Differential Interference Contrast (DIC) Microscope microbiologystudy

Differential Interference Contrast (DIC) Microscope is widely used to image unstained and transparent living specimens and observe the structure and motion of isolated organelles, making it an alternative to conventional brightfield illumination requiring specimens’ staining.

The invention of the Differential Interference Contrast (DIC) microscope dates back to 1947 when Francis Smith first discovered the DIC microscopy technique. Later, in the mid-1950s, it was further developed as a practical microscopy method by a French optic theoretician Georges Nomarski. The modification of the Wollaston prism by Normarski, previously used to detect optical gradients and convert them into intercity variances led to its several implementations, collectively called DIC.

To make unstained cells visible, this technique utilizes variations in the optical path length changing the phase of light and rendering necessary resolution and contrast. In turn, it produces monochromatic shadow-cast images of the structures in the specimen. As the optical paths increase along the direction of reference, the part of the specimen becomes visibly brighter, and when the optical path decreases, the regions give the opposite contrast. The contrast of the image increases remarkably as the gradients in the optical path become steeper.  One of the important aspects of DIC microscopy is its ability to achieve highly efficient optical sectioning as it employs high numerical aperture (NA) objectives in combination with NA condenser illumination. 

Principle of Differential Interference Contrast (DIC) Microscope

Polarized light is the sole illumination used for DIC microscopy to observe the specimen. Two distinct light rays, polarized in orthogonal planes, are formed when polarized light is dispersed. These closely aligned light rays undergo refraction or scattering in different ways within the specimen, leading to different phase shifts. When these light rays come back together, they interfere with each other, resulting in elliptical polarization. An analyzer can then convert this polarization into an amplitude shift. This process allows phase shifts with wavelength variances ranging from 1/200 of a wavelength to a whole wavelength (or even 1/1000 of a wavelength when using a camera) to be visualized.

The working principle of the DIC microscope is based on the splitting and interference of light waves on the specimen. When the polarized illumination light wave enters an objective specific prism, it splits into two component waves perpendicular to each other which are spatially shifted along a particular direction. The rays pass parallel through the condenser. Further, the light waves recombine after passing through adjacent points on the specimen plate. The interference of the light wave depends on the phase shifts of the two component waves. The difference in the optical path length of the two adjoining points causes the phase variation, therefore, an adjustable bias retardation is attached to it. This technique of microscopy is termed as differential interference which operates on the conversion of the optical path length gradient of two points along the shortened direction to create visible intensity variations. 

Parts of Differential Interference Contrast (DIC) Microscope

The DIC microscopy technique requires four major optical components: a Polarizer, a Condenser prism (beam-splitting modified Wollaston prism below the condenser), an Objective prism (beam-recombining modified Wollaston prism above the objective), and an analyzer installed in an inverted or upright microscope, more precisely in the light path.

Polarizer: The polarizer above the condenser changes the illuminated light from the lamp source into plane-polarized light. It is conventionally set with its light transmission axis in an east-west direction when facing the microscope.

Condenser prism: The condenser prism located below the condenser splits the single beam of polarized light into two component waves of light. Through the condenser lens, the light waves are focused onto the specimen. This optic system utilizing a dual beam is the foundation of DIC microscopy for the identification of the gradients in the optical path length within the specimen. Two different DIC prisms called DIC II and DIC III are typically employed based on the NA objectives. 

Objective prism: The beam-recombining objective prism is placed above the objective. The objective prism must be matched with the objective and condenser prism’s magnification. After the light waves pass through the specimen and the objective, the prism recombines the two components of waves produced. The adjustment of the objective prism in a linear direction allows adjustment in the production of shadow-cast images. 

Analyzer: In the microscope, the analyzer is placed below the objective nose-piece that transforms the elliptically polarized light emerging from the objective prism into plane-polarized light. This transformation forms an image at the image plane. The analyzer’s light transmission axis is aligned in a north-south direction when viewing through the microscope, as per convention.

Sample Preparation for Differential Interference Contrast (DIC) Microscope

Usually, smears of fluid samples, living cell cultures, unstained tissues, isolated subcellular organelles, blood cells, embryos, chromosomes, protozoan cells, and ultra-thin sections of tissues are suitable specimens for observation under DIC microscopy. Similarly, amplitude specimens like naturally pigmented protists, algae, and histological specimens are also examined to obtain additional information.

Operating Procedure of Differential Interference Contrast (DIC) Microscope

The DIC technique is used on any inverted or upright microscope that can install polarizers and prisms. Before proceeding with the DIC setup it is important to ensure that all the components are clean and dust-free for an excellent-quality image. 

• Initially, the Koehler illumination must be set to allow enough light to pass from the condenser to the objective ensuring no DIC optics are used. 
• Adjust the polarizer and the analyzer, the two polarizing filters perpendicular to each other. To obtain this orientation of the polarizing filters: 
  • First, the analyzer is moved into the light path through the motorized internal filter turret position so that the analyzer’s transmission axis in the filter cube turret is permanently oriented north-south. 
  • Then, the plastic filter holder above the condenser is swung into the plate to place the polarizer in the light path. The rotation of the polarizer is carried out until it is perpendicular to the analyzer. 
• The two DIC prisms, the condenser and objective prism are placed in the light path. 
  • To anchor the condenser prism, press one of the advance buttons beside the condenser turret until the display reads DIC II or DIC III. DIC II prism is used for low numerical aperture air objectives while the DIC III prism is used for high numerical aperture oil immersion objectives. 
  • The placement of the objective prism is accompanied by rotating to a position that is accessible with the objective nosepiece.
• The mounted specimen is focused on the stage with the proper adjustment of Koehler illumination. 
• The lateral position of the objective prism is adjusted until the shadow-cast relief image with a 3-D appearance is obtained by moving the screw desirably positioned on the objective prism holder.
• At last, the image contrast is improved by adjusting the numerical aperture of the condenser.

Applications of Differential Interference Contrast (DIC) Microscope

• DIC microscopy is commonly used to observe and study biomolecules and exogenous particles diffusing on synthetic and live-cell membranes.
• In combination with fluorescence microscopy, this technique is used to observe the morphological structures of cells. Similarly, when combined with enhanced video techniques, termed VEC-DIC is used to image structures smaller than the optical resolution of a microscope. 
• It is also used in electrophysiology to observe and study the cells in tissues with the use of infrared light as it can penetrate deeper in the tissue. 
• DIC can also be used in imaging the surface features in semiconductors and metallurgy.
• It is an important microscopic tool used in microbiology, cell biology, and neuroscience, providing detailed, three-dimensional images for the study of biological processes. 

Advantages of Differential Interference Contrast (DIC) Microscope

• DIC provides optical staining with striking colors and details in 3-D appearance to the image with improved visibility. 
• Quality images with high resolution, both lateral and axial, and contrast are obtained from the DIC technique as it utilizes full objective and condenser apertures. 
• They do not exhibit halo artifacts in the image. Relatively thick samples can also be observed through optical sectioning. 
• The numerical aperture of the microscopic system can be fully utilized. 
• It is used to observe living unstained cells and nanoprobes at the same time for a longer period. Moreover, the dynamic changes in the cell morphology can be observed and recorded without staining.  

Limitations of Differential Interference Contrast (DIC) Microscope

• The DIC technique requires expensive equipment including the prisms.  
• DIC microscopy can be used to observe isolated organelles, however, it is comparatively difficult to image cytoplasm and cell membrane. 
• Apochromatic lenses might not be appropriate as they can have a considerable impact on polarized light.
• Similarly, specimens with birefringence, commonly present in various types of crystals, may not be appropriate due to their impact on polarized light.

Precautions and Safety Considerations

• The optical instruments should be handled with care to avoid physical shocks and any kind of deformities in the objective and condenser. 
• Clean the lenses and mirrors and ensure that the lenses are free from dust, dirt, and fingerprints. 
• Avoid using plastic vessels, plastic coverslips and slides, and birefringent specimens as they produce low-quality images in DIC. 
• The system should be installed in a location free from dust, with no direct exposure to sunlight or high temperature, and with little vibration. 
• The prism should be adjusted in an accurate combination with the objective and condenser lenses. 
• Avoid creating air bubbles in the oil immersion. 
• Make sure to remove the objective prism, polarizer, and analyzer before starting the microscopy from the optical path. 

Conclusion

As an alternative to a conventional brightfield microscope that is unable to view living cells, the technique of DIC microscopy has been widely used since the 1950s to observe unstained living cells. This is considered the most advantageous factor of using DIC microscopy as it is non-invasive to the cells and does not require the use of toxic chemicals. The fundamental feature of this technique is the use of combination prisms mirrored to each other in the optical system. Based on the principle of differential interference, it produces relief-like images with high resolution and contrast possessing a 3-D appearance. Its sensitivity to viewing minute details and features as well as the dynamics of the cells is considered a remarkable feature compared to the traditional phase contrast microscopes. 

References