Lecture Notes on Microscopy and Cell Structure

Lecture Presentations for Campbell Biology, Ninth Edition

Authors: Jane B. Reece, Lisa A. Urry, Michael L. Cain, Steven A. Wasserman, Peter V. Minorsky, Robert B. Jackson

Lecturers: Erin Barley, Kathleen Fitzpatrick

Topic: Microscopy & the Cell Structure

Aim & Objectives of the Lecturer

  • Aim: To determine the correlation between the microscopes and the cell structure.
  • Objectives:
    • To identify types of microscopes used to view specimens.
    • To differentiate between eukaryotes and prokaryotes.
    • To identify the functions of various cell organelles.

Importance of Microscopy in Biology

  • Microscopes are essential tools in biology, allowing scientists to study cells that are typically too small to be seen by the naked eye.
  • Cells can be complex structures, requiring detailed examination.

Anatomy of a Microscope

  • Eyepieces (Ocular Lens): Lenses through which the viewer looks.
  • Head: Supports the ocular lens.
  • Digital LCD Screen: Displays images of the specimen.
  • Arm (Frame): Supports the optical components.
  • Diopter Adjustment: Allows for fine-tuning of the focus for different eyes.
  • Nose Piece: Holds the objective lenses.
  • Objective Lenses: Different lenses with varying magnifications.
  • Stage Clip: Holds the slides in place on the stage.
  • Aperture: Opening that allows light to reach the specimen.
  • Stage Controls: Adjust the position of the stage.
  • Mechanical Stage: Platform for the specimen slide that can move.
  • Condenser: Focuses the light onto the specimen.
  • Illumination Light Switch: Turns the light on/off.
  • Base: Supports the entire microscope.
  • Brightness Adjustment: Controls the intensity of the light.
  • Coarse Adjustment: Provides initial focusing.
  • Fine Adjustment: Allows for precise focusing.

Principles of Microscopy

  • Purpose of Microscopy: To visualize cells that are too small to see with the naked eye.
  • Light Microscopes (LM):
    • Work by passing visible light through a specimen and glass lenses.
    • Lenses refract (bend) the light, effectively magnifying the image.

Important Parameters of Microscopy

  • Magnification: The ratio of an object’s image size compared to its real size.
  • Resolution: The measure of image clarity, specifically the minimum distance between two distinguishable points.
  • Contrast: Visible differences in parts of the sample, which aids in distinguishing structures.

Microscopy Scale

  • Size scales for visualization:
    • Atoms: 0.1 nm
    • Small molecules: 1 nm to 10 nm
    • Ribosomes: 20 nm
    • Proteins: Approx. 5 nm to 10 nm
    • Lipids: 1 nm to 1 μm
    • Mitochondrion: 1 μm

Types of Light Microscopy Techniques

  • Brightfield Microscopy: Can be used for unstained and stained specimens.
  • Phase-contrast Microscopy: Increases contrast in transparent specimens.
  • Fluorescence Microscopy: Uses fluorescent dyes to visualize cellular components.
  • Confocal Microscopy: Produces sharper images of thick tissues using laser illumination.
  • Differential Interference Contrast (Nomarski): Enhances contrast in unstained samples.

Limitations of Light Microscopy

  • LMs can magnify effectively up to about 1,000 times the actual specimen size.
  • Most subcellular structures, including organelles, are too small to be resolved using a light microscope.

Electron Microscopy (EM)

  • Types of EM:
    • Scanning Electron Microscopes (SEMs):
    • Focus a beam of electrons on the surface of a specimen.
    • Produce 3-D images.
    • Transmission Electron Microscopes (TEMs):
    • Focus a beam of electrons through a specimen.
    • Primarily used to study internal cell structures.

Advances in Light Microscopy Technologies

  • Recent Techniques:
    • Deconvolution Microscopy: Improves resolution through computational techniques.
    • Confocal Microscopy: Enhances 3D imaging of tissues and cells.

Cell Fractionation

  • Definition: A technique that separates cells into their organelle components.
  • Process:
    • Cells are broken apart through homogenization
    • Centrifuges are used to increase gravitational force to separate cell components.
    • Allows scientists to study the functions of organelles and enables correlation of cell function with structure.

Cell Fractionation Techniques

  • Homogenization: Mixing tissue cells to create a homogenate.
  • Centrifugation: Functionally separates components by spinning the homogenate at various forces (g) for specific times.
    • Differential Centrifugation Process:
    • Centrifuged at 1,000 g for 10 min, separates debris and nuclei.
    • Further centrifugation to 20,000 g for 20 min yields a pellet rich in mitochondria.
    • Subsequent spins at 80,000 g and 150,000 g yield pellets rich in other organelles, such as microsomes and ribosomes.

Overview of Eukaryotic vs. Prokaryotic Cells

  • Eukaryotic Cells:

    • Characteristics:
    • Contain DNA in a membrane-bound nucleus.
    • Have membrane-bound organelles.
    • Generally larger in size compared to prokaryotic cells.
    • Examples: Protists, fungi, animals, and plants.

  • Prokaryotic Cells:

    • Characteristics:
    • Lack a nucleus; contain DNA in an unbound nucleoid region.
    • No membrane-bound organelles.
    • Bound cytoplasm by a plasma membrane.
    • Examples: Organisms in the domains Bacteria and Archaea.

Basic Features Shared by All Cells

  • Plasma Membrane (Cell Membrane): Serves as a selective barrier regulating entry and exit of substances.
  • Cytosol: Semifluid substance within cells.
  • Chromosomes: Carry genetic information.
  • Ribosomes: Sites of protein synthesis.

Structure of the Plasma Membrane

  • Composition:
    • Consists of a double layer of phospholipids.
    • Hydrophilic (water-attracting) regions face the inside and outside of the cell, while hydrophobic (water-repelling) regions are tucked away toward each other inside the bilayer.
    • Associated proteins and carbohydrate side chains play roles in signaling and structural functions.

![Structure of Plasma Membrane](Figure 6.6)