What is Microscopy?
Microscopy is a field of science involving microscopes to look at things and parts of things that the human eye cannot see. It includes looking at things and parts of things that cannot be seen with the naked eye. There are three well-known types of microscopy: optical, electron, and scanning probe. X-ray microscopy is a new type.
Electromagnetic radiation or electron beams that have been diffracted, refracted, or reflected by the specimen are used in optical and electron microscopy. The scattered radiation or another signal is then used to make an image. In transmission electron microscopy and conventional light microscopy, this can be done by either moving a narrow beam across the sample or shining a wide field of light on the sample. For example, confocal laser scanning microscopy and scanning electron microscopy).
What are the types of Microscopes?
Optical microscope
In optical or light microscopy, the sample is viewed more clearly by passing visible light that has passed through it or been reflected off it through one or more lenses. The image obtained can be seen with the naked eye or captured on a photographic plate. The virtual light microscope consists of a single lens and its attachments or a system of imaging and lens apparatus coupled with the proper lighting apparatus, sample stage, and support. The most recent innovation is the digital microscope, which focuses on the target object using a CCD camera. There is no need for eyepieces because the image is displayed on a computer screen.
Disadvantages
Bright field microscopy, an ordinary optical microscope, has three Disadvantages.
- Only dark or refracting objects can be is simple imaged by using this method.
- There is a limited diffraction resolution that depends on the wavelength of the incident light. In the visible range, optical microscopy has a practical limit of about 1500x magnification and a resolution of about 0.2 micrometers.
- Image clarity is compromised by unfocused light from areas outside the focal plane.
Live cells, in particular, do not usually have enough contrast to be studied well because a cell inside is colorless and see-through. Staying structures with certain dyes is the most common way to make structures stand out. However, this usually means fixing and killing the sample. Also, staining can cause artifacts, which are visible structural features that are not part of the material but are caused by how the sample is handled. These methods often rely on variations in the refractive indices of cell structures. Bright-field microscopy is like seeing through a glass window; all that is seen is the dirt on the glass, not the glass itself.
Bright field Microscope
The simplest method for light microscopy is known as bright field microscopy. Transmitted white light is used to illuminate the sample, which is then lighted from below and seen from above. Most biological samples have poor contrast, and the blur and unfocused images result in low apparent resolution. Significant benefits include how simple the process is and how little sample preparation is needed.
Dark field Microscope
A method for enhancing the contrast of translucent, unstained specimens is known as dark field microscopy. Darkfield illumination uses a positioned light source to reduce the amount of direct transmitted light that gets into the image plane. The light that an object scatters is captured. Dark fields can make a significant difference while requiring the least amount of setup or sample preparation, especially for transparent objects. However, many biological samples’ final images have low light intensity, and the method is still limited by low apparent resolution.
Phase contrast Microscope
Proportional variations in optical density will be visible with more advanced techniques. A popular technique for displaying differences in refractive indices as contrast in phase contrast microscope. For example, the nucleus of a cell will stand out darkly against the cytoplasm surrounding it. Excellent contrast should not be used with thick items, however. Even around little objects, a halo frequently forms, obscuring detail. The system consists of a condenser with a circular annulus emitting a light cone. Within the phase objective, this cone is overlaid on a ring of comparable size. Since each objective’s ring varies in size, a distinct condenser setting must be selected. Phase contrast imaging is produced due to interference with the diffracted light caused by the direct light’s altered physical properties.
Fluorescence microscope
When certain substances are exposed to intense light, they release light with a lower frequency. This effect is called fluorescence. Depending on their chemical composition, specimens frequently display their distinctive autofluorescence appearance.
Due to its potential for being exceedingly sensitive and enabling single-molecule detection, this technique is crucial in modern life sciences. Structures or chemical compounds can be stained using a variety of fluorescent dyes. Immunostaining, a strong technique, involves combining antibodies with a fluorophore. Fluorescein and rhodamine are two examples of frequently used fluorophores.
Confocal Microscope
Confocal laser scanning microscopy scans a focused laser beam (for example, one with a wavelength of 488 nm) over a sample to activate fluorescence point by point. Light is sent through a pinhole so that light that is not focused does not reach the detector. The image is made by a computer that maps the measured fluorescence intensities to the laser’s position. Confocal microscopy has a higher lateral resolution than complete sample illumination, making optical sectioning a lot better (axial resolution). Therefore, confocal microscopy is usually applied when the 3D structure is crucial.
Electron microscopy
Before the invention of sub-diffraction microscopy, The wavelength of the light restricted conventional microscopy’s resolution to about 0.2 micrometers. An electron beam with a much lower wavelength is used in electron microscopes to achieve better resolution.
Transmission electron microscopy (TEM)
Similar to the compound light microscope, transmission electron microscopy (TEM) works by passing an electron beam through a very thin slice of the material.
Limitation
The transmission electron microscope’s requirement for very thin specimen sections is the most significant drawback—typically around 100 nanometers. It is not easy to produce these thin sections for objects. A highly focused ion beam can be used to create thin slices of semiconductors. To make biological tissue specimens sufficiently stable for ultrathin sectioning, they are chemically fixed, dried, and embedded in a polymer resin. To acquire the necessary picture contrast, sections of biological specimens, organic polymers, and related materials might need to be stained with strong atom labels.
Scanning electron microscopy (SEM)
The 3D image provided by scanning electron microscopy (SEM) allows for visualizing surface details on specimens. It produces outcomes that closely resemble those of a stereo light microscope. In 2011, SEM’s top resolution was 0.4 nanometers.
Generally, an SEM’s image resolution is lower than a TEM’s. The electrons do not need to pass through the sample because the SEM only captures photos of a sample’s surface rather than its interior. As a result, there is less need for labor-intensive sample preparation to make the specimen thin enough to see through with an electron microscope. The SEM can take pictures of large objects that can move around on its stage and fit within the working distance, which is usually 4 millimeters for high-resolution images. It can make images that show the sample’s three-dimensional surface structure. Because the SEM has a good depth of field,
