Light Optical Microscopy TechniquesV Trung Kin - 20071634Materials Science & Engineering, Center for Training Excellent Students, Hanoi University of Science and Technology, Dai Co Viet Street No.1, Hai Ba Trung, Ha Noi, Viet Nam
Abstract: Light optical microscope is known as a type of microscope which uses the seeing-light and a system of lenses to magnify images of small samples. Nowadays, inventors over the world be in success with many kind of useful microscope, such as, Scanning Electron Microscope, Transmission Electron Microscope, Scanning Tunneling Microscope but these are very, very expensive and only used by scientists, and rarely used by students. In Vietnam, not only students cannot be user but also Vietnamese scientists, whom be hardly allowed to use them because of many troubles. Its too for using light optical microscope. In this situation, Vietnamese young students should think about how to solve this problems. This document will tell you all about light optical microscopy techniques, in details, and then readers should have the ideas and skills to make a simple light optical microscope - own microscope. Keywords: Light optical microscopy techniques, LOM, own microscope.
Credit for the first microscope is usually given to Zacharias Jansen, in Middleburg, Holland, around the year 1595. Since Zacharias was very young at that time, it's possible that his father Hans made the first one, but young Zach took over the production. Another favorite for the title of 'inventor of the microscope' was Galileo Galilei. He developed an occhiolino or compound microscope with a convex and a concave lens in 1609 and the first such device to be given the name "microscope" in 1625 by fellow Lincean Giovanni Faber. Over the past decades the number of applications of optical microscopy has grown enormously, and is now found in almost any field of science and industry, such as microelectronics, nanophysics, biotechnology, pharmaceutic industry and microbiology. These microscopes use visible light (or UV light in the case of fluorescence microscopy) to make an image. Optical microscopes can be further subdivided into several categories: Compound Microscope - These microscopes are composed of two lens systems, an objective and an ocular (eye piece). The maximum useful magnification of a compound microscope is about 1000x. Stereo Microscope (dissecting microscope) - These microscopes magnify up to about maximum 100x and supply a 3-dimensional view of the specimen. They are useful for observing opaque objects. Confocal Laser scanning microscope - Unlike compound and stereo microscopes, these devices are reserved for research organizations. They are able to scan a sample also in depth. A computer is then able to assemble
the data to make a 3D image. Following this document, we concentrate in very simple light optical microscope that all readers may build up. The paper is structured in the following fashion. Section II describes the theory and basic principles. Its very important for optical microscope engineering, with a discussion of Abbes theory, resolution limit, magnification, and some used techniques in light optical microscope. In Sec. III, the four main systems of light optical microscope be cracked in details. Finally, Sec. IV gives the way to solve regular problems when building a microscope. Tricks and helpful hints for light optical microscopy are also putted in that section. II. Theoretical formalism and basic principles 1. Theory and basic principles + Refraction The lens of an optical magnifying glass forms an image of an object because the refractive index of glass is much greater than that of the atmosphere, and reduces the wavelength of the light passing through the glass. A parallel beam of light incident at an angle on a polished block of glass is deflected, and the ratio of the angle of incidence on the surface to the angle of transmission through the glass is determined by the refractive index of the glass (Figure 3.3).
In the case of a convex glass lens (a lens having positive curvature), the spherical curvature of the front and back surfaces of the lens results in the angle of deflection of a parallel beam of light varying with the distance of the beam from the axis of the lens, and bringing the parallel light beam to a point focus at a distance f that, for a given wavelength, is a characteristic of the lens, and is termed its focal length
If the lens curvature is negative, then the lens is concave and a parallel beam incident on the lens will be made to diverge. The beam of light will now appear to originate at a point in front of the lens: an imaginary focus corresponding to a negative focal length -f.
For symmetric focal length f, an object at -u gives an image at v which is magnified by a factor M, where:
Note: asymmetric lenses can have multiple focal lengths.
There is no reason why the front and back surfaces of the lens should have the same curvature, nor even why one surface should not have a curvature of opposite sign to the other. It is the net curvature of the two surfaces taken together which determine whether the lens is convex or concave. Similarly, there is no reason why the refractive index should be the same for all the lenses in an optical system, and different grades of optical glass possess different refractive indices. The lenses used in the optical microscope are always assemblies of convex and concave lens components with refractive indices selected to optimize the performance of the lens assembly. Depending on their position in the microscope, these lens assemblies are referred to as the objective lens, the intermediate or tube lens and the eyepiece. It is also quite common for the medium between the sample and the near side of an objective lens to be a liquid, rather than air. An immersion lens is one that is designed to be used with such an inert, high refractive index liquid between the sample and the objective lens. + Abbes theory of image formation The image of a light absorbing specimen is formed due to diffraction. The specimen is seen by the light as a complex superposition of gratings with varying grating constants and holes. Some of the light will pass through the specimen undeviated and will only give rise to a uniformly bright image. The deviated (diffracted) light carries the information about the structures in the specimen. To simplify things, consider the grating specimen schematically shown in Figure 1. Parallel light (i.e. a plane wave) which enters from below along the optical axis, will be diffracted and the different diffraction orders will emerge at different angles. The smaller the distance between the grids, i.e. the smaller the periodicity, the bigger the angles will be.
In the rear focal plane of the objective we find the diffraction pattern, which is the Fourier transform of the image. The grid will appear as bright spots on a line. The central spot is the zeroth order. All light that go through the sample undeviated will pass this spot in the back aperture of the objective. The spots next to the zeroth order are the first diffraction orders and so on. As illustrated in Figure 2, blocking all spots but the zeroth order will result in an evenly intense image in the image plane. Blocking all light but the first order spots will result in an image with an intensity variation having the same frequency as the grid. The second and third diffraction order alone gives a false period. However, by adding the four orders (zeroth to third) we get a reasonable image of the specimen. Ernst Abbe, a German microscopist of the 19th hundred century, stated that an image will be formed only if at least two of the diffraction orders are captured by the objective. The more diffraction orders that can be captured by the objective the finer details can be resolved.
+ The resolution limit Knowing that at least the first diffraction order is needed to obtain an image we can calculate the resolution limit. Consider a grating with the grating constant d. The grating equation gives:
where is the angle to the optical axis, m is the diffraction order and is the wavelength of light. According to this equation the smaller the distance d, the higher the angle for the
same diffraction order. Hence, the smaller the spatial distances in the specimen, the more the light will bend off . Resolution is therefore dependent on how many of the diffraction orders we can capture with the objective. For the first order m=1, the grating equation gives:
This would be the smallest distance which can be resolved with the microscope. This limit could be decreased by using an oil or water immersion objective. If only air is in between the cover slide and the objective the light will bend off in a higher angle due to refraction. See Figure 3. Oil and water have a higher refractive index than air (approximately 1.5 and 1.3 compared to 1). By using oil or water in between the cover slide the light will travel to the objective at a lower angle Hence, the resolution limit will decrease.
According to Snells law of refraction:
where n is the refraction index. If n2 =1, as for air, we get:
The resolution limit could now be written:
where NA is the numerical aperture. The NA is given on all objectives and therefore we could calculate the resolution limit of the microscope. However, this expression is not true
unless the light is emerging from one point. Also the NA of the condenser has to be taken into account. Consider the following figure:
Parallel light from the condenser travels from refractive index n 1 and into the specimen, which has a lower refractive index of n2. Assume NAcondenser = n1sin. According to refractive and diffraction laws
From the equations we can calculate the smallest distance d to be r