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Optical microscopy laboratory practice 2012 Construction of the microscope The main parts of the microscopes are similar, but there are application dependent parts too. The biological microscope (Fig.1.) illuminates the transparent subject through the condenser under the stage. Since the metal samples are not transparent, the metallurgical microscope illuminates the subject from above, parallel to the viewing direction. 1 eyepiece lens 2 binocular head 3 revolving objective lenses 4 filter support 5 truss 6 subject-table 7 condenser height adjustment 8 condenser 9 aperture adjustment 10 condenser centralizer 11 subject movement (x-y direction) 13 illumination aperture 14 and 15 rough and fine contrast adjustment 16 lamp housing Fig.1. The main parts of the biological microscopes [4] In Fig. 1. the binocular head (2) shows the same picture for both eyes: it is for comfort viewing, and it gives no stereo picture. The interpupillary distance of the eyes can be adjusted via rail mechanism. 1 Construction of the metallurgical microscope Metallurgical samples, and other not-transparent subject can only be observed with the aid of reflected light. Since the metallurgical microscope illuminates the subject from above, parallel to the viewing direction, the examination light is reflected to the sample via mirrors. Most cases the illuminating mirrors are integrated into the objective lens. The main construction of the metallurgical microscope can be observes on Fig.2.: it can based on bright-field or dark-field illumination principle. Our Zeiss metallurgical microscope (Fig 4.) uses dark-field illumination. Fig 2. bright-field (left) or dark-field (right) illumination principle. [3] Microscope Parts and Functions [7] 1. Eyepiece: The eyepiece (sometimes called the 'ocular') is the lens of the microscope closest to the eye that you look through. It is half of the magnification equation (eyepiece power multiplied by objective power equals magnification), and magnifies the image made by the objective lens... sometimes called the virtual image. Eyepieces come in many different powers. One can identify which power any given eyepiece is by the inscription on the eyecup of the lens, such as "5x", "10x", or "15X". Oculars are also designed with different angles of view; the most common is the wide field (W.F.). 2. Eyepiece Holder: This simply connects the eyepiece to the microscope body, usually with a setscrew to allow the user to easily change the eyepiece to vary magnifying power. 3. Body: The main structural support of the microscope which connects the lens apparatus to the base. 4. Nose Piece: This connects the objective lens to the microscope body. With a turret, or rotating nose piece as many as five objectives can be attached to create different powers of magnification when rotated into position and used with the existing eyepiece. 5. Objective: The lens closest to the object being viewed which creates a magnified image in an area called the "primary image plane". This is the other half of the microscope magnification equation (eyepiece power times objective power equals magnification). Objective lenses have many designs and qualities which differ with each manufacturer. Usually inscribed on the barrel of the objective lens is the magnification power and the numerical aperture (a measure of the limit of resolution of the lens). 6. Focusing Mechanism: Adjustment knobs to allow coarse or fine (hundredths of a millimeter) variations in the focusing of the stage or objective lens of the microscope. 7. Stage: The platform on which the prepared slide or object to be viewed is placed. A slide is usually held in place by spring-loaded metal stage clips. More sophisticated high-powered 2 microscopes have mechanical stages which allow the viewer to smoothly move the stage along the X (horizontal path) and Y (vertical path) axis. A mechanical stage is a must for high-power observing. 8. Illumination Source: The means employed to light the object to be viewed. The simplest is the illuminating mirror which reflects an ambient light source to light the object. Many microscopes have an electrical light source for easier and more consistent lighting. Generally electrical light sources are either tungsten or fluorescent, the fluorescent being preferred because it operates at a cooler temperature. Most microscopes illuminate from underneath, through the object, to the objective lens. On the other hand, stereo microscopes use both top and bottom illumination. 9. Base: The bottom or stand upon which the entire microscope rests or is connected. 10. Photography unit with CMOS or CCD sensor able to make pictures via microscope. Stage Camera Eyepiece Illumination Source Objective Illumination Source Focusing Mechanism Objective Stage Focusing Mechanism Fig. 3. Zeiss metallurgical microscope [3] Fig 4.. Inverse-system metallurgical microscope [3] 3 Microscope Objective Specifications [8] Identification of the properties of individual objectives is usually very easy because important parameters are often inscribed on the outer housing (or barrel) of the objective itself as illustrated in Figure 5. Manufacturer - The name of the objective manufacturer is almost always included on the objective. Linear Magnification - In the case of the apochromatic objective in Figure 5, the linear magnification is 60x, although the manufacturers produce objectives ranging in linear magnification from 0.5x to 250x with many sizes in between. Optical Corrections - These are usually listed as Ac Achromat (achromatic), Field curvature corrections are abbreviated Plan, and as Apo (apochromatic) for the highest degree of correction for spherical and chromatic aberrations. Numerical Aperture - This is a critical value that indicates the light acceptance angle, which in turn determines the light gathering power, the resolving power, and depth of field of the objective. Fig 5. Objective Specifications [8] Characterization of microscope picture [9] Definition of Magnification in Microscopy Microscope magnification is how large the object will appear compared to its actual size. Finding the Overall Magnification To find the overall magnification of the microscope, multiply the magnification of the eyepiece by the magnification of the objective lens you are using. For example, if the magnification of the eyepiece is 10x and the magnification of the objective lens is 40x, the overall magnification is 400x. What is Microscope Resolution? Resolution refers to the ability of a microscope to distinguish two separate points. Magnifying an object without good resolution will simply produce a large image of the object where details cannot be identified. What is Depth of Focus? The depth of focus of an optical microscope is the range of image plane position at which the image may be viewed without appearing out of focus for an object or specimen The bigger the overall magnification, the lower the depth of focus. 4 The numerical aperture of the objective lens In most areas of optics, and especially in microscopy, the numerical aperture of an optical system such as an objective lens is defined by where n is the index of refraction of the medium in which the lens is, and θ is the half-angle of the maximum cone of light that can enter or exit the lens. There are several equations that have been derived to express the relationship between numerical aperture, wavelength, and resolution: Resolution (r) = Where /(2NA) is the average wavelength of the illuminating light Fig 6. Objectives with Low and High numerical aperture The table 1 describes the relationship between the NA and the resolution, and between the NA end the limits of useful magnification. NA Resolution Resolution pairs / mm 0,04 0,12 0,25 0,5 0,65 0,75 0,95 1,3 1,4 6,9 2,3 1,1 0,55 0,42 0,37 0,29 0,21 0,19 145 436 910 1820 2380 2730 3450 4750 5090 Table 1 5 line Lower and Upper limits of useful magnification 500*NA 1000*NA 20 40 60 120 125 250 250 500 325 650 375 750 475 950 650 1300 700 1400 Stereoscopic microscope A stereo microscope is an optical microscope fitted with two sets of lenses, each positioned to view an object from a slightly different angle. The result is a three-dimensional image. It is widely used in industry for quality control of small pieces, or for manual SMD soldering. Estimating the average grain size diameter as determine from a random cross section. The average grain size diameter (d) can be calculated by the following equation, where L0 is a straight line crossed N grains : d= Lo N Monitoring questions 1. 2. 3. 4. 5. 6. What are the main parts, and its functions of a metallurgical microscope? Bright-field and dark-field illumination principle Microscope Objective Specifications Definition of the numerical aperture Definition of the Overall Magnification, Resolution and Depth of focus Estimating the average grain size diameter as determine from a random cross section. Literature: [1]. Bernolák-Szabó-Szilas: A mikroszkóp (zsebkönyv). 1979, Mőszaki Könyvkiadó, Budapest [2]. Lovas Béla: Mikroszkóp-mikrokozmosz. 1995. Gondolat Kiadó, Budapest [3]. A korábbi laboratóriumi útmutató: Dr. Csiszár Sándor: Mikroszkóp Villamosipari anyagismeret (Laboratóriumi gyakorlat) [4]. Determann – Lepusch: Das Mikroskop und seine Anwendung Ernst Leitz Wetzlar GmbH [5]. www.zeiss.de/micro [6]. http://micro.magnet.fsu.edu/ [7]. http://wiki.answers.com/Q/What_are_the_microscope%27s_parts_and_functions [8]. http://www.microscopyu.com [9]. www.ehow.com 6 Report of the optical microscopy Made by: …………………………………… ……… laboratory practice Name neptun code, group identification Leader of laboratory practice Date: Note: 1. What are the main parts, and its functions of a metallurgical microscope? [ ] Zeiss metallurgical microscope [ ] Inverse-system metallurgical microscope 2. Calculation of the Overall Magnification of the microscope using the data of a given objective lens: 3. Measuring of the field of view using the given objective lens: : measuring instrument: field of view diameter in millimeters: 4. Microscope Objective Specifications (don’t touch the surface of the lens!): Data Explanation Manufacturer Optical corrections Magnification Numerical aperture Cover glass thickness Infinity correction Antireflex layer color 7 5. Based on NA look out the resolution and the Lower and Upper limits of useful magnification values from Table 1: • Resolution: • Lower and Upper limits of useful magnification: 6. What is the relation between the of the Overall Magnification (Point 2) and the Lower and Upper limits of useful magnification. 7. Identify 3 different metallurgical cross sections: 8. Estimating the average grain size diameter as determine from a random cross section. The horizontal measure of the field of view (millimeters): How many grains are on the given horizontal line of the field of view : Calculation of the average grain size diameter: 9. Measuring 2 holes on printed circuit board via image analysis software 10. Specimens inspected with Stereomicroscope: 8