A microscope is an instrument used to investigate tiny objects which cannot be seen by naked eyes. There exist three types of microscopes which are optical microscopes, electron microscopes, and scanning probe microscopes. (1) Six types of microscopes talked in this report are reflected and transmitted light microscope, scanning electron microscopes (SEM), transmission electron microscopes (TEM), focused ion beam (FIB), and atomic force microscope (AFM).
1.1 Reflected light microscopes
Reflected light microscope is a type of microscope using visible light and a system of lenses to magnify images of small samples. It is used to examine opaque specimens which will not transmit light and other materials such as ceramics.The reflected light travels through the objective lens, which in this arrangement acts as both a condenser and an objective, and strikes the specimen.It is then reflected off the specimen back up through the objective lens, the head, the eyepieces, and finally to the eye.(2)
1.2 Transmitted light microscope
Transmitted light microscope is a type of microscope where the light transmits from a source on the opposite side of the specimen from the objective. Usually the light is passed through a condenser to focus it on the specimen to get very high illumination. (3)After the light passes through the specimen, the image of the specimen goes through the objective lens and to the oculars where the enlarged image is viewed.
1.3 Scanning electron microscope
The scanning electron microscope (SEM) is one kind of electron microscope. The SEM utilizes a very fine probing beam of electrons scanning over the specimen to emit a variety of radiations. The signal which is proportional to the amount of radiation leaves an individual point of the sample at any time. The signal obtained from one point will display the information of that point. In practice, the points follow one another with very high speed so that the image of each point becomes an image of a line, and the line move down the screen so rapidly that the naked eye sees a complete image on the computer. SEMs are patterned after reflecting light microscopes and will yield similar information
1.4 Transmission electron microscope
A transmission electron microscope (TEM) works much like a slide projector. A projector shines a beam of light through the slide, as the light passes through it is affected by the structures and objects on the slide. These effects result in only certain parts of the light beam being transmitted through certain parts of the slide. This transmitted beam is then projected onto the viewing screen, forming an enlarged image of the slide. TEMs work the same way except that they shine a beam of electrons through the specimen. Whatever part is transmitted is projected onto a screen for the user to see. TEMs are patterned after transmission light microscopes and will yield similar information.
1.5 Focused ion beam
A focused ion beam system (FIB) is a relatively new tool that has a high degree of analogy with a focused electron beam system such as a scanning electron microscope or a transmission electron microscope. In SEM and TEM the electron beam is directed towards the sample generating signals that are used to create high magnification images of the sample. The major difference with a focused ion beam system is the use of a different particle to create the primary beam that interacts with the sample. A highly focused ion beam is used instead of electrons in FIB. As the beam scans the surface of the sample, a highly magnified image is created, which allows the system operator to view the samples microscopic features clearly.
1.6 Atomic force microscope
The AFM is one of the foremost tools for imaging, measuring and manipulating matter at the nanoscale. The information is gathered by ‘feeling’ the surface with a mechanical probe. To achieve atomic scale resolution, a sharp stylus (radius ~1-2 nm) attached to a cantilever is used in the AFM to scan an object point by point and contouring it while a constant small force is applied to the stylus. Piezoelectric elements that facilitate tiny but accurate and precise movements enable the very precise scanning. (4)
2. Study of comparison among six kinds of microscopes
2.1 Optical microscopes
Optical microscopes, which use visible wavelengths of light, are the simplest and most used. Both transmitted light microscopy and reflected light microscopy need low energy and the microscope itself is much cheaper and smaller than electron microscopes. Compared to electron microscopes, the optical microscopes have another advantage that the image obtained from them is in color.
Comparing to reflected light microscope, the transmitted light microscope only works on light transparent specimens but not metal, ceramics and some polymers such as rubber. However sample preparation of transmitted light microscope is relatively complicated. As it requires sample thin enough for the light to go through. This can be done by using a microtome to slice at lower temperature; as well the distortion of the section due to the sample preparation is a problem for observing. (5)
The SEM has allowed researchers to examine a much bigger variety of specimens no matter it is bulk or thin layer. The scanning electron microscope has many advantages over optical microscopes.The SEM has a large depth of field, which allows more of a specimen to be in focus at one time.The SEM has much higher resolution (~1-5nm). (5)Because the SEM uses electromagnets rather than lenses, much more control in the degree of magnification can be done.All of these advantages, as well as the actual strikingly clear images, make the scanning electron microscope one of the most useful instruments in research today.
However, materials that can be examined in the SEM must be vacuum compatible, clean and electrically conducting such as metal. But for non-conducting materials such as ceramic and polymers, gold or carbon coating on the surface of the sample is essential.
TEM is a technology using a high energy (80-200kV) beam of electrons to transmit through an ultra thin specimen (50-200nm). High resolution (~0.2nm) is the most significant advantage of TEM. (5)
However, there are a number of drawbacks to the TEM technique. Many materials require extensive sample preparation to produce a sample thin enough to be electron transparent, which makes TEM analysis a relatively time consuming process. The structure of the sample may be changed during the preparation process. Also the field of view is relatively small, which leads to the region analyzed may not be characteristic of the whole sample. There is potential that the sample may be damaged by the electron beam, particularly in the case of biological materials.
FIB is usually used to examine metal surfaces. If the sample is non-conductive, a low energy electron flood gun can be used to provide charge neutralization.
FIB is inherently destructive to the specimen because when the high-energy gallium ions strike the sample, they will sputter atoms from the surface. Ga atoms will also be implanted into the top few nanometers of the surface making the surface amorphous. (6) These limitations produce noticeable effects when using techniques such as high-resolution ‘lattice imaging’ TEM or electron energy loss spectroscopy.
The AFM is a very high-resolution type of scanning probe microscope, with demonstrated resolution of fractions of 1 nm. (4)
AFM provides a true three-dimensional surface profile. Additionally, samples viewed by AFM do not require any special treatments such as coating. Most AFM modes can work perfectly in air or even a liquid environment without a need of vacuum. This makes it possible to study not only metal, ceramic, polymer but also biological macromolecules and even living organisms. In principle, AFM can provide higher resolution than SEM. It has been shown to give true atomic resolution in ultra-high vacuum and in liquid environments. High resolution AFM is comparable in resolution to TEM.
A disadvantage of AFM compared with the scanning electron microscope (SEM) is the image size. The AFM can only image a maximum height on the order of 10-20 micrometers and a maximum scanning area of around 150 by 150 micrometers. (4)
Another inconvenience is that the AFM could not scan images as fast as an SEM, requiring several minutes for a typical scan, while a SEM is capable of scanning at near real-time after the chamber is evacuated. The relatively slow rate of scanning during AFM imaging often leads to thermal drift in the image making the AFM microscope less suited for measuring accurate distances between topographical features on the image. (4)
3. Example of applications
3.1 Reflected light microscope
Normally, reflected light microscope is used to image metal, ceramic and rubber. That’s the reason why it is also called metallurgical microscope. Nowadays it becomes a fast growing interest; especially in regard to its increasing usefulness in the fluorescence microscopy as well as the rapidly growing semiconductor industry had also led to an increase in the use of reflected light microscopes. (7)
3.2 Transmitted light microscope
Polymers can commonly be looked at under the transmitted light microscope, because most of them are transparent or translucent. It can also analyze cell slices obtained from organism. Most of the lab can afford a transmitted light microscope since it is relatively cheap.
About any scientific field can use an SEM as a research tool. It can be used to look at the crystalline structures of chemical compounds and how their bonds form. A scanning electron microscope is especially useful for looking at the surfaces of materials at an atomic level.
TEM can do diffraction analysis of small areas by selected area diffraction. High resolution x-ray microanalysis and analysis of crystal defects such as dislocations, stacking faults using diffraction contrast can also be done by using TEM. Another important application is it can image lattice of crystalline materials. (8)
FIB can be used as Ion beam imaging. The FIB also offers the ability to perform nanopatterning and micromachining respectively, and by instructing the machine to add or remove pertinent features, operator can design and prototype a new micro or nanostructure, modify integrated circuits and cross section specific features to allow failure analysis even in the 3D (TEM sample preparation). FIB is also used for Secondary ion mass spectrometry (SIMS). (7) The ejected secondary ions are collected and analyzed after the surface of the specimen has been sputtered with a primary focused ion beam.
The atomic force microscope (AFM) is one of the most powerful tools for determining the surface topography of native biomolecules at subnanometer resolution. AFM allows biomolecules to be imaged not only under physiological conditions, but also while biological processes are at work. The AFM can also provide insight into the binding properties of biological systems.
Characteristics of six different types of microscopes are compared in this article, including sample preparation and technique limitations. Each one has its advantage and disadvantage, so it is necessary to consider comprehensively before choosing, for example, the type of the material, needed information, vacuum compatible, conductivity and sample preparation, etc.
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