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practical microscopy

17 June 2006. 2. Aids TH1 - McDonald. Microscopes

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practical microscopy

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    1. 17 June 2006 Aids TH1 - McDonaldMcDonald 1 Practical Microscopy Dark Field Microscopes Live Blood Microscopy

    2. 17 June 2006 2 Aids TH1 - McDonald

    3. 17 June 2006 3 Aids TH1 - McDonald TH1 Patient Blood

    4. 17 June 2006 4 Aids TH1 - McDonald Lyme Patient

    5. 17 June 2006 5 Aids TH1 - McDonald Your Presenter ~25 years in optical instrument design. Around 12 or 15 years of microscope design, mostly exotic models for semiconductor applications. I am not a biologist. I am an expert on the design and function of microscopes including brightfield, darkfield, phase contrast, fluorescence, etc, but I cannot interpret biological images or comment much on the preparation of specimens or slides. I am on the MP since Sept. 2005 for rheumatoid arthritis.

    6. 17 June 2006 6 Aids TH1 - McDonald Goals To Describe Practical Requirements for fielding a Microscope that may be useful for seeing TH1 microbes. To Suggest Configurations and Price if purchased from Olympus, Zeiss or on eBay. To discuss practical aspects of darkfield, phase contrast & fluorescence methods. To discuss resolution limits and other microscopy concepts, e.g., “How small are these things?” My goal is to equip you or your doctor to buy and use no more than the microscope that you need following the examples of Wright and Sanders.

    7. 17 June 2006 7 Aids TH1 - McDonald Presentation Content (43 slides) Light and Energy (1 slide) Simple Microscope (1 slide) Compound Microscopes (4 slides) Cutaway high end Microscope (1 slide) Low Cost Chinese Microscopes (2 slides) Darkfield Information (11 slides) N.A., Resolution, Lenses (8 slides) Other Contrast Methods (6 slides) Other Resources (2 slides)

    8. 17 June 2006 8 Aids TH1 - McDonald Light & Energy Short Wavelengths have greater energy Long Wavelengths have lesser energy

    9. 17 June 2006 9 Aids TH1 - McDonald Simplest Possible Microscopes Single lens magnifier makes the image appear larger. Our brain processes the light as though coming in a straight line so the image appears larger.

    10. 17 June 2006 10 Aids TH1 - McDonald Compound Microscope In a compound microscope the first lens projects a magnified image of the object, and the second lens is like a hand lens, it magnifies the projected image.

    11. 17 June 2006 11 Aids TH1 - McDonald Another Look at the Ray Trace

    12. 17 June 2006 12 Aids TH1 - McDonald Some Compound Microscopes

    13. 17 June 2006 13 Aids TH1 - McDonald Modern Research Grade Microscope has those same essential elements plus a few other features

    14. 17 June 2006 14 Aids TH1 - McDonald Microscopes for Semiconductor Labs These are a couple of the microscopes that I design and peddle to the semiconductor industry for failure analysis. They work pretty much the same way as the preceding microscopes but with much more expensive detectors.

    15. 17 June 2006 15 Aids TH1 - McDonald Low Cost Chinese, Dark Field Microscope This new Chinese microscope is listed on eBay for US$1,200.00 (buy now category) and purports to be a darkfield “Live Blood” microscope. The specs look quite reasonable. The Vendor is bargainmicroscopes

    16. 17 June 2006 16 Aids TH1 - McDonald Another Chinese Microscope Same supplier, Mr. Michael Le. This one includes darkfield & phase contrast, plus camera & Monitor. Listed at $3,500.

    17. 17 June 2006 17 Aids TH1 - McDonald Darkfield Illumination Darkfield microscopy isn’t new. All of the earliest microscopes were darkfield because they hadn’t mastered brightfield microscopy yet. It is a contrast enhancement technique. It does not offer finer resolution, but for some specimens it offers better contrast.

    18. 17 June 2006 18 Aids TH1 - McDonald Brightfield Illumination

    19. 17 June 2006 19 Aids TH1 - McDonald Darkfield Illumination

    20. 17 June 2006 20 Aids TH1 - McDonald Compare Brightfield and Darkfield

    21. 17 June 2006 21 Aids TH1 - McDonald Metallurgical Bright & Dark Field Images

    22. 17 June 2006 22 Aids TH1 - McDonald Why Darkfield? Biological specimens are often watery materials held together by transparent gels and suspended in water. It is like the grade school joke about seeing a polar bear in a snow storm, nothing stands out. However some specimens show up well with darkfield illumination. Arguably, removing the blinding glare of the brightfield light probably allows such contrast as there is to be seen. Other contrast techniques are also useful such as “Phase Contrast”, “DIC”, “Fluorescence” or “Killing and Staining”.

    23. 17 June 2006 23 Aids TH1 - McDonald Epi Darkfield Requires as little as a Desk Lamp In Dark Field illumination the light must come from outside the objective. For top lit specimens at low magnification I have used a simple desk lamp.

    24. 17 June 2006 24 Aids TH1 - McDonald More Elaborate means are required for Transmitted Darkfield

    25. 17 June 2006 25 Aids TH1 - McDonald Lower Cost Abbe Condensers B1 Brightfield condenser B2 Darkfield Dry B3 & B4 Immersed Darkfield Condenser under stage in lower photo Guangdongoptics.com

    26. 17 June 2006 26 Aids TH1 - McDonald Darkfield Cone of light entering the objective So a darkfield microscope must have a special condenser. The collection cone of the objective must be the same size or smaller than the hollow cone shape dark output from the condenser so that no light enters the objective unless deflected by the specimen. These input and output cones of light are known as the numerical apertures of the lens and condenser.

    27. 17 June 2006 27 Aids TH1 - McDonald Choosing Darkfield Condensers Cardioid Darkfield Condenser for expensive Research Grade Microscopes. Abbe Darkfield Condenser also quite good and more affordable. Choose a condenser that can be “oiled” to the bottom of the slide. Condenser must have an NA greater than that stamped on the objective.

    28. 17 June 2006 28 Aids TH1 - McDonald Definition of Numerical Aperture Numerical Aperture (NA) is the sine of the collection half angle, multiplied by the index of the intervening medium. NA is the size of the blue cone.

    29. 17 June 2006 29 Aids TH1 - McDonald Numerical Aperture The Numerical Aperture has everything to do with how well the lens performs. This number is stamped on the side of the objective and ranges from about 0.1 to 1.4. Larger NA’s are more expensive and better.

    30. 17 June 2006 30 Aids TH1 - McDonald Numerical Aperture Is Stamped on the Lens & Governs the Lens Performance & Cost High NA = Best Resolution High NA = Best light gathering power of the lens. High NA = Difficult to design and manufacture and expensive. Buy the highest NA you can afford.

    31. 17 June 2006 31 Aids TH1 - McDonald Rayleigh & Sparrow Resolution Criteria depend on NA & ?

    32. 17 June 2006 32 Aids TH1 - McDonald About Resolution Using the Sparrow formula, a visible light microscope with an oil immersion lens can just resolve about 0.18 microns. A human hair is about 100 microns. The darkfield technique limits the NA to about 1.25, so the finest resolution on a good day for a dark field microscope is about a fifth of a micron, (0.2 microns). Several web sites suggest that red blood cells are 6 to 8 microns in diameter. That should put the mobile dots and pearl stringers at about a third to a half a micron diameter.

    33. 17 June 2006 33 Aids TH1 - McDonald Things to look for in Objective Lenses For high resolution live blood work, you need: An oil immersion objective, NA about 1.25. An oil immersion condenser, NA about 1.4. Buy lenses that are marked Plan Apo meaning they have excellent aberration correction and a flat field. If budget is an issue, Apochromats or Fluoride lenses may work, and they are cheaper, but try before you buy.

    34. 17 June 2006 34 Aids TH1 - McDonald Good Objectives are Expensive Recall our simple 2 lens compound microscope? A high quality objective is full of glass, and may take a year for an engineer to design and great care to manufacture.

    35. 17 June 2006 35 Aids TH1 - McDonald Objective Lens Price & Performance

    36. 17 June 2006 36 Aids TH1 - McDonald Other Contrast Techniques

    37. 17 June 2006 37 Aids TH1 - McDonald Phase Contrast & DIC The amplitude of light may not change in transparent materials but light does slow down in these materials. Differential Interference optics use polarization and prisms to make a more dramatic effect. Phase Contrast uses special apertures and a retarding glass plate to make these differences visible.

    38. 17 June 2006 38 Aids TH1 - McDonald Phase Contrast Phase Contrast uses complementary pair of annuli, in the condenser and in the back of the objectives.

    39. 17 June 2006 39 Aids TH1 - McDonald Differential Interference Contrast DIC produces a more striking image but the DIC optics are more complicated and more expensive.

    40. 17 June 2006 40 Aids TH1 - McDonald Fluorescence Microscopy a) phase contrast b) fluorescence c) combined (www.molecularexpressions.com)

    41. 17 June 2006 41 Aids TH1 - McDonald Fluorescence Technology Specimens are ‘tagged’ with fluorescing chemical. The chemicals only tag specific targets, e.g. a particular antibody or virus. The specimen is illuminated with UV light. The fluorescing chemical responds by emitting a longer wavelength visible light (like the fluorescent lights in this room). The image seems to float in a black field. The technique only works if a fluorophore has been developed ($$$) for the target specimen.

    42. 17 June 2006 42 Aids TH1 - McDonald Setting up the Microscope Molecularexpressions.com has an excellent ‘how-to’ description for applying oil to the condenser and objective on this page (page bottom): http://tinyurl.com/zltga Setting up Koehler illumination (very important). http://tinyurl.com/qxlvg http://www.microscopyu.com/tutorials/java/kohler/index.html Olympusmicro.com has this ‘how-to’ for setting up your darkfield condenser: http://tinyurl.com/h7fqt

    43. 17 June 2006 43 Aids TH1 - McDonald More Information There are fantastic web resources: Best microscopy site on the WWW: www.molecularexpressions.com http://www.olympusmicro.com/primer/index.html http://www.microscopyu.com/ Printed Photography through the Microscope, Kodak Books (my favorite, short but excellent plus great photos) Video Microscopy by Shinya Inoue (comprehensive) 40 page PDF primer on Microscopy http://www.olympusmicro.com/primer/microscopy.pdf

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