Microscopy Fundamentals

Microscope Fundamentals

• Smallest distinguishable distance between two points is measured in micrometers (μm).
• Contrast enables detection of different specimen regions based on intensity or color.

Magnification

• Apparent size increase achieved by convex lenses; a compound microscope uses two lens sets for magnification.
• Total magnification formula: Total Magnification = Objective Magnification x Eyepiece Magnification.
• Example: Objective Mag = 40X, Eyepiece Mag = 10X, resulting in Total Mag = 400X.

Objective Lenses

• Scanning Objective Lens (4x): Lowest magnification; provides a general overview of the slide.
• Low Power Objective (10x): Greater magnification; ideal for observing slide samples without excessive detail.
• High Power Objective Lens (40x): Known as “high dry” lens; best for fine detail analysis of specimen samples.
• Oil Immersion Objective Lens (100x): Highest magnification; requires immersion oil to improve clarity due to refractive index differences.

Magnification Calculations

• Magnification can be calculated if either image size or actual size is known.
• Formulas include:
  • Magnification = Image Size ÷ Actual Size
  • Actual Size = Image Size ÷ Magnification
  • Image Size = Actual Size × Magnification

Microscopy Techniques

• Dark Field Microscopy: Illuminates unstained samples against a dark background for enhanced visibility; uses opaque disc to scatter only reflected light.
• Effective for viewing small aquatic organisms, diatoms, unstained bacteria, and protozoa.

Differential Interference Contrast (DIC)

• Generates a 3D image using polarized light split into two beams.
• Beams interact with specimen thickness, leading to brightness variations showing refractive index differences.

Fluorescence Microscopy

• Utilizes fluorescence and phosphorescence for imaging; allows observation of substances that absorb and emit light differently.
• Light sources include xenon arc lamps, mercury-vapor lamps, lasers, and high-power LEDs.
• Employs dichroic mirrors and filters to isolate specific wavelengths of emitted light.

Digital Holographic Microscopy (DHM)

• Utilizes interference patterns generated by overlapping laser beams to create a hologram of the sample.
• Advantages include static and dynamic 3D imaging, auto digital focus for quick scanning, and non-invasive techniques for live cells.

Scanning Tunneling Microscopy (STM)

• Based on tunneling electrons between a conductive probe and a conductive sample surface.
• The probe is moved closely to the sample; the resulting tunneling current provides detailed surface images.

Tunneling Phenomenon

• Describes how electrons move across gaps between objects with different electric potentials when in close proximity.