🦠Microbiology Unit 2 – How We See the Invisible World

Key Concepts and Definitions

• Microscopy involves using microscopes to view objects that are too small to be seen with the naked eye
• Resolution measures the ability to distinguish two separate points as distinct and is limited by the wavelength of light used
• Magnification increases the apparent size of an object but does not necessarily increase resolution
• Contrast enhances the difference between the object and the background, making it easier to observe details
• Numerical aperture (NA) is a measure of a microscope's ability to gather light and resolve fine details, calculated using the formula: $NA = n \sin θ$
  • $n$ represents the refractive index of the medium between the objective lens and the specimen
  • $θ$ represents the half-angle of the maximum cone of light that can enter the objective lens
• Working distance is the distance between the objective lens and the specimen when the specimen is in focus
• Depth of field refers to the range of distances over which the specimen remains in acceptable focus

History of Microscopy

• The first microscopes were simple magnifying glasses developed in the late 16th century by Dutch spectacle makers Hans and Zacharias Janssen
• Robert Hooke coined the term "cell" in 1665 after observing plant cells using a compound microscope
• Antonie van Leeuwenhoek advanced microscopy in the late 17th century by creating high-quality single-lens microscopes capable of magnifying up to 300 times
• Ernst Abbe and Carl Zeiss collaborated in the 19th century to develop improved lenses and illumination systems, leading to the creation of the first modern compound microscopes
• August Köhler introduced Köhler illumination in 1893, a method that provides even illumination of the specimen and is still used in modern microscopes
• The development of electron microscopy in the 1930s allowed for the visualization of structures smaller than the wavelength of visible light
• The invention of the scanning tunneling microscope in 1981 and the atomic force microscope in 1986 enabled the imaging of individual atoms and molecules

Types of Microscopes

• Light microscopes use visible light to magnify specimens and include:
  • Bright-field microscopes, which produce dark objects on a bright background
  • Dark-field microscopes, which produce bright objects on a dark background
  • Phase-contrast microscopes, which convert phase shifts in light passing through a specimen into brightness differences
  • Differential interference contrast (DIC) microscopes, which use polarized light to enhance contrast
• Electron microscopes use electron beams to magnify specimens and achieve higher resolution than light microscopes
  • Transmission electron microscopes (TEM) pass electrons through thin sections of specimens to create images
  • Scanning electron microscopes (SEM) scan the surface of specimens with electron beams to generate detailed 3D images
• Scanning probe microscopes use physical probes to scan and interact with the surface of a specimen, providing information about its topography and properties
  • Scanning tunneling microscopes (STM) use an electrically conductive tip to measure the electron density of a conductive surface
  • Atomic force microscopes (AFM) use a fine tip attached to a cantilever to scan the surface of a specimen and measure its topography

Principles of Light and Optics

• Light behaves as both a wave and a particle (photons), a concept known as wave-particle duality
• Refraction occurs when light passes through materials of different densities, causing it to bend and change velocity
• Lenses use refraction to focus light and form images
  • Convex lenses converge light rays to a focal point
  • Concave lenses diverge light rays
• Aberrations are imperfections in the image formed by a lens system, such as:
  • Spherical aberration, caused by the difference in refraction between the center and edges of a lens
  • Chromatic aberration, caused by the different refractive indices of various wavelengths of light
• Numerical aperture (NA) determines the light-gathering ability and resolution of a microscope objective
• Köhler illumination provides optimal contrast and even illumination by focusing the light source onto the specimen and the condenser aperture diaphragm onto the objective back focal plane

Sample Preparation Techniques

• Fixation preserves the structure of specimens by cross-linking proteins and stabilizing membranes (formaldehyde, glutaraldehyde)
• Dehydration removes water from specimens using a series of increasing concentrations of ethanol or acetone
• Embedding infiltrates specimens with a supportive medium (paraffin wax, epoxy resin) to allow for thin sectioning
• Sectioning cuts specimens into thin slices using a microtome or ultramicrotome
  • Paraffin-embedded samples are typically cut into 5-10 μm thick sections
  • Resin-embedded samples for electron microscopy are cut into 50-100 nm thick sections
• Staining enhances contrast and highlights specific structures or molecules
  • Hematoxylin and eosin (H&E) staining is commonly used for tissue sections, staining nuclei blue and cytoplasm pink
  • Immunohistochemistry uses antibodies conjugated to enzymes or fluorophores to label specific proteins
• Mounting places the stained specimen on a microscope slide and covers it with a coverslip and mounting medium for optimal viewing

Microscopy in Action: Observing Microorganisms

• Brightfield microscopy is the most common technique for observing bacteria, fungi, and parasites in clinical samples
• Gram staining differentiates bacteria based on cell wall composition, with Gram-positive bacteria appearing purple and Gram-negative bacteria appearing pink
• Acid-fast staining identifies Mycobacterium species, which retain the carbol fuchsin stain after an acid-alcohol decolorization step
• Wet mounts allow for the observation of live microorganisms in a drop of water or saline solution
• Darkfield microscopy enhances the contrast of unstained, transparent specimens like spirochetes (Treponema pallidum)
• Fluorescence microscopy uses fluorescent dyes to label specific structures or molecules, such as the auramine-rhodamine stain for Mycobacterium tuberculosis
• Electron microscopy provides high-resolution images of viruses, bacteria, and cellular ultrastructure
  • Negative staining with heavy metal salts (uranyl acetate) enhances contrast in TEM images
  • SEM reveals the surface morphology of microorganisms and biofilms

Limitations and Challenges

• Resolution is limited by the wavelength of light used, with visible light microscopes having a maximum resolution of approximately 200 nm
• Sample preparation can introduce artifacts or alter the native structure of specimens
  • Fixation and dehydration can cause shrinkage or distortion of tissues
  • Sectioning can result in incomplete or damaged samples
• Staining can be nonspecific or variable, leading to misinterpretation of results
• Live cell imaging is challenging due to the need to maintain appropriate environmental conditions (temperature, pH, osmolarity) and minimize phototoxicity
• Electron microscopy requires extensive sample preparation and can only image dead, fixed specimens
• Three-dimensional reconstruction from 2D images can be computationally intensive and may not fully capture the complexity of biological structures

Future Developments in Microscopy

• Super-resolution microscopy techniques (STED, PALM, STORM) overcome the diffraction limit of light, achieving resolutions of 20-100 nm
  • Stimulated emission depletion (STED) microscopy uses a depletion laser to selectively turn off fluorophores around a focal point
  • Photoactivated localization microscopy (PALM) and stochastic optical reconstruction microscopy (STORM) use photoswitchable fluorophores to localize individual molecules
• Cryo-electron microscopy (cryo-EM) enables the high-resolution imaging of macromolecular complexes and viruses in their native state
  • Specimens are rapidly frozen in liquid ethane to preserve their structure
  • Single-particle analysis averages multiple 2D projections to reconstruct a 3D model
• Correlative light and electron microscopy (CLEM) combines the advantages of fluorescence microscopy and electron microscopy to study the same specimen
• Expansion microscopy physically expands specimens using a swellable polymer, allowing for super-resolution imaging with conventional microscopes
• Advances in computational methods, such as deep learning and artificial intelligence, will improve image analysis, segmentation, and interpretation