treatment
    • SEM:
      • Pros: Large depth of field
      • Cons: Can only see the surface; cells must be fixed (no live imaging)
  • Fluorescence (light) microscopy
    • Pros:
      • Allows visualization of specific structures in cells.
      • Can do live cell imaging.
      • Confocal: Sharp images, ideal for co-localization studies.
    • Cons: Signal may fade due to photobleaching.

The Green Fluorescent Protein (GFP)

  • Allows for live imaging of fluorescence.
  • Origin: Aequorea victoria (jellyfish).
  • Purified as a protein in the 1970s.
  • DNA (gene) isolated in 1992.
  • Expressed in live E. coli and C. elegans in 1994.
  • The Nobel Prize in Chemistry, 2008 was awarded for the discovery and development of GFP.
    • Osamu Shimomura
    • Martin Chalfie
    • Roger Tsien developed different "flavors" of fluorescent proteins.

How to Use GFP for Live Imaging

  • Express under a cell-type specific DNA regulatory element to allow visualization or marking of a particular type of cell.
    • In this mode, GFP is used as a reporter of gene expression, showing when and in which cells a gene is expressed (e.g., in a multicellular organism).
  • Express as a protein fusion to examine the localization of a particular protein within a cell.
    • In this mode, the distribution of a protein within a single cell is analyzed.
Examples of GFP Use
  • Cell-type specific expression:
    • Neurons: GFP expressed only in neurons using a neuron-specific gene regulatory region.
    • Skin cells: GFP expressed in all skin cells using a skin-specific gene regulatory region.
  • Reporter genes
    • Can reveal the activity of specific DNA regions.
  • Protein Fusion:
    • GFP can be fused to a normal protein to examine that protein’s subcellular localization.
    • The tagged protein X with GFP can signal for a specific location inside the cell (e.g., nucleus, microtubules, etc.).

Time-Lapse Microscopy with GFP Fusion Proteins

  • Used to visualize sub-cellular localization over time.
  • Examples:
    • GFP fused to a nuclear protein marks gastrulation movements in Drosophila embryos.
    • GFP fused to a microtubule-specific protein marks mitotic spindles in Drosophila embryos.