Microscopes
The microscope is a laboratory instrument used to observe small objects that are invisible to the naked eye. Microscopy is the science of investigating small objects and structures using a microscope. Microscopic means being invisible to the eye unless aided by a microscope.
There are different types of microscopes.
A simple microscope uses one lens and magnifies the sample from 70 to 250 times. A compound microscope has two lenses and magnifies the sample 2000 times. In the electron microscope, electron beams produce images hundreds of times.
History:
Four thousand years ago, there were objects resembling lenses. Even though Greek descriptions and a lot of writing are there for the optics and optical properties of water-filled spheres, the widespread use of lenses in eyeglasses came in the 13th century. The earliest microscopes were single-lens magnifying glasses with limited magnification.
In the 14th century, Italians made the first eyeglass lens.
In 1590, Zacharias Janssen and his father, Hans Martens, claimed that the microscope was their contribution.
In 1620, a compound microscope appeared in Europe
with an objective lens and an eye-piece. It was possible to view the image of an object using this microscope.
Robert Hooke published Micrographia in 1667. Micrographia outlines Hooke's various studies using the microscope.
Anton van Leeuwenhoek used a microscope with one lens to observe insects and other specimens in 1675. Leeuwenhoek was the first to view bacteria using a microscope.
18th century, as technology improved, microscopy became more popular among scientists. Part of this was due to the discovery that combining two types of glass reduced the chromatic effect.
1830: Joseph Jackson Lister discovered that using weak lenses together at varying distances provided good magnification.
1878: Ernst Abbe invented a mathematical theory that linked resolution to light wavelength.
1903: Richard Zsigmondy invented the ultramicroscope, which allowed the observation of specimens below the wavelength of light.
Transparent biological materials were studied for the first time using Frits Zernike's invention of the phase-contrast microscope in 1932.
1938: Six years after the invention of the phase contrast microscope came the electron microscope, developed by Ernst Ruska, who realized that using electrons in microscopy enhanced resolution.
1981: 3-D specimen images were possible with the invention of the scanning tunneling microscope by Gerd Binnig and Heinrich Rohrer.
Gerd Binnig and Heinrich Rohrer worked for the period 1981-1983 at IBM Zurich to study the quantum tunneling technique. They developed an instrument called a scanning probe microscope. It is capable of detecting even a minute force between the probe and the sample.
In 1985, scanning probe microscopes became popular commercially. In 1986, Gerd Binnig and Quate Gerber were awarded the Nobel Prize for this invention.
In 2013, Australian engineers developed a precision-measuring quantum microscope.
In 2014, for developing stimulated emission depletion (STED), Stefan Hel shared the Nobel Prize with Eric Betsingh and William Morner in Chemistry for imaging single molecules in fluorescence microscopy.
Types:
There are different classes of microscopes. They interact with the sample to generate the image, i.e. Light or photons (optical microscopes), electrons (electron microscopes), and a probe (scanning probe microscopes).
General features:
Optical microscope:
- Generate images using light or photons.
- Uses optical lenses.
- The magnification of the image depends on the wavelength of the light received from the sample.
- Resolution is high for small wavelengths.
Electron microscope:
- Generate images using electrons.
- Uses electromagnetic lenses.
Alternatively, microscopes can be classified based on whether they analyze the sample via a scanning point (confocal optical microscopes, scanning electron microscopes, and scanning probe microscopes) or analyze all the samples together (wide-field optical microscopes and transmission electron microscopes).
Let us discuss one by one.
The most common type of microscope (and the first invented) is the optical microscope. Microscopes have a refractive glass (occasionally plastic or quartz) to focus light on the eye or onto another light detector. Mirror-based optical microscopes operate in the same manner. The typical magnification of a light microscope, assuming visible range light, is up to 1,250× with a theoretical resolution limit of around 0.250 micrometers or 250 nanometres. Shorter wavelengths of light, such as ultraviolet, are one way to improve the spatial resolution of the optical microscope, as are devices such as the near-field scanning optical microscope [1].
Features:
- Instrument containing one or more lenses
- Produces an enlarged image of a sample
- Microscopes have refractive glass.
- Using ultraviolet light is one way to improve the spatial resolution of the optical microscope.
Sarfus is a recent optical technique that increases the sensitivity of a standard optical microscope to a point where it is possible to directly visualize nanometric films (down to 0.3 nanometres) and isolated nano-objects (down to 2 nm-diameter).
Ultraviolet light enables the resolution of microscopic features and the imaging of transparent objects. To visualize circuitry embedded in bonded silicon, we can use near-infrared light.
Many wavelengths of light are used in fluorescence microscopy, ranging from ultraviolet to visible, to cause samples to fluoresce.
Phase-contrast microscopy is an optical microscopic illumination technique in which small phase shifts in the light passing through a transparent specimen. Here, the conversion of phase shifts into amplitude [1]. This technique made it possible to study the cell cycle in live cells.
The traditional optical microscope has more recently evolved into the digital microscope. Instead of directly viewing the object through the eyepieces, a type of sensor similar to those used in a digital camera produces an image. These sensors may use CMOS or charge-coupled device (CCD) technology.
Digital microscopy with low light levels to avoid damaging vulnerable biological samples is popular.
Scanning optical and electron microscopes (such as the confocal and scanning electron microscope) use lenses to focus a spot of light or electrons onto the sample and then analyze the signals generated by the beam interacting with the sample. We obtain the magnification of the image by displaying the data from scanning a physically small sample area on a relatively large screen. These microscopes have the same resolution limit as wide-field optical, probe, and electron microscopes.
Electron microscope:
The two types of electron microscopes are transmission electron microscopes (TEMs) and scanning electron microscopes (SEMs)[1][2]. They both have a series of electromagnetic and electrostatic lenses to focus a high-energy beam of electrons on a sample. In a TEM, the electrons pass through the sample, analogous to basic optical microscopy[1]. The samples must also be thin (below 100 nm) for the electrons to pass through them.[1][2] Cross-sections of cells stained with osmium and heavy metals reveal clear organelle membranes and proteins such as ribosomes[2]. A detailed view of viruses (20 – 300 nm) and a strand of DNA (2 nm in width) is possible [2] with a 0.1 nm level of resolution.
Features:
- It has a series of electromagnetic and electrostatic lenses to focus a high-energy beam of electrons on a sample.
- The samples must be thin.
- With a 0.1 nm level of resolution, detailed views of viruses (20 – 300 nm) and a strand of DNA (2 nm in width)
In contrast, the SEM has raster coils to scan the surface of bulk objects with a fine electron beam. So, do not section the specimen but coat it with a nanometric metal or carbon layer, needed for non-conductive samples [1]. SEM allows fast surface imaging of samples.
Features:
- It has a series of electromagnetic and electrostatic lenses.
- SEM has raster coils to scan the surface of bulk objects.
- Sectioning of specimens is not necessary for scanning.
- Coating a nanometric metal or carbon is necessary for non-conductive samples.
Scanning probe microscope:
There are three most common types of scanning probe microscopes:
1) Atomic force microscope (AFM),
2) Near-field scanning optical microscopes (NSOM or SNOM),
3) Scanning near-field optical microscopy and scanning tunneling microscopes (STMs) [3].
An atomic force microscope has a fine probe, usually made of silicon or silicon nitride, attached to a cantilever. The probe scans over the surface of the sample, and the forces that cause an interaction between the probe and the surface of the sample are measured and mapped.
A near-field scanning optical microscope is similar to an AFM, but its probe consists of a light source in an optical fiber covered with a tip and an aperture for the light to pass through. The microscope can capture either transmitted or reflected light to measure very localized optical properties of the surface, commonly of a biological specimen.
Scanning tunneling microscopes have a metal tip attached to a tube through which current flows [4]. The tip scans over the surface of a conductive sample until a tunneling current flows. The current is kept constant by the computer-controlled movement of the tip, and an image is formed by the recorded movements of the tip[3].
Other types:
Scanning acoustic microscopes use sound waves to measure variations in acoustic impedance. Similar to Sonar in principle for detecting defects in the subsurfaces of materials, including those found in integrated circuits. On February 4, 2013, Australian engineers built a quantum microscope with unparalleled precision [5].
Mobile apps:
We can use mobile app microscopes as optical microscopes optionally when we activate the flashlight. However, these microscopes are difficult to use due to visual noise and are often limited to 40x resolution.
Computational microscope:
A joint venture of (the US National Institute of Health, Chicago University, and Zhejiang University in China) scientists have developed a new image processing technique. With this technique, we can reduce the processing time by a thousand-fold. Algorithmic reconstruction and sensing are combined to obtain tiny images of objects using different light rays. Using these pictures, we can construct 2D or 3D images with the help of iterative techniques or machine learning [6].
The multi-focus microscope gives clear pictures in different layers. So we can view and study the samples in all directions.
X-ray microscope:
We get images in this microscope using soft X-rays. We use it in tomography to get 3D images of biological samples, including those not treated by chemicals. Research is ongoing to improve the technology using hard X-rays.
The scanning helium microscope:
It is the next-generation microscope based on the quantum gas jet. Research is ongoing to address the challenges related to the production of neutral beams to improve the quality of the image.
References:
1. Lodish, Harvey; Berk, Arnold; Zipursky, S. Lawrence; Matsudaira, Paul; Baltimore, David; Darnell, James (2000). "Microscopy and Cell Architecture". Molecular Cell Biology. 4th Edition.
2. ^ Jump up to a b c d e f g Alberts, Bruce; Johnson, Alexander; Lewis, Julian; Raff, Martin; Roberts, Keith; Walter, Peter (2002). "Looking at the Structure of Cells in the Microscope". Molecular Biology of the Cell. 4th Edition.
3. ^ Jump up to a b Bhushan, Bharat, ed. (2010). Springer Handbook of Nanotechnology (3rd rev. & extended ed.). Berlin: Springer. p. 620. ISBN 978-3-642-02525-9.
4. ^ Sakurai, T.; Watanabe, Y., eds. (2000). Advances in scanning probe microscopy. Berlin: Springer. ISBN 978-3-642-56949-4.
5. ^ "Quantum Microscope for Living Biology". Science Daily. 4 February 2013. Retrieved 5 February 2013.
6. Wikipedia
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