Published on November 16, 2018
Compound Light Microscope : Compound Light Microscope DR. QAZI IMTIAZ RASOOL OBJECTIVES: OBJECTIVES After completing this experiment, you should be able to : Name the different parts and their functions Explain the physical basis of microscopy Describe the mechanism of image formation and the type of image seen. Explain how to get different magnifcations . Describe the procedure (protocol) that must be followed when useing a microscope. TYPES OF OPTICAL MICROSCOPES: TYPES OF OPTICAL MICROSCOPES Simple microscope Compound Microscope Dissection Microscope Electron Microscope Fluorescence Microscope , Scanning Electron Microscope (SEM) Transmission Electron Microscope (TEM) Types of Compound Microscope : Types of Compound Microscope Monocular - restricted if you want to use a CCD camera because this would occupy the eyepiece. are light weight and are inexpensive . Binocular - 2 eyepieces which proves to be more comfortable. It is the most common choice. Trinocular - 3 eyepiece tube that can be used by another person simultaneously or by a CCD camera. is more expensive than the other two types. 18th-century microscopes : 18th-century microscopes lenses in eyeglasses in the 13th century : lenses in eyeglasses in the 13th century Definitions: Definitions Absorption When light passes through an object the intensity is reduced depending upon the color absorbed. Thus the selective absorption of white light produces colored ligh t. Refraction Direction change of a ray of light passing from one transparent medium to another with different optical density. A ray from less to more dense medium is bent perpendicular to the surface, with greater deviation for shorter wavelengths Diffraction Light rays bend around edges - new wavefronts are generated at sharp edges - the smaller the aperture the lower the diffraction Dispersion Separation of light into its constituent wavelengths when entering a transparent medium - prism or a rainbow Antony van Leeuwenhoek, Town clerk owner of a dry goods store in the city of Delft : Antony van Leeuwenhoek, Town clerk owner of a dry goods store in the city of Delft A. Visual acuity B. Resolving power C. Magnifcation D. Numerical aperture E. Image formation F. Working distance G. Calculation of total magnifcation Physical Basis of Microscopy LIMIT OF RESOLUTION OR RESOLVING POWER: LIMIT OF RESOLUTION OR RESOLVING POWER 2 closely placed dots as two separate dots. Resolving power human eye is 200 µm. compound microscope is 0.2 µm electron microscope is 1-10 nm. Resolution increases with the decreasing wavelength of light. Violet light offers more then red coloured Electron beams, which have very low wavelength offers maximum resolution. LR = 0.61 x wavelength of light Numerical aperture Numerical Aperture: Numerical Aperture Resolving power is directly related to numerical aperture. The higher the NA the greater the resolution Resolving power: The ability of an objective to resolve two distinct lines very close together NA = n sin θ (n=the lowest refractive index between the object and first objective element) (hopefully 1) θ is 1/2 the angular aperture of the objective Slide12: Refractive Index Objective n=1.52 n = 1.52 n = 1.52 Specimen Coverslip Oil n=1.33 n = 1.52 n = 1.0 n = 1.5 Water n=1.52 Air IMAGE FORMATION: IMAGE FORMATION PARTS: PARTS Base. It is a heavy metallic, U- or horseshoe-shaped base or foot ,. Pillars. Two upright pillars project up from the base and are attached to the C-shaped handle. The hinge joint tilted Handle (the arm or limb). The curved handle , supports focusing and magnifying systems Body Tube. Fitted at the upper end of the handle,either vertically or at an angle, through which light passes to eyepiece,. It is 16–17 cm in length, Fixed stage. Isquare platform with aperture in center , and fitted to pillars. Mechanical stage. It is a calibrated metal frame ftted on the right edge of the fxed stage . Slide16: N osepiece . at the lower end of the body tube and has two parts: fixed and revolving objective lenses. O bjectives , or;simply ‘lenses ’ 3-4 varying magnifying powers each has a cover glass covering. each lens numerical aperture (NA) rather than its focal length , are etched on each. The objective lenses are Low-power (LP) Objective (10 × ; NA = 0.25; focal length = 16 mm). Objectives: Objectives PLAN- APO -40X 1.30 N.A. 160/ 0.22 Flat field Apochromat Magnification Numerical Tube Coverglass Factor Aperture Length Thickness - Infinity corrected OBJECTIVES LENSES: OBJECTIVES LENSES 1. lenses designed to be parfocal , in focus . 2. characterized 2 parameters, magnification and numerical aperture . former 5× to 100× latter from 0.14 to 0.7, 3. F ocal lengths is 40 to 2 mm, 4.With higher magnifications normally have a higher NA and a shorter depth of field in the resulting image. . Slide19: Source of light. External light source. is a distant light Internal light source. electric lamp housed in a lamp box with a frosted glass window, to provide uniform white light . M irror . double-sided mirror, plane mirror is used with a distant source of light (natural, or daylight ).Concave mirror, when the light source is near microscope. I llumination system Slide20: of lenses fitted in a short cylinder helps in resolving the image . l ens system. 2 lenses which should be corrected for spherical and chromatic aberrations.NA equal objective lens. Iris diaphragm. small lever on the side can adjust aperture narrowing the aperture decreases the NA of the condenser. Filter . metal ring can accommodate a pale blue or green filter(monochromatic light) Condenser (‘ Substage ’ a system EYEPIECES APOCHROMATIC Ocular lens, is a cylinder containing two or more lenses Working Distance distance between(objective and the slide) 8–13mm LP, 1–3 mm HP, 0.5–1.5 mm OiI lenses Calculation of Total Magnifcation combination of objective lens and eyepiece. eyepiece of 10×, LP (10 ×) = 10 × 10 = 100 times. HP(45 ×) = 45 × = 450 times. Oil immersion objective (100 × ) = 100 × 10 = 1000 times. : EYEPIECES APOCHROMATIC Ocular lens, is a cylinder containing two or more lenses Working Distance distance between(objective and the slide) 8–13mm LP, 1–3 mm HP, 0.5–1.5 mm OiI lenses Calculation of Total Magnifcation combination of objective lens and eyepiece. eyepiece of 10×, LP (10 ×) = 10 × 10 = 100 times. HP(45 ×) = 45 × = 450 times. Oil immersion objective (100 × ) = 100 × 10 = 1000 times . PRECAUTIONS AND ROUTINE CARE: PRECAUTIONS AND ROUTINE CARE 1. Eyes are at a level slightly above eyepiece.. 2. Lenses are clean and free from dust and smudges. Do not touch with your fIngers , 3. Check the position of the objective, condenser, and diaphragm, to ensure optimal illumination . 4. Never lower any objective while looking into the microscope . 5. If the objectives are not parfocal , check the working distance of each objective separately by using fne focusing . 6.Remove oil with lens paper, then xylene to clean the lens.