Phaos Optic Science Educational Series
March 29, 2021
12:00 PM (GMT)
20 Minutes
Reflected Light Microscopy
Reflected light microscopy is often referred to as incident light, epi-illumination, or metallurgical microscopy, and is the method of choice for fluorescence and imaging specimens that remain opaque even when ground to a thickness of 30 microns such as metals, ores, ceramics, polymers, semiconductors and many more!
Because light is unable to pass through these specimens, it must be directed onto the surface and eventually returned to the microscope objective by either specular or diffused reflection.
Objectives
The resolving power in reflected light is based on the same relationship between the wavelength of light and numerical aperture (the Abbe equation) as in transmitted light. Optical performance is achieved in reflected light illumination when the instrument is adjusted to operate under Köhler illumination. A function of Köhler illumination (aside from providing evenly dispersed illumination) is to ensure that the objective will be able to deliver excellent resolution and good contrast even if the source of light is a coiled filament lamp.
In many cases, modern reflected light microscopes may also be operated using transmitted light because the parfocal length is maintained in all objectives.
Transmitted Light Microscopy
Transmitted light microscopy is the general term used for any type of microscopy where the light is transmitted from a source on the opposite side of the specimen to the objective lens. Usually, the light is passed through a condenser to focus it on the specimen to get maximum illumination. After the light passes through the specimen it goes through the objective lens to magnify the image of the sample and then to the oculars, where the enlarged image is viewed.
In order to get a usable image in the microscope, the specimen must be properly illuminated. The light path of the microscope must be correctly set up for each optical method and the components used for image generation. The condenser was invented to concentrate the light on the specimen in order to obtain a bright enough image to be useful.
The microscope techniques requiring a transmitted light path includes;
1. Bright-field
Bright Field is the most common technique for illuminating diffuse, non-reflective objects. The term bright field refers to the mounting position of the illuminator.
It helps to observe tissues because it makes the object appear against a bright background. This is caused by the absorption of part of the transmitted light in dense areas.
2. Dark-field
Dark field illumination are normally flat ring lights that must be mounted very close to the test object. Unlike bright field lights, most of the light is reflected away from the camera.
It is mostly used for biological samples such as bacteria and micro-organisms. As the entrance of the light is bigger, it permits the diffraction of the lights rays and will illuminate obliquely. As a result, the field around the specimen is generally dark to allow clear observation of the bright parts.
Visual Difference between Bright and Dark Field Techniques.
3. Phase contrast
Phase contrast is used to enhance the contrast of light microscopy images of transparent and colourless specimens. It enables visualisation of cells and cell components that would be difficult to see using an ordinary light microscope.
Phase contrast microscopy translates small changes in the phase into changes in amplitude (brightness), which are then seen as differences in image contrast. This characteristic enables background light to be separated from specimen diffracted light. The difference of the light phase is increased by slowing down (or advancing) the background light by a ¼ wavelength, with a phase plate just before the image plane. When the light is focused on the image plane, the diffracted and background light cause destructive (or constructive) interference which decreases (or increases) the brightness of the areas that contain the sample, in comparison to the background light.
Some of the light that passes through the specimen will not be diffracted (Illustrated as bright yellow in the figure below). These light waves form a bright image on the rear aperture of the objective. The light waves that are diffracted by the specimen pass the diffracted plane and focus on the image plane only. This allows the background light and the diffracted light to be separated.
4. Polarisation
Polarising microscopy involves the use of polarised light to investigate the optical properties of various specimens.
It is a contrast-enhancing technique that allows you to evaluate the composition and three-dimensional structure of anisotropic specimens. It uses polarising filters to make use of polarised light, configuring the movement of light waves and forcing their vibration in a single direction.
5. Differential Interference Contrast (DIC) optics
Differential Interference Contrast (DIC) is a microscopy technique that introduces contrast to images of specimens which have little or no contrast when viewed using bright field microscopy. The images produced using DIC have a pseudo 3D-effect, making the technique ideal for electrophysiology experiments.
In DIC, light emitted from the source is linearly polarised by passing through a polariser. The linearly polarised beam of light enters an objective-specific prism, which splits it into two rays that vibrate perpendicular to each other. The rays are parallel as they pass through a condenser, but as they are vibrating perpendicular to each other, they are unable to cause interference.
The split beams pass through the specimen. The specimen’s varying thickness and refractive indices alter the wave paths of the beams. They then enter the objective, where they are focussed above the rear focal plane. The two beams enter a second prism, in the nosepiece, which combines them. Because the beams passed through different parts of the specimen, they have different lengths.
The analyser, which is a second polarizer, brings the vibrations of the beams into the same plane and axis, causing destructive and constructive interference to occur between the two wavefronts. The light then travels to the eyepiece or camera, where a DIC image with differences in intensity and colour, can be seen.
Overall, What’s The Difference?
Reflection is the process by which electromagnetic radiation is returned either at the boundary between two media (surface reflection) or at the interior of a medium (volume reflection), whereas transmission is the passage of electromagnetic radiation through a medium.
Both processes can be accompanied by diffusion (also called scattering), which is the process of deflecting a unidirectional beam into many directions.
