Microscopy and Staining Techniques

Biology Club and Course Announcements

• Biology Club Kickoff Meeting Details:

  • Date & Time: Tuesday, September 15th, from $4:00\,\text{PM}$ to $5:00\,\text{PM}$.

  • Location: Room 352.

  • Faculty Adviser: Professor Heather Cadogan.

  • Eligibility: Open to all students regardless of major (includes Biology, Psychology, Pre-Nursing, Environmental Science, Biomedical Science, etc.).

  • Activities & History: Guest speakers, designing and installing campus gardens, certifying the campus as Baywise (meeting specific environmental standard requirements), pizza parties, game parties, and field trips.

• Course Logistics & Reminders:

  • The History of Microbiology Quiz is due by the end of the day.

  • Upcoming schedule: Finishing Chapter 2 (Gram staining details, estimated $30\,\text{min}$) during the next class prior to commencing Chapter 3.

Fundamentals of Microscopy Concepts

• Refraction:

  • The bending of light rays as they pass from one medium into another medium of a different density (such as air into water).

• Resolution (Resolving Power):

  • The ability of an optical system to distinguish two adjacent points or objects as separate and distinct entities.

  • Overall clarity of an image depends directly on resolving power.

• Relationship Between Resolution and Wavelength:

  • Resolution and wavelength are inversely related.

  • Shorter wavelengths of radiation provide higher (better) resolving power.

Microscopy Scale and Specimen Magnification Range

• Unaided Human Eye:

  • Resolves large biological specimens (e.g., ticks, human egg cells, frog egg cells).

• Light Microscopy:

  • Resolves eukaryotic cells and bacteria (e.g., red blood cells, Escherichia coli).

• Electron Microscopy:

  • Resolves cellular ultrastructure, viruses (e.g., bacteriophages, herpes virus, influenza virus), and biological macromolecules (e.g., proteins, DNA).

• Probe Microscopy:

  • Resolves atomic-level structures and molecular bonds (e.g., individual gold atoms, nanocellulose molecules).

Light Microscopy

Light microscopy utilizes visible light within the electromagnetic spectrum (positioned between short gamma rays and long radio waves). Modern laboratory light microscopes are binocular (containing two eyepieces), which combine light beams to form a single circular view called the field of view.

Bright Field Microscopy

• Characteristics:

  • The background field of view is brightly illuminated.

  • Specimens are frequently stained with synthetic dyes to increase contrast against the bright background, as unstained biological specimens are often translucent.

• Clinical Applications & Examples:

  • Gram Stain: Used to visualize bacterial morphology and cell wall characteristics.

  • White Blood Cells: Visualized with pale pink cytoplasm and dark internal nuclei.

  • Neisseria gonorrhoeae: Gram-negative diplococci causing gonorrhea. This bacterium reproduces inside host white blood cells rather than being destroyed by them.

Dark Field Microscopy

• Mechanism:

  • Utilizes a specialized stop in the condenser that blocks central light rays, directing light at an oblique angle toward the specimen.

  • Only light reflected or scattered off the specimen enters the objective lens, producing a bright sample against a dark background (resembling a photographic negative).

• Advantages:

  • Specimens do not require staining, preserving delicate living cells that might be damaged or distorted by chemical dyes.

• Clinical Applications & Examples:

  • Spirochetes: Flexible, corkscrew-shaped bacteria easily damaged by heat fixing and staining.

  • Borrelia burgdorferi: Corkscrew-shaped bacterium that causes Lyme disease (named after Lyme, Connecticut).

  • Treponema pallidum: Spirochete that causes syphilis (historically treated with Salvarsan prior to antibiotic development).

Phase Contrast Microscopy

• Mechanism:

  • Splits a light beam into two pathways: one path passes directly through the medium, while the other passes through the specimen.

  • Light travels faster through less dense regions (cytoplasm) and slower through dense regions (nucleus or inclusions).

  • When the light beams are recombined, phase shifts (refraction differences) create variations in light intensity and shading.

• Characteristics:

  • Produces detailed, high-contrast images of internal structures in living, unstained specimens, giving a pseudo-three-dimensional appearance.

• Comparative Example (Amoeba proteus):

  • Bright Field (Unstained): Difficult to discern cell boundaries unless dense internal granules are present.

  • Dark Field: Specimen outline pops out clearly, but fine internal details are obscured.

  • Phase Contrast: Superior clarity showing sharp internal structures, including the nucleus and inclusion granules.

Differential Interference Contrast (DIC) Microscopy

• Mechanism:

  • Similar to phase contrast (uses two separated light beams recombined to generate image contrast).

  • Employs prisms to split light beams into component wavelengths.

• Characteristics:

  • Diffracts light into colors, producing a vibrant, highly detailed image with pronounced three-dimensional depth and color variations rather than grayscale shading.

Fluorescent Microscopy

• Mechanism:

  • Uses ultraviolet (UV) light sources to illuminate specimens stained with fluorescent dyes called fluorochromes.

  • UV light excites electrons within the fluorochrome molecules; as electrons return to their ground state, they release energy as visible light.

• Immunofluorescence Applications:

  • Fluorochromes are conjugated to specific antibodies.

  • When applied to a sample, the labeled antibody binds to its target pathogen. Excess antibody is washed off, leaving the target fluorescing under UV light.

  • Used in diagnostic testing for pathogens such as Legionella (causes Legionnaires' disease) and Bordetella pertussis (causes whooping cough / pertussis).

Confocal Microscopy

• Mechanism:

  • Uses focused laser light to excite fluorochromes within a single focal plane.

  • Incorporates pinhole apertures to eliminate out-of-focus light scattering above and below the focal plane.

• Characteristics & Features:

  • Computers capture optical sections across multiple planes and construct stackable, three-dimensional digital reconstructions of complex specimens.

Electron Microscopy

Invented in $1932$ by Ernst Ruska (awarded the Nobel Prize in Physics in $1986$, $54\,\text{years}$ later). Electron microscopes bypass the limits of light refraction by utilizing electron beams focused by electromagnets instead of glass lenses.

• Key Physics Principles:

  • Wavelength of shortest visible light: $400\,\text{nm}$.

  • Wavelength of an electron: $0.01\,\text{nm}$ to $0.001\,\text{nm}$.

  • Extremely short wavelengths yield vastly superior resolution and high magnification capabilities.

• Operational Constraints:

  • Electrons travel through a vacuum chamber; therefore, live specimens cannot be observed.

Transmission Electron Microscopy (TEM)

• Mechanism:

  • A beam of electrons passes (transmits) directly through an ultrathin section of a specimen.

• Features & Specifications:

  • Magnification: $100,000\times$ to $500,000\times$.

  • Generates two-dimensional, ultrastructural views of internal cellular organelles, viral cores, and molecular complexes.

• Examples:

  • Internal structures of Paramecium.

  • Herpesvirus capsids and nucleic acid cores.

  • Dividing Escherichia coli cells showing fimbriae and replicated genetic material moving toward division sites.

Scanning Electron Microscopy (SEM)

• Mechanism:

  • A primary electron beam scans back and forth over the surface of a specimen coated in a thin layer of heavy metal.

  • Secondary electrons knocked off the surface are collected to build an image.

• Features & Specifications:

  • Magnification: $10,000\times$ to $50,000\times$.

  • Generates vivid three-dimensional topological images of specimen surface structures.

• Examples:

  • Surface cilia of Paramecium.

  • Bacterial biofilms in dental plaque.

  • Avian influenza virus surface morphology.

Probe Microscopy

Developed in the $1980$\text{s}, probe microscopy uses physical probes with tips measuring just one atom in diameter (frequently constructed from tungsten) to achieve atomic-level resolution without requiring a vacuum, allowing live specimen analysis.

Scanning Tunneling Microscopy (STM)

• Development:

  • Invented in $1980$ by Gerd Binnig and Heinrich Rohrer (awarded Nobel Prize in Physics in $1986$, $6\,\text{years}$ after invention).

• Mechanism:

  • The metallic probe hovers extremely close to the specimen surface without making physical contact.

  • An electrical voltage induces electron flow ("tunneling current") between the probe tip and surface atoms.

  • A computer translates changes in current intensity into an atomic-scale topographical map.

• Example: Visualizing individual columns of gold atoms.

Atomic Force Microscopy (AFM)

• Development:

  • Invented in $1982$ by IBM scientists.

• Mechanism:

  • The metallic probe tip maintains direct contact with the specimen surface.

  • The probe is mounted on a flexible cantilever arm. As the tip moves over surface contours, the cantilever deflects up and down.

  • A laser beam reflected off the top of the cantilever tracks deflection angles; a computer processes these angular shifts into a three-dimensional surface image.

• Example: Visualizing nanocellulose molecules derived from plants.

Sample Preparation and Staining Techniques

Specimen Mounting Techniques

• Wet Mount:

  • Specimen is suspended in a drop of liquid (water or saline) on a slide and covered with a glass or plastic coverslip.

  • Keeps specimens moist, allowing real-time observation of living, motile organisms.

• Smear Preparation & Heat Fixing:

  • A thin film of liquid specimen is spread across a glass slide and allowed to dry.

  • The slide is placed on a slide warmer plate for heat fixing.

  • Heat fixing kills organisms, fixes cell structures in place, and firmly adheres cells to the glass slide so they do not wash off during staining procedures.

Staining Dyes by Charge Characteristics

• Positive Stains (Basic Dyes):

  • Possess positively charged chromophores.

  • Bacterial cell surfaces naturally carry a negative charge; opposite charges attract, causing the dye to bind directly to the cells.

  • Result: Colored cells against a clear/bright field of view.

• Negative Stains (Acidic Dyes):

  • Possess negatively charged chromophores.

  • Repelled by the negative charges on bacterial cell surfaces.

  • Result: Dyes color the surrounding background, leaving cells clear and uncolored.

Staining Procedures by Complexity

• Simple Staining:

  • Employs a single basic dye (e.g., methylene blue).

  • Used primarily to highlight cell morphology, size, and arrangement.

• Differential Staining:

  • Employs two or more contrasting dyes (e.g., Gram stain).

  • Differentiates different types of bacteria or structures based on distinct biochemical and structural features (such as cell wall thickness and composition).