The general biological microscope is mainly composed of the objective lens, eyepiece, lens tube, object platform, and mirror. Objects placed on the stage are magnified through an objective lens. When the target is in focus, the magnified image can be viewed through the eyepiece. Digital microscopes, on the other hand, use cameras and magnifying optics to output real-time images to monitors.
Telescopes have a similar structure. However, they are used to observe distant objects. A telescope receives light from a star or other distant object through an objective lens and adjusts the refraction to focus through an eyepiece. Microscopes, on the other hand, are designed to shine a light on or through objects and to amplify transmitted or reflected light through objective lenses and eyepieces.
1. Microscopic ability
The biological microscope has multiple objectives with different magnifications that can accurately image a sample. The magnification of a microscope is the product of the magnification of the objective lens and the eyepiece.
However, the performance of a microscope does not depend solely on magnification. Resolution is another important factor. Resolution is the ability to identify two points of light separately, expressed as the shortest distance between two points that can still be distinguished as distinct entities. While high magnification is required to visualize small objects, the resolution will determine how sharp they are. For optical microscopes, the maximum resolution is theoretically limited to about 100 to 200 nm due to the visible wavelength (400 to 800 nm). If you need higher resolution, consider using an electron microscope.
The numerical aperture (NA) is an essential factor when considering objective performance. As NA increases, so does the resolution and brightness of the lens. When selecting a microscope, the user must check the NA, magnification, and resolution of the lens against the sample used for imaging.
Aberration, which is the distortion or blurring of the image caused by a lens shape defect, is another factor that affects the performance of a lens. The more thoroughly the aberration correction, the better the performance of the lens.
2. The main types of microscopes
The following table describes the major microscope types in the optical, electron, and scanning probe categories.
(1) Optical microscope
|Digital microscope||Microscopes that use a camera and magnified optics. It enables the output of a live image to a monitor.|
|Binocular stereoscopic microscope||A microscope that allows easy observation of 3D objects at low magnification.|
|Brightfield microscope||A typical microscope uses transmitted light to observe targets at high magnification.|
|Polarizing microscope||A microscope uses different light transmission characteristics of materials, such as crystalline structures, to produce an image.|
|Phase-contrast microscope||A microscope that visualizes minute surface irregularities by using light interference. It is commonly used to observe living cells without staining them.|
What is a phase-contrast microscope?
|Differential interference contrast microscope||This microscope, similar to the phase contrast, is used to observe minute surface irregularities but at a higher resolution. However, the use of polarized light limits the variety of observable specimen containers.|
|Fluorescence microscope||A biological microscope that observes fluorescence emitted by samples by using special light sources such as mercury lamps. When combined with additional equipment, brightfield microscopes can also perform fluorescence imaging.|
|Total internal reflection fluorescence microscope||A fluorescence microscope that uses an evanescent wave to only illuminate near the surface of a specimen. The region that is viewed is generally very thin compared to conventional microscopes. Observation is possible in molecular units due to reduced background light.|
(Laser scanning confocal microscope)
|This microscope uses laser beams to observe thick samples with different focal distances.|
|Multiphoton excitation microscope||The use of multiple excitation lasers reduces damage to cells and allows high-resolution observation of deep areas. This type of microscope is used to observe nerve cells and blood flow in the brain.|
|Structured illumination microscope||A high-resolution microscope with advanced technology to overcome limited resolution found in optical microscopes that are caused by the diffraction of light.|
What is a structured illumination microscope? 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.PrincipleConventionally, 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.FeaturesProvides much higher resolution than conventional optical microscopes, approximately twofold, both in the horizontal and vertical directions. The ability to process multiple captured images at high speed makes live imaging of cells possible.StructureStructured 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.
(2) Electron microscope
|Transmission electron microscope (TEM), scanning electron microscope (SEM), etc.||These microscopes emit electron beams, not light beams, toward targets to magnify them.|
(3) Scanning probe microscope (SPM)
|Atomic force microscope (AFM), scanning near-field optical microscope (SNOM), etc.||This microscope scans the surface of samples with a probe and this interaction is used to measure fine surface shapes or properties.|
|X-ray microscope, ultrasonic microscope, etc.||–|