Transmitted Light Microscopy – Bright field, Phase contrast and DIC imaging

Transmitted light microscopy is the general term used for any type of microscopy where light is transmitted, or passed through a sample. A transmitted light microscope use light that passes through a condenser to focus it on the specimen to get very high illumination. After the light passes through the specimen, the image of the specimen goes through the objective lens and to the eyepieces where the enlarged image is viewed. The light path of the microscope must be properly aligned allowing the specimen to be evenly illuminated. The condenser can concentrate the light on the specimen to obtain a bright enough image. The best setup for proper specimen illumination and image acquisition is known as Köhler illumination after the man who invented it.

There are several different techniques for transmitted light; bright-field, dark-field, phase contrast and differential interference contrast (or Nomarski) optics microscopy.

Bright field Microscopy

This is the simplest of a range of techniques used for illumination of samples in light microscopy. The typical appearance of Bright-Field (BF) microscopy image is a darker sample on a bright field or background, thus the name. In BF microscopy, illumination light is transmitted through the sample and the contrast is generated by the absorption of light in dense areas of the specimen. The light path of BF microscopy is very simple, no additional components are required beyond the normal light microscopy setup, which consist of a transmitted light source, a condenser lens, and objective lens and eyepiece or camera. The BF microscopy has low contrast for weakly light absorbing samples. Staining is often required to increase contrast for colorless and transparent samples, e.g. bacterial cells.

 

Dark field Microscopy

Dark-Field (DF) illumination technique can enhance the contrast, thus being useful application in the study of materials of low contrast and effective light scattering, such as unstained biological samples, small particles or internal inclusions and pores in thin sections. In DF illumination, a central circular disc blocks some light from light source direct condenser rays from entering the objective lens. While the directly transmitted light is blocked, only the scattered light by the sample (by reflection, refraction or diffraction) enters the objective lens to generate the image. The object details appear bright on a dark background of field. The limitation of DF microscopy technique is the low light levels.

 

Phase contrast Microscopy

Phase contrast microscopy is a widely used technique capable of high visibility and contrast, in especially living cells. In phase contrast microscopy, the basic principle to making phase changes is to separate the background light from the specimen-diffracted light and to manipulate these differently. When the light is focused on the image plane, the background and diffracted light will cause destructive or constructive interference, which changes the brightness of the area including the sample in comparison to the background light. This optical technique can be carried out on a traditional bright-field microscope with the addition of two components; a phase contrast condenser with a condenser annulus (also known as a phase ring) and a set of phase contrast objectives, each of which contains a phase plate. Annulus or a ring also limit the aperture to some extent, which decreases resolution. The limitation of phase contrast microscopy technique is the low light levels since this technique is based on a diminishment of brightness of most object.

 

Differential Interference Contrast (DIC) Microscopy

Differential interference contrast microscopy is also known as Nomalski microscopy. The DIC microscopy technique produces high contrast images with a 3D shadowed effect. This optical technique is based on an interference principle involving two coherent beams of light and image contrast achieved with gradients in optical path. DIC has two Nomarski (or modified Wollaston) prisms in its optical configuration, one of which has an adjustable position. Additionally, the main components of a DIC microscope include a polarizer and an analyzer. In a DIC microscope, light from the source first passes through a polarizer. The linearly polarized beam of light is split into two rays by a Nomarski prism located near the condenser diaphragm plane. The two rays are focused by passing through the condenser and travel to the specimen, in which the wave path will be changed base on its thickness and refractive index. The two rays pass through the objective lens and are recombined by the second Nomarski prism. Because the beams passed through different parts of the specimen, they have different optical lengths. The analyzer brings the rays into the same plane and axis, allowing wave interference to increase or decrease intensity, thus generating contrast. An advantage of DIC over phase contrast is the ability to utilize the device at full numerical aperture without the masking effects of phase plates or condenser annulus.

  • Köhler illumination is critical in order to get the best image possible from bight-field, phase contrast, or DIC microscopy technique.

Equipment and Software:

Olympus BX53

CellSens dimension

 

<Bright Field Image with x60w objectives>

Images was acquired with x60w objectives of Olympus BX53 using a Multiple Image Alignment (MIA-tiling) method of Cellsens software.

Transverse section of the lily of valley rhizome. Imaged by UBC BIF-EunKyoung Lee