Practical Microscopy

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Practical Microscopy

• Dark Field Microscopes
• Live Blood Microscopy

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Microscopes TH1 Disease

• Wirotsko, Wright, Cantwell, Mattman all published
  images of microbes in blood and tissues of TH1
  patients.
• Amateur MP patient James Sanders has also
  demonstrated impressive dark field images of
  suspicious objects in TH1 disease blood.

Simple, inexpensive light microscopes may be a
key to persuading medicine that CWD microbes
cause TH1 disease
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TH1 Patient Blood
Courtesy of James Sanders
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Lyme Patient
Courtesy of James Sanders
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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.

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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.

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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)

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Light Energy

• Short Wavelengths have greater energy
• Long Wavelengths have lesser energy

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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.

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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.

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Another Look at the Ray Trace
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Some Compound Microscopes
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Modern Research Grade Microscope has those same
essential elements plus a few other features
US15,000 to US30,000
Image courtesy www.molecularexpressions.com
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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.

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Low Cost Chinese, Dark Field Microscope

• This new Chinese microscope is listed on eBay for
  US1,200.00 (buy now category) and purports to be
  a darkfield Live Blood microscope.
• The specs look quite reasonable.
• The Vendor is bargainmicroscopes

eBay Buy now at US1,200
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Another Chinese Microscope

• Same supplier, Mr. Michael Le.
• This one includes darkfield phase contrast,
  plus camera Monitor.
• Listed at 3,500.

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Darkfield Illumination

• Darkfield microscopy isnt new.
• All of the earliest microscopes were darkfield
  because they hadnt mastered brightfield
  microscopy yet.
• It is a contrast enhancement technique.
• It does not offer finer resolution, but for some
  specimens it offers better contrast.

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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.

image from www. microscopy-uk.org.uk
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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
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Compare Brightfield and Darkfield
image from www. microscopy-uk.org.uk
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Metallurgical Bright Dark Field Images
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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.

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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.

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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
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Lower Cost Abbe Condensers

• B1 Brightfield condenser
• B2 Darkfield Dry
• B3 B4 Immersed Darkfield
• Condenser under stage in lower photo
• Guangdongoptics.com

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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.

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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.

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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.

Lens
?
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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 NAs are more
  expensive and better.

Large (Fast) NA
Small (Slow) NA
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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.

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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
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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.

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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.

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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.

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Objective Lens Price Performance
Achromats Most common Lowest price Poorly corrected, bad for demanding applications.
Fluorites or Semi Plan Apochromats Mid-grade lenses, better correction, flat field.
Plan Apochromats Best grade, most expensive (gt3,000 for some) , very well corrected.
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Other Contrast Techniques
Differential Interference Contrast
Phase Contrast
http//www.molecularexpressions.com/primer/techniq
ues/dic/dicphasecomparison.html
These Items would likely be invisible without PC
or DIC
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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.

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Phase Contrast

• 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
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Differential Interference Contrast

• DIC produces a more striking image but the DIC
  optics are more complicated and more expensive.

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Fluorescence Microscopy

• a) phase contrast b) fluorescence c) combined
• (www.molecularexpressions.com)

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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.

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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/i
  ndex.html
• Olympusmicro.com has this how-to for setting up
  your darkfield condenser
• http//tinyurl.com/h7fqt

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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