With a conventional biological microscope, it is difficult to observe colorless, transparent cells while they are alive. A phase contrast microscope makes it possible by utilizing two characteristics of light, diffraction and interference, to visualize specimens based on brightness differences (contrast).

Principle

With regard to periodic movements, such as sinusoidal waves, the phase represents the portion of the wave that has elapsed relative to the origin. Light is also an oscillation and the phase changes, when passing through an object, between the light that has passed through (diffracted light) and the remaining light (direct light). Even if the object is colorless and transparent, there is still a change in phase when light pass through it. This phase contrast is converted into brightness differences to observe specimens.

Features

• Transparent cells can be observed without staining them because the phase contrast can be converted into brightness differences.
• Because it is not necessary to stain cells, cell division and other processes can be observed in a living state.

Structure

Because diffracted light is too weak to be normally observed by the eye, a phase plate is located at the focal point of light between the objective lens and the image surface so that only the phase of the direct light changes. This generates contrast on the image surface.
Structural features include a ring aperture, instead of a pinhole, on the focal plane of the converging lens and a phase plate on the rear focal plane of the objective lens.

A fluorescence microscope enables cells and proteins to be observed by using a fluorescent protein or antibody as a label. This type of microscope is indispensable for modern cell biology.

Principle

Fluorescent materials absorb a specific wavelength of light (excitation light) and emit light of a longer wavelength (fluorescence), which is based on Stokes’ law. For example, the fluorescence emitted by a target molecule can be observed by adding a specific fluorescent reagent to cells and then applying excitation light. A fluorescent microscope has all of the components to induce this fluorescence and capture the resulting image.

Features

• Allows observation of fluorescence images, in addition to observation with transmitted light.
• It is possible to only observe specific areas by using different fluorescent labels.
• Fluorescent dyes enable users to view localization of particular proteins in cells.
• The use of fluorescent proteins such as GFP allows for observation of living cells.

Structure

Generally, a fluorescence microscope is a combination of a biological microscope and fluorescent incident illumination equipment. The structure includes a focusing knob, an XY stage handle for positioning specimens, and a revolver for switching objective lenses. For lighting, it is also equipped with a cube turret that adjusts the wavelength of the excitation light, a shutter that prevents photobleaching of samples, and an ND (neutral density) filter that adjusts the strength of the excitation light.

This type of microscope is characterized by using laser beams as the light source. Laser scanning allows high-resolution observation as well as accurate 3D measurement.

Principle

These systems scan the surface of an object with a laser(s), record the spatial distribution of fluorescence and reflected light from the focal plane, and then visualize the resulting data with a computer to allow observation of high-resolution images.
As the name implies, this microscope uses a confocal optical system.

Features

• By scanning the laser across the surface of an object in the X, Y, and Z planes, a high-resolution image with corresponding height data can be captured. With a biological sample, for example, this allows users to understand its 3D structure as well as to obtain clear fluorescence images.
• While general optical microscopes use an image-forming optical system, laser microscopes use the confocal optical system. The former illuminates a specific area entirely while the latter focuses light on a single point with a point source light. Furthermore, a pinhole is provided at the image position to receive only the focused light. This results in better contrast with no unnecessary scattered light entering from the surrounding areas.

Structure

Laser beams emitted from the laser light source go through the objective lens to scan a sample. The fluorescence of the sample excited by the laser beams is returned to the objective lens, processed as an image, filtered by the pinhole, and displayed on the monitor.

A type of high-resolution microscope based on technology that has overcome the limited resolution of optical microscopes caused by the diffraction limit of light.

Principle

Conventionally, the resolution of optical microscopes was limited to 200 nm or larger due to the diffraction limit of light. This limit has been overcome by a high-resolution microscope developed in the United States that is based on structured illumination. Structured illumination microscopy enables high-resolution images to be obtained by using the moire effect of a grid or other patterned illumination (structured illumination) to capture diffracted light, which is impossible with conventional optical microscopes.

Features

• Provides much higher resolution than conventional optical microscopes, approximately twofold, both in the horizontal and vertical directions.
• Ability to process multiple captured images at high speed makes live imaging of cells possible.

Structure

Structured illumination microscopes do not have a new structure but use a new way to capture light. More specifically, this type of microscope is based on moire fringes, which are caused by interference of light, and is designed to emit a specific pattern of light (structured illumination) to generate moire effects. Because images captured through this technology contain detailed information about the object, high-resolution images can be composed through computerized analysis of multiple images.