History and Types of Microscopes

History and Types of MicroscopesWhat is a microscope?thither is so many little objects that human hearts angle be able to see. The microscope is a tool to see minute objects consisting of lens or combination of lenses1. Due to their highly-improved lenses, we potful observe high-quality images and these geezerhood this images can be transferred to computers. Todays microscopes atomic number 18 so advanced that they can show objects which are sized of the millionth part of a meter called micron2.The science of searching small objects with microscopes is called microscopy. microscopical means that impossible to see, without a help of a microscope, with a naked eye3.History of Microscope after(prenominal) the glass is graduation exercise made in the first century, Romans was attempt to tiller objects to be seen bigger. The first and innocent forms were called flea glasses and they were able to show 6 periods bigger4.The microscope is developed in Netherlands at the 1590s only its inventor is not easy to identify. Some proofs are leading to Cornelis Drebbel5. But others insist that Zacharias Jansen and his father Hans were working with lenses, they combined about lenses and put them into a tube and invented the microscope. Few others believed that Galileo Galilei was the first discoverer of microscope6.First microscopes were not good enough to use at researches because it can only enlarge by 9 times bigger7.First, the real microscope was utilise by Anton van Leeuwenhoek in the late 17th century which was made by pipes, simple lens, plate and screw( presage1). general anatomy 1Unlike the others, his microscope could show objects millionth of a meter bigger of its sizes(270x). Others best achievement was 50x elaboration. With this microscope, he saw and identified bacteria, erythrocyte, and sperm cells. He published their drawings on Philosophical Transactions of the majestic Society of London at 1674.These drawings were forgotten until in that locatio n were huge developments in science8.In 1665 Van Leeuwenhoeks work was a guide to Robert Hooke and he wrote Micrographia. It is the first make that provi diethylstilbestrol microscopic pictures of insects, plants etc. 9 (Figure 2).Figure 2-Drawing of an insect by Robert Hooke10After 200 years from Robert Hooke, German engineer called Carl Zeiss improved lenses of the microscope and he established a fellowship named Zeiss. After that, he hired Ernst Abbe to the company. Abbe improved the microscopes and lenses11.Types of microscopesStereoscopeDis character microscope is employ with visible baseless. It is used to see dissection better.It has 3-dimensional images and it has low magnification.Figure 3 earthworm captured by StereoscopeConfocal MicroscopeConfocal laser see microscopy (CLSM) plays the most significant role on imaging tiny samples in three-dimensional form. CLSM works like an optical microscope with some differences. It uses monochromatic laser calorie-free instead of visible light-colored 12.CLSM has widely used from cell biology, genetics, microbiology and development biology to quantum optics, nanocrystal imaging and spectroscopy13.History of Confocal MicroscopeEarly in 1940, Hans Goldmann from Switzerland invented a slit lamp to make documentation of eye examinations. Some researchers believe it might be first confocal optical system 14.Marvin Minsky invented first confocal scanning microscope in 1955 and in 1957 got its patent.Figure 4 Marvin Minskys patent application that shows the principle of CLSM 15.By moving the stage, illumination point in focal plane could be scanned 16.In 1969 M. David Egger and Paul Davidovits draw the first CLSM in two pages and published. Only one illumination spot generated with this point scanner. It was used for the imaging of the nerve tissue 17, 18.In 1983 confocal microscope was first used and controlled by a computer after the publication of first work by I. J. Cox and C. Sheppard from Oxford Universi ty. Based on Oxford groups designs, first CLSM was offered from 1982 19.At the Laboratory of molecular(a) Biology in Cambridge, William Bradshaw Amos and John Graham White and colleagues invented the first confocal beam scanning microscope in the middle of 1980s.This time the illumination spot was moving but not the stage. This technique allowed faster image acquisition, cardinal images per second 20.Working dominion of Confocal MicroscopeFor getting higher intensities a laser is used. The laser light reflects from the dichroic reverberate. After that it hits mirrors on motors and across the sample lasers get scanned by these mirrors. And emitted light passes through and through the dichroic mirror and gets focused onto pinhole. Finally, the detector measures that light. As it appears the complete image of the sample cannot be observed just one point can be observed. The photomultiplier detector is connected to a computer and one pixel at a time it builds an image 21.Figure 5 Pri ncipal Light Pathways in Confocal Microscopy 22.What is the advantage of using a confocal microscope?By scanning lots of thin parts of a sample, it is easy to build a very good three-dimensional image. Confocal microscope has better resolution swimmingly and vertically. The best resolution can be obtained at 0.2 microns for horizontal and 0.5 microns for vertical 23.Examples there are some examples of imaging with the confocal microscope. Figure 6 Nematode. Miami University in Oxford, Ohio 24.Figure 7 Example image of confocal microscope 25.scan Electron Microscope (SEM)SEM is an negatron microscope that uses the focused beam of electrons to images of the sample. Electrons interact with atoms in the sample and gives information about external morphology (texture), chemical composition, and crystalline structure and orientation of materials making up the sample 26.A beam of electrons uses raster scan pattern which is a rectangular pattern of an image and reconstruction in the scre en. Most computers use bitmap image systems to hive away the image 27.The image is created by matching the position with the perceived signal. SEM can get better than 1 nm resolution. Standard SEM microscopes are generally suitable for dry and conductive surfaces in high vacuum. Also, there are specialized machines that work under changeable conditions from low temperature to high temperature and in low vacuum. There is environmental SEM for wet conditions.McMullan presented the history of SEM 28. Manfred von Ardenne invented SEM in 1937. In the early 1960s, Cambridge groups marketed as Stereoscan in 196528, 29.After interaction of high energized beam of electrons and outer orbit electrons of samples atoms plumbers snake electrons which curb low electrons will be formed. These electrons carry information about sample surface.After interactions, there will be electron beams which have lower energy, become to the surface of the sample and will gather there.These electrons called a s secondary electrons. For imaging for SEM, mostly secondary electrons are being used. Change of secondary electrons numbers depends on the topography of surface and angle of the point where the beam hits the surface 30.Figure 7 Blood image by SEM 31.Transmission Electron MicroscopeHigh energized electrons pass through the very thin sample. After interaction of electrons, images are enlarged and focused on fluorescence screen, photographic film layer or CCD camera 32.In 1930 slime Knoll and Ernst Ruska invented TEM 33. It allows us to see smaller objects than the optical microscope.TEM is used in cancer research, virology, materials science, nanotechnology, and semiconductor.TEMs contrast depends on absorption of electrons, ponderousness, and composition of the sample. Complex wave interactions at higher magnifications modulate the intensity of the image with analysis of an expert for the image. The resolution trap is up to 0.2 nm for TEM.Compared to SEM, TEM has troublesome work to get the sample coiffure and the user must have a very good background about it 34.Figure 8 Example of TEM of a plant cell 35. composite Light MicroscopesCompound microscopes are 2-dimensional light microscopes and they are most used microscopes. Even though it has low resolution it has high magnification.Figure 9-meiosis seen by involved microscope36.Figure 10-Microscope view of plant cells37.Parts of optical MicroscopeFigure 10 Parts of a microscope38Eyepiece Lens The lens that allows us to see through.Tubes It helps ocular to connect to lenses.Arm Holds the tube.Base Supports the microscope at the bottom.Illuminator Light source or a mirror that helps us to see a sample from the tube. If it is a mirror it can reflect outer light to use.Stage This platform is used to put samples and it has clips to prevent the sample from moving.Revolving Nosepiece or Turret This part is for memory lenses together and it can rotate to switch between lenses.Objective Lenses These lenses are most commonly can be put three or four lenses on the microscope. They have 4,10,40 or light speed times bigger magnification. They are color coded and should build to DIN standards.Rack Stop It is used to protect the physical object lens from breaking39.DIN StandardsThe real image is formed 160mm away from the object glass lens.Parfocal distance should be 45 mm.Eyepiece lens should be 170mm40.Working Principle of Optical MicroscopeFigure 11 41As shown in Figure 9 light starts its journey from illuminator and with a mirror it reaches to sample. Then it goes to prism through objective lenses. It reflects from the prism and comes to eye in the tube. When light passes through the objective lens makes the image of sample bigger and focuses 160 mm inside the tube and then ocular lenses magnifies the image of sample 25cm away from the eye. This image is a virtual image of the sample (Figure 10). Typical microscopes have four different objective lenses. Scanning (5x), low power(10x), m edium power (20x) and high power lenses (40x). We can easily calculate the magnifying of the microscope with multiplying objective lens and ocular lens. For example, after image magnified by objective lenses 40 times of original image of the sample, will magnify second time 20 times bigger by ocular lenses. So, our eye can see 4020=800 times bigger image of an original image of the sample.Figure 12 42Differences Between Electron and Light MicroscopeLight microscopes techniques are simple but for electron microscope high-level technical skill needed.Preparation time of the sample is few minutes to few hours for light microscopes but several days for electron microscopes.Live or dead samples can be seen in light microscopes but for electron microscopes only dead and dried samples can be seen.Light microscopes have low resolution than electron microscope and the resolution limit for the light microscope is 200 nm but for SEM 1nm and for TEM 0.2 nm.Light rays are used to illume for lig ht microscope but for electron microscope electrons are being used.Lenses are made of glass for light microscope but for electron microscope all lenses are electromagnets.Magnification of light microscope is 500x to 1500x but for EM 160,000x and photographic magnification is 1000,000x or more.Light microscopes are cheap but electron microscopes are expensive 43.Calculation of ResolutionIf we postulate to get good details of very small objects like cells, we need to increase the resolution. It can be described as to see different between two small and very near objects. It can be affected of the wavelength of light and power of lenses. Mathematical formula of separating two different small objects which have the smallest distance (dmin)Dmin = 1.22 x wavelength / N.A. objective + N.A. condenserDifferent then the theoretical power, in practice samples quality affects its resolving power44.Definition of Numerical Aperture(N.A.) is a encourage of objectives defined by Abbe.Numerical Ap erture (NA)=n-sin() or n-sin()Figure 13 Numerical ApertureAs shown in Figure 11 light waves go through a sample to the objective lens. But when it comes to practice it is nearly impossible to get the value of aperture above 0.95 with dry objective lenses. When the light cones get the bigger stagecoach of starts to increase from 7 to 60 and N.A. increases from 0.12 to 0.87. In todays world, it is possible to use alternative media to make images in water (refractive index = 1.33), glycerin (refractive index = 1.47), and immersion oil color (refractive index = 1.51) by the objective lens. We can clearly see Figure 12 and Table 1 highly corrected objectives have bigger N.A.Figure 14Table 1 Numerical Aperture versus Optical Correction45There is a limit of resolution in optical microscopes as shown belowLet N.A. be 1.4 and resolution is different for lights wavelength.A minimum distance of two points of the image is 0.61 /N.A.As we know visible light wavelength is between 400-700 nm. There will be no resolution between two objects if distance is 1/3 .If we choose green light = 500nm and r=0.61 x 500nm / 1.4 =218 nm.If we choose blue light = 400nm and r=0.61 x 400nm / 1.4 =174 nm.If we choose green light = 700nm and r=0.61 x 700nm / 1.4 =305 nm46.Diffraction Limit of Electron MicroscopeElectron microscope has diffraction limit and it is 1nm for SEM, 0.3nm for TEM. This limit occurs because of wave nature of electrons. Electrons has a phenomenon called wave-particle duality. Particle of matter (incident electron) can be explained as wave. We can assimilate to sound or water waves.Louis de Broglie says that the wavelength of a particle can be calculated as following formula=h/p wavelength of a particleh Plancks constant (62610-34)p momentum of a particlepulsing is the product of mass and the velocity of a particle and equation can be written as= h / mvAccelerating voltage determines the velocity of the electrons we can use following formulaeV = mv2/2We can calcu late the velocity of electrons byDue to these formulae, we can show the wavelength of propagating electrons at a given(p) accelerating voltageSince the mass of an electron is 9.1 x 10-31 kg and e = 1.6 x 10-19So, the wavelength of electrons is 3.88pm when the microscope is operating at 100 keV, 2.74 pm at 200 keV, and 2.24 pm at 300 keV.We know electrons in an electron microscope reach %70 of speed of the light wit accelerating voltage of 200 keV, there are effects which are significant length contraction, time dilation, and an increase in mass. By these changesc speed of the light (299 792 458 mps)So, wavelength of an electron at 100 keV, 200 keV, 300 keV in electron microscopes is 3.70 pm ,2.51 pm, and 1.96 pm, respectively 47.Another reason for limitation for TEM is, sample transparency has to be proper for electron transparency. To be more precise its thickness has to be 100nm or less.Electrons can be deflected in magnetic fields by the Lorentz force. This problem may make crys tal structure determination closely impossible 48, 49.Diffraction Limit of Optical MicroscopeThere is a limit for imaging with an optical microscope called Abbe diffraction limit. This limit is /2( is imaging radiations free-space wavelength) 50. Modern works show us that this limit can be passed and can make optical microscopes lenses to have a high resolution51.But with diffraction limit even though the lens is corrected there will be blur image of the point. This called Airy disk or diffraction. British mathematician Lord George Biddel Airy has found it. We can see its cross section and appearance below (Figure 13).Figure 15Diameter of the disk isBdiff =2.44 (f/)52With f/ limitation can be controlled and wavelength of the light. The maximum resolving power of the lens is driven by this limitation. If we want to calculate diffraction limit we can use following formulaIf we reach the limit lens will become unable to break apart greater frequencies. In theory, if the contrast is %0 the diffraction limit will appear to be as shown in Table 2 at different f/s for 0.520 m light as known as green light.Table 253Different Ways to Break Resolution Limit of Optical MicroscopeThere are several ways to break resolution limit of optical microscope. To do that researchers change lenses or different parts of microscopes. Here are some examplesBy employing stimulated emission to inhibit the fluorescence process in the outer regions of the excitation point-spread function54.By using laterally structured illumination in a wide-field, non-confocal microscope(This method claims that spatially structured excitation light illuminates the sample) 55.By improving the lenses with ZrO2.Synthesis of ZrO2 NanoparticlesZirconium(IV) isopropoxide2-propanol complex (5.6 g) and anhydrous benzyl alcohol (55mL) were charged into a 100 mL Teflon-lined autoclave. This Teflon-lined autoclave was sealed and placed into an oven at 240 C for 4 days and then cooled to obtain a white turbid sus pension. 56.Figure 1657.Figure 16 is a schematic of hSIL integrated with an Olympus optical microscope for super-resolution imaging of the underlying nanopattern. The hSIL collects near-field information on the nanopattern and forms a virtual image that can be captured by the objective lens57.Figure 17 -Super-resolution optical imaging through hSIL on 45 nm gaps. SEM images of the chip with periodic structures of 50 nm gaps (a) and the gold-coated chip with 45 nm gaps (b). (c, d) Optical images of the chip with 50 nm gaps under white and filtered blue light (max 470 nm) without SILs. (e1, e2) Optical images of the chip with hSIL of h/d = 0.8 (d = 11.5 m). (f1, f2) Optical images of the gold-coated chips through SIL of h/d = 0.78 (d = 10.5 m) and (g1, g2) with hSIL of higher h/d = 0.84 (d = 11.3 m). Optical images of e1g1 and e2g2 were taken under white light and filtered blue light, respectively. The corresponding image magnification factors of e2, f2, and g2 are 3.1, 2.9, and 3.6. 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