Microscopy & Cell Visualization

• Microscopy Skills


  Microscopy is a fundamental technique in biology that allows us to observe and study cells and tissues that are too small to see with the naked eye
• Staining


  Staining helps to increase contrast and highlight specific cellular structures.
• Common stains include

  • Methylene blue for animal cells
  • Iodine for plant cells (stains starch dark blue-black)
  • Safranin for plant cell walls
• Making Temporary Mounts


  This involves preparing a specimen for viewing under a microscope.
• Measuring with an Eyepiece Graticule

  An eyepiece graticule is a small ruler in the eyepiece of the microscope.
• Focusing

  • Use the coarse adjustment to bring the specimen into rough focus
  • Use the fine adjustment for precise focusing
• Calculating Actual Size and Magnification

  Actual size = (Size in eyepiece units) × (Calibration factor)

  Magnification = (Size of image) ÷ (Actual size of object)
• Producing a Scale Bar


  A scale bar provides a visual reference for the size of the specimen in your microscope image.
• The process of measurement in microscopy follows these steps:

  1. Calibration of instruments
  2. Observation of specimen
  3. Recording of measurements
  4. Calculation of actual sizes

  This process demonstrates how scientists transform raw observations into meaningful data.
• Developments in Microscopy


  Microscopy has come a long way since the first simple light microscopes. Modern techniques allow us to visualize cellular structures with unprecedented detail and even observe molecular interactions in living cells.
• Electron Microscopy

  Electron microscopes use beams of electrons instead of light to create images.
• Advantages of Electron Microscopy:

  1. Much higher magnification and resolution than light microscopes
  2. Ability to see internal cellular structures in great detail
• There are two main types of Electron Microscopy:

  1. Transmission Electron Microscopy (TEM)
  2. Scanning Electron Microscopy (SEM)
• TEM can achieve magnifications up to 2,000,000x, compared to about 1,500x for light microscopes.
• Freeze Fracture


  This technique involves rapidly freezing a sample and then fracturing it.
• Advantages of Freeze Fracture:

  1. Reveals the interior of membranes
  2. Preserves the natural state of the sample better than chemical fixation
• Cryogenic Electron Microscopy (Cryo-EM)


  Cryo-EM involves flash-freezing samples in their native state and viewing them with an electron microscope.
• Advantages of Cryogenic Electron Microscopy (Cryo-EM):

  1. Allows visualization of biological molecules in their native state
  2. Doesn't require sample staining or fixation
  3. Can be used to determine 3D structures of proteins
• Cryo-EM has revolutionized structural biology, earning its developers the 2017 Nobel Prize in Chemistry.
• Fluorescent Stains in Light Microscopy


  Fluorescent stains absorb light at one wavelength and emit it at another, allowing specific cellular components to be visualized.
• Advantages of Fluorescent Stains in Light Microscopy:

  1. Can label specific cellular structures or molecules
  2. Allows visualization of multiple components simultaneously using different colors
  3. Can be used in living cells
• Green Fluorescent Protein (GFP) can be genetically engineered into organisms to make specific proteins fluoresce green.
• Immunofluorescence


  This technique uses fluorescently labeled antibodies to detect specific proteins in cells.
• Advantages of Immunofluorescence: 


  1. Highly specific - can locate exact proteins within a cell
  2. Can be used to track changes in protein location or abundance
• Identifying Cell Types - Prokaryotic Cells


  • Smaller size (typically 0.1-5 μm)
  • No membrane-bound organelles
  • Nucleoid region instead of a nucleus
• Identifying Cell Types - Plant Cells

  • Rectangular shape
  • Thick cell wall
  • Large central vacuole
  • Presence of chloroplasts
• Identifying Cell Types - Animal Cells


  • Irregular shape
  • No cell wall
  • Small or no vacuoles
  • Presence of centrioles
• What is the nucleoid region in prokaryotes?

  It appears as a light area in the cell and contains the bacterial DNA.
• What does the prokaryotic cell wall consist of?

  A thick layer outside the plasma membrane.
• What characterizes the nucleus in eukaryotes?


  It is a large, membrane-bound structure that contains darker-staining chromatin.
• What is the shape and structure of a mitochondrion?

  It is oval or rod-shaped with a double membrane and inner folds called cristae.
• What is the structure of a chloroplast in plants?


  It is oval-shaped with an internal membrane system called thylakoids.
• What is the sap vacuole in plants?


  It is a large, membrane-bound structure that is often centrally located.
• What is the Golgi apparatus?


  It is a stack of flattened membrane sacs.
• What distinguishes rough ER from smooth ER?


  Rough ER is studded with ribosomes, while smooth ER does not have ribosomes.
• How are chromosomes visible in the nucleus?

  They are visible as condensed structures within the nucleus.
• What do ribosomes look like in cells?


  They appear as small, dark dots, often on rough ER or free in the cytoplasm.
• What is the function of the cell wall in plants and fungi?


  It is a thick layer outside the plasma membrane.
• What is the plasma membrane?

  It is a thin line surrounding the cell contents.
• What are microvilli in some animal cells?

  They are small, finger-like projections on the cell surface.