Practical Microscopy Dark Field Microscopes Live Blood Microscopy

Practical Microscopy Dark Field Microscopes Live Blood Microscopy 17 June 2006 Aids TH 1 - Mc. Donald 1

Microscopes & TH 1 Disease n n Wirotsko, Wright, Cantwell, Mattman all published images of microbes in blood and tissues of TH 1 patients. Amateur MP patient James Sanders has also demonstrated impressive dark field images of suspicious objects in TH 1 disease blood. 17 June 2006 Aids TH 1 - Mc. Donald Simple, inexpensive light microscopes may be a key to persuading medicine that CWD microbes cause TH 1 disease 2

TH 1 Patient Blood Courtesy of James Sanders 17 June 2006 Aids TH 1 - Mc. Donald 3

Lyme Patient Courtesy of James Sanders 17 June 2006 Aids TH 1 - Mc. Donald 4

Your Presenter n n n ~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. n n 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. 17 June 2006 Aids TH 1 - Mc. Donald 5

Goals n n n To Describe Practical Requirements for fielding a Microscope that may be useful for seeing TH 1 microbes. To Suggest Configurations and Price if purchased from Olympus, Zeiss or on e. Bay. 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. 17 June 2006 Aids TH 1 - Mc. Donald 6

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

Light & Energy n n Short Wavelengths have greater energy Long Wavelengths have lesser energy 17 June 2006 Aids TH 1 - Mc. Donald 8

Simplest Possible Microscopes n n 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. 17 June 2006 Aids TH 1 - Mc. Donald 9

Compound Microscope n 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. 17 June 2006 Aids TH 1 - Mc. Donald 10

Another Look at the Ray Trace 17 June 2006 Aids TH 1 - Mc. Donald 11

Some Compound Microscopes 17 June 2006 Aids TH 1 - Mc. Donald 12

Modern Research Grade Microscope has those same essential elements plus a few other features 17 June 2006 US$15, 000 to US$30, 000 Image courtesy www. molecularexpressions. com Aids TH 1 - Mc. Donald 13

Microscopes for Semiconductor Labs n 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. 17 June 2006 Aids TH 1 - Mc. Donald 14

Low Cost Chinese, Dark Field Microscope n n n This new Chinese microscope is listed on e. Bay 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 17 June 2006 Aids TH 1 - Mc. Donald e. Bay Buy now at US$1, 200 15

Another Chinese Microscope n n n Same supplier, Mr. Michael Le. This one includes darkfield & phase contrast, plus camera & Monitor. Listed at $3, 500. 17 June 2006 Aids TH 1 - Mc. Donald 16

Darkfield Illumination Darkfield microscopy isn’t new. n All of the earliest microscopes were darkfield because they hadn’t mastered brightfield microscopy yet. n It is a contrast enhancement technique. n It does not offer finer resolution, but for some specimens it offers better contrast. n 17 June 2006 Aids TH 1 - Mc. Donald 17

Brightfield Illumination • In Brightfield Illumination, light from inside the Field floods the objective, making the field bright. • Objects deflect light out of the field, making the objects dark against the bright background. 17 June 2006 image from www. microscopy-uk. org. uk Aids TH 1 - Mc. Donald 18

Darkfield Illumination • In Darkfield Illumination, light from outside the Field, does not normally enter the objective, making the field dark. • Light striking objects is displaced into the objective. • Objects appear bright against dark background. image from www. microscopy-uk. org. uk 17 June 2006 Aids TH 1 - Mc. Donald 19

Compare Brightfield and Darkfield image from www. microscopy-uk. org. uk 17 June 2006 Aids TH 1 - Mc. Donald 20

Metallurgical Bright & Dark Field Images 17 June 2006 Aids TH 1 - Mc. Donald 21

Why Darkfield? n n n 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”. 17 June 2006 Aids TH 1 - Mc. Donald 22

Epi Darkfield Requires as little as a Desk Lamp n 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. 17 June 2006 Aids TH 1 - Mc. Donald 23

More Elaborate means are required for Transmitted Darkfield This very high end cardioid darkfield condenser creates a hollow cone of upward directed light that does not enter the objective unless deflected by the sample. www. olympus-micro. com 17 June 2006 Aids TH 1 - Mc. Donald 24

Lower Cost Abbe Condensers B 1 Brightfield condenser n B 2 Darkfield Dry n B 3 & B 4 Immersed Darkfield n n Condenser under stage in lower photo n Guangdongoptics. com 17 June 2006 Aids TH 1 - Mc. Donald 25

Darkfield Cone of light entering the objective n n n 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. 17 June 2006 Aids TH 1 - Mc. Donald 26

Choosing Darkfield Condensers Cardioid Darkfield Condenser for expensive Research Grade Microscopes. n Abbe Darkfield Condenser also quite good and more affordable. n Choose a condenser that can be “oiled” to the bottom of the slide. n Condenser must have an NA greater than that stamped on the objective. n 17 June 2006 Aids TH 1 - Mc. Donald 27

Definition of Numerical Aperture n n 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. 17 June 2006 Aids TH 1 - Mc. Donald Lens 28

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. Large (Fast) NA Small (Slow) NA 17 June 2006 Aids TH 1 - Mc. Donald 29

Numerical Aperture Is Stamped on the Lens & Governs the Lens Performance & Cost n High NA = Best Resolution n High NA = Best light gathering power of the lens. n High NA = Difficult to design and manufacture and expensive. n Buy the highest NA you can afford. 17 June 2006 Aids TH 1 - Mc. Donald 30

Rayleigh & Sparrow Resolution Criteria depend on NA & Rayleigh (better known) Sparrow (more accurate) Minimum Separation to resolve point sources Spatial Resolution does not depend on Magnification 17 June 2006 Aids TH 1 - Mc. Donald 31

About Resolution n 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. 17 June 2006 Aids TH 1 - Mc. Donald 32

Things to look for in Objective Lenses n n n 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. 17 June 2006 Aids TH 1 - Mc. Donald 33

Good Objectives are Expensive n n 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. 17 June 2006 Aids TH 1 - Mc. Donald 34

Objective Lens Price & Performance Achromats Fluorites or Semi Plan Apochromats 17 June 2006 Most common Lowest price Poorly corrected, bad for demanding applications. Mid-grade lenses, better correction, flat field. Best grade, most expensive (>$3, 000 for some) , very well corrected. Aids TH 1 - Mc. Donald 35

Other Contrast Techniques Differential Interference Contrast Phase Contrast http: //www. molecularexpressions. com/primer/techniques/dicphasecomparison. html These Items would likely be invisible without PC or DIC 17 June 2006 Aids TH 1 - Mc. Donald 36

Phase Contrast & DIC n n n 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. 17 June 2006 Aids TH 1 - Mc. Donald 37

Phase Contrast n Phase Contrast uses complementary pair of annuli, in the condenser and in the back of the objectives. http: //www. microscopyu. com/articles/phasecontrast/phasemicroscopy. html 17 June 2006 Aids TH 1 - Mc. Donald 38

Differential Interference Contrast n DIC produces a more striking image but the DIC optics are more complicated and more expensive. 17 June 2006 Aids TH 1 - Mc. Donald 39

Fluorescence Microscopy a) phase contrast b) fluorescence c) combined (www. molecularexpressions. com) 17 June 2006 Aids TH 1 - Mc. Donald 40

Fluorescence Technology n n n 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. 17 June 2006 Aids TH 1 - Mc. Donald 41

Setting up the Microscope n Molecularexpressions. com has an excellent ‘how-to’ description for applying oil to the condenser and objective on this page (page bottom): n http: //tinyurl. com/zltga n Setting up Koehler illumination (very important). n n n 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: n http: //tinyurl. com/h 7 fqt 17 June 2006 Aids TH 1 - Mc. Donald 42

More Information n There are fantastic web resources: n Best microscopy site on the WWW: www. molecularexpressions. com n http: //www. olympusmicro. com/primer/index. html http: //www. microscopyu. com/ n n Printed Photography through the Microscope, Kodak Books (my favorite, short but excellent plus great photos) n Video Microscopy by Shinya Inoue (comprehensive) n 40 page PDF primer on Microscopy http: //www. olympusmicro. com/primer/microscopy. pdf n 17 June 2006 Aids TH 1 - Mc. Donald 43