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Optical Microscopes

Optical Microscopes. Justification History Components of the Optical Microscope Theory of operation Basic Microscope Magnification Refraction of light and Snell’s Law The thin lens equation. Imaging and resolution Confocal Microscopy Vendors. Outline. Why Optical Microscopes?.

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Optical Microscopes

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  1. Optical Microscopes FNI 2B OM

  2. Justification History Components of the Optical Microscope Theory of operation Basic Microscope Magnification Refraction of light and Snell’s Law The thin lens equation Imaging and resolution Confocal Microscopy Vendors Outline FNI 2B OM

  3. Why Optical Microscopes? • Still one of the most commonly used methods of inspection due to ease of use and sample preparation. • Often used to prepare samples for electron microscopy or scanning probe microscopy FNI 2B OM

  4. Labomed Stereo Zoom Scope Zoom Eye piece Focus Top Lamp Main power Top Illumination Power Bottom Lamp With removable glass cover Bottom Illumination Power FNI 2B OM

  5. Nikon Optiphot Future camera Bright field Dark field Eyepiece Lamp Nosepiece Use this to change the objectives never touch the actual objective lenses Coarse focus Objective Lenses Fine focus Stage Base FNI 2B OM

  6. Nikon Optiphot Apertures Beamsplitter Coarse Focus Fine Focus Stage X, Y Lamp Intensity Control FNI 2B OM

  7. Camera Used to capture images Zeiss Axiotron Objective lenses Used to change magnification Never touch these Eyepieces you look through these Fiber optics Light Source Be sure to turn this off when you are finished using it Brightfield Normal lighting Darkfield Indirect lighting Used to quickly lower the stage so the objectives do not become damaged Coarse Focus Fine Focus FNI 2B OM

  8. Zeiss Axiotron Beam splitter For camera Motorized objective Always use these buttons to change objectives Always use the front button to increase the magnification and the back button to decrease magnification. Otherwise you may damage the objective lens Vacuum Chuck Not connected to vacuum Stage X and Y Stage Rotation FNI 2B OM Stage handle

  9. Filters Yellow filter should be used for viewing photoresist The other filter can be used to enhance contrast Zeiss Axiotron Aperture Adjust so it is just beyond the field of view Polarizer Can be used to enhance contrast Optional filters Not used FNI 2B OM

  10. Magnification • Objective lenses up to 100x or 150x • Eyepieces 10x – 15x • Magnification = OL x EP • The limit of optical magnification is about 2000x. FNI 2B OM

  11. θt θi This part slows down first Refractive Index Waves do not have the same speed in all materials. The speed of light in vacuum is c=2.998 x 108 m/s. The speed of light in glass is slower, only about v=2.000 x 108 m/s The refractive index is the ratio of these two speeds FNI 2B OM

  12. c v θt θt θi θi This part slows down first Refraction and Snell’s Law Snell’s law describes refraction of sound ( a change in direction as it passes through a boundary) If the incident angle is not zero and the velocities are not equal. http://hyperphysics.phy-astr.gsu.edu/hbase/geoopt/refr.html FNI 2B OM

  13. c v θt θi Snell’s Law A light wave traveling through a air with propagation speed 2.998 x 108 m/s passes into water with propagation speed 2.308 x 108 m/s with an incident angle of 50˚. What is the angle of transmittance? FNI 2B OM

  14. The focal point of a lens A Convex Lens FNI 2B OM

  15. f i o f Theory of Operation f is focal point distance o is object distance i is image distance M is magnification FNI 2B OM

  16. Thin Lens Calculations If a lens has a 4.5 cm focal length and a specimen is 5.3 cm from it where will the image appear to be and what will be the magnification? FNI 2B OM

  17. Imaging • Light field • Dark field • Resolution • Depth of Field • Polarizing filters, Nomarski • Yellow Filters • Apertures • Beam splitter • Digital image capture • Limits of resolution FNI 2B OM

  18. Sample Preparation Biological sample may be placed on a glass slide with a cover slip. Various stains can be used to highlight different parts of the sample. When gold particles are small enough they no longer appear as yellow shiny metal but can have a variety of colors which depends on their size. These are called quantum dots. These can be combined with antibodies and used to stain different specific proteins different vibrant colors. http://www.invitrogen.com/site/us/en/home/Products-and-Services/Applications/Cell-and-Tissue-Analysis/qdots-microspheres-nanospheres.html FNI 2B OM

  19. Inspection The optical microscope is the most common method of inspection because they are relatively easy to use and easy to prepare samples for. They are also commonly used when preparing samples for the electron and scanning probe microscopes. FNI 2B OM

  20. Confocal Microscope FNI 2B OM

  21. Optical Microscope Vendors • Olympus www.olympus.com • Zeiss www.zeiss.com/micro • Nikon www.nikonusa.com • Leica www.leica-microsystems.com • Microscope U http://www.microscopyu.com/ FNI 2B OM

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