Research Instruments Microscopy Workshop - Olympus


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RI have been working with and supporting IVF clinics set up and operate serveral makes and models of microscopes for general lab use. This session was run in association with Olympus, Nikon and Leica.

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Research Instruments Microscopy Workshop - Olympus

  1. 1. Microscopy Workshop University Hospital of Wales, Cardiff Wales Heart Research Institute 10 November, 2011 Adam Westmacott
  2. 2. Introduction • Light travels in straight lines • In the form of a Sine wave
  3. 3. Introduction • There are only 2 things that the eye can see • Colour-Represented by Wavelength
  4. 4. Introduction • Intensity-Represented by Amplitude • So we can only see our specimen if it has a different Colour or Intensity than the background
  5. 5. Jargon • Resolution- Resolution can be defined as the least distance between 2 points at which they can still be recognised as 2 separate entities • For the eye, this is 70 microns, when the object is 250mm away • For light microscopy, this is 0.24 microns • Contrast-The phenomena that allows you to distinguish relevant information from irrelevant. Either by colour or intensity • Contrast and Resolution are inversely proportional
  6. 6. Jargon • Working Distance- The distance between the specimen and the front lens of the objective • Depth of Field –Depth of field in a microscope is the area in front of and behind the specimen that will be in acceptable focus • Field of View- This is the area of the specimen in view down the eyepieces. It is dependant on the magnification and the F.N.( field number) of the eyepiece.
  7. 7. Jargon • Field of View-A F.N. of 22 indicates a F.O.V. diameter of 22mm when using a 1x objective. F.O.V. when using 20x objective = 1.1mm F.O.V. when using 100x objective = 0.22mm • Numerical Aperture- This indicates the resolving ability of an objective. Larger N.A.= Greater resolution and also brighter fluorescence signal. However larger N.A.= less depth of field and shorter working distance. NA= nSinA, where n= refractive index of medium and A is the half angle at which light enters the objective.
  8. 8. Jargon • Chromatic Aberration- When white light passes through a convex lens, the colours split and focus at different points causing colour fringing. Objectives have additional elements to overcome this problem. • Apochromatic objectives are fully corrected and Achromatic objectives are corrected for red/blue. • Plan objectives are designed, assuming a flat specimen, to provide a focused image across the whole field of view
  9. 9. Contrast Techniques • • • • • • Brightfield Darkfield Phase Contrast Differential Interference Contrast Hoffman Modulation Contrast Fluorescence
  10. 10. Contrast-Enhancing Techniques Specimen Type Imaging Technique TRANSMITTED LIGHT Transparent Specimens Phase Objects Bacteria, Spermatozoa, Cells in Glass Containers, Protozoa, Mites, Fibers, etc. Phase Contrast Differential Interference Contrast (DIC) Hoffman Modulation Contrast Oblique Illumination Light Scattering Objects Diatoms, Fibers, Hairs, Fresh Water Microorganisms, Radiolarians, etc. Rheinberg Illumination Darkfield Illumination Phase Contrast and DIC Light Refracting Specimens Colloidal Suspensions powders and minerals Liquids Phase Contrast Dispersion Staining DIC Amplitude Specimens Stained Tissue Naturally Colored Specimens Hair and Fibers Insects and Marine Algae Brightfield Illumination Fluorescent Specimens Cells in Tissue Culture Fluorochrome-Stained Sections Smears and Spreads Fluorescence Illumination Birefringent Specimens Mineral Thin Sections Liquid Crystals Melted and Recrystallized Chemicals Hairs and Fibers Bones and Feathers Polarized Illumination
  11. 11. Microscope • 3 different types • Stereo • Inverted Compound • Upright Compound
  12. 12. Stereo Microscopes • 2 separate optical paths at an angle to each other • Brain merges the 2 images to give a 3D image • Magnification range 4x- 200x • Magnification should not exceed 1000x NA • 2 principal types
  13. 13. Greenhough Optics 10 Degree Converging light path Great depth of focus
  14. 14. Galilean Optics Parallel Optics Allows build up of other accessories
  15. 15. Stereo Microscopes • Variety of Illumination options • Transmitted Brightfield, Darkfield, Oblique and Polarised Light • Fluorescence • Reflected through the optics or by separate source • Fibre optic goose neck or ring lights
  16. 16. Inverted Microscopes • Predominately used for looking at specimens in suspension- Live cells • Long working distance optics • Whole variety of contrast techniques • Environmental and temperature control • Sample Manipulation/Injection • Magnifications 40x – 1000x
  17. 17. Inverted Microscopes
  18. 18. Inverted Microscopes Optical Workbenches
  19. 19. Upright Microscopes • Samples on glass slides • Variety of Contrast techniques • Magnification range 10x – 1000x
  20. 20. Upright Microscopes Fully Motorised and computer controlled with advanced imaging system Student Teaching Microscope
  21. 21. The MicroscopeWhat is Important • Location • Avoid direct lighting - not next to a window. • Comfortable working area • Adjustable chair • Isolate external vibration
  22. 22. The MicroscopeIllumination • Ensure that the illumination is matched to the application • 6v 30W, 12v 100W and now true colour LED • Fluorescence- HG, XE, Metal Halide or LED
  23. 23. Condensers • The condenser presents the light for the objective to do its work • Remember that a compromise is just that • A swing out condenser will not give as even an illumination at low power as a Ultra Low Condenser. You may not see it, but the camera will. Make sure software has shading correction.
  24. 24. Condensers
  25. 25. Microscopy Workshop Micro_Series_Part_5.pdf Thank You