IVF Laboratory Technology - Week 1 Lecture 3: Contrast Enhancing Techniques

Microscope Components

  • Function of each part of the light microscope and its location:
    • Illumination Source
    • Sub-stage condenser
    • Diaphragms
    • Specimen Stage
    • Objectives
    • Eyepiece
  • Basic design of the inverted microscope and its advantages and disadvantages.
  • Basic structure and operation of the stereomicroscope.
  • Basic principle for different microscopic techniques to improve specimen contrast or imaging:
    • Darkfield
    • Polarized
    • Phase contrast
    • Hofman Modulation Contrast

Contrast Enhancing Techniques

Dark Field

  • Darkfield microscopy uses oblique illumination to enhance contrast in specimens not well-imaged under normal brightfield conditions.
  • Direct light is blocked by an opaque stop in the sub-stage condenser.
  • Light passing through the specimen from oblique angles enters the objective lens.
  • The field of view is dark because the direct light path to the objective lens is blocked.
  • Only light deflected by structures within the specimen enters the objective lens and these structures appear light against a dark background.
  • Contrast arises from light scattering; the specimen seems to light up over a dark background.

Polarized Light

  • When light travels through a polarizing material, a selected vibration plane is passed, while other orientations are blocked, resulting in linearly polarized light.
  • This linearly polarized light can be passed or absorbed by a second polarizer, depending on its orientation.
  • When linearly polarized light passes through certain specimens, the plane of the waves is rotated, exhibiting birefringence, where the extent of rotation varies with wavelength or color.
  • To use this, a second filter (analyzer) is inserted before viewing.
  • With a blank slide, rotating the analyzer until its "pickets" are at right angles to the polarizer prevents light from reaching the eye (crossed polars).
  • When a birefringent specimen is viewed under crossed polars, the rotated light passes through the analyzer, resulting in a black background with a colored or silvery subject.
  • The microscope needs a polarizer (before the specimen) and an analyzer (between the objectives and observation tubes or camera port).

Phase Contrast

  • Phase contrast microscopy enhances contrast to produce high-contrast images of transparent specimens like living cells, microorganisms, thin tissue slices, lithographic patterns, fibers, latex dispersions, glass fragments, and subcellular particles.
  • The wavelength of a light wave determines its color.
  • The height or amplitude of the light wave determines its brightness.
  • Phase contrast manipulates phase relationships between light rays from the specimen and translates them into brightness changes.
  • Light passing through a transparent part of the specimen travels slower and is shifted in phase compared to light that doesn't pass through the specimen.
  • Interference of Light Waves:
    • Waves in phase interfere constructively, producing a wave with greater amplitude and increased brightness: 2 waves add together = wave > amplitude\text{2 waves add together = wave > amplitude}
    • Waves out of phase interfere destructively, producing a wave with smaller amplitude and reduced brightness: 2 waves subtract = wave < amplitude\text{2 waves subtract = wave < amplitude}
  • Phase Contrast Microscope Configuration
    • Condenser Annulus
    • Specimen-Phase Plate
    • Objective
    • Digital Camera System

Hoffman Modulation Contrast

  • The Hoffman Modulation Contrast system increases visibility and contrast in unstained, living material by detecting optical gradients (slopes) and converting them into variations of light intensity.
  • Invented by Dr. Robert Hoffman in 1975, it uses accessories adapted to commercial microscopes.
  • Similar to DIC, but uses plates with small slits in both the axis and off-axis of the light path to produce two sets of light waves passing through the specimen, forming a 3-D-like image.
  • A modulator is inserted on the back focal plane of an achromat or planachromat objective.
  • Light intensity varies above and below an average value (modulated).
  • Modulators have three zones:
    • A small, dark zone transmits 1% of light (D).
    • A narrow gray zone transmits 15% (G).
    • A clear zone transmits 100% of the light (B).
  • A condenser with a rotating turret holding the components of the system is used below the stage.
  • There is an off-center slit partially covered with a small rectangular polarizer.
  • Opposite gradients deflect the slit image to either the dark or bright part of the modulator.
  • A specimen with positive and negative phase gradients and a flat area is imaged using modulation contrast optical components.
  • The Hoffman image appears brighter on one side, gray in the central portion, and darker on the other side.
  • Rotation of the polarizer alters the contrast and orientation of the specimen to improve or degrade contrast.
  • Rotation may bring other gradients into proper orientation, revealing new details about the specimen.
  • Advantages over other techniques:
    • Creates images without halo effects (unlike phase-contrast).
    • Can be performed on specimens in plastic vessels (unlike DIC), making it preferred in tissue culture.
    • Provides well-defined edges, preferred where reliable measurements are necessary.