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Tutorial on Microscopy September 15, 2007

Tutorial on Microscopy September 15, 2007. Why the need to study microscopy ? It is a tool complementary to molecular biology It has become an indispensable tool for many biologists and pathologists to check sterility of cultures to study the histology of biopsies

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Tutorial on Microscopy September 15, 2007

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  1. Tutorial on Microscopy September 15, 2007

  2. Why the need to study microscopy? • It is a tool complementary to molecular biology • It has become an indispensable tool for many • biologists and pathologists • to check sterility of cultures • to study the histology of biopsies • to study the developmental program of organ • to follow the movement of a protein from • the cytoplasm to the chloroplast

  3. Students need to understand the microscope • to get a sense of size, • to get the best image possible, • to learn how to enjoy using it, • to use it for cool and fun purposes, • to keep it in good shape, • to be able to share the instrument with others.

  4. MOTIC ZEISS

  5. Eye-piece or ocular Revolving nose with objectives

  6. Eye-piece or ocular Revolving nose with objectives Stage and its controls Focussing knob

  7. Eye-piece or ocular Revolving nose with objectives Stage and its controls Condenser Light source Focussing knob

  8. Light and lenses are the two pieces of equipment which are used to manipulate the light. Both are inherent to the microscope you used; they cannot be changed.

  9. The lenses: three types in your microscope • The condenser • The objective • The eye-piece or ocular

  10. The lenses: three types in your microscope • The condenser • The objective • The eye-piece or ocular

  11. The condenser: a combination of lenses http://micro.magnet.fsu.edu/primer/anatomy/condensers.html

  12. The condenser: a combination of lenses • The simplest condenser • Role: to condense and • focus light onto the • specimen Also called an iris diaphragm Allows more or less light to enter the condenser http://micro.magnet.fsu.edu/primer/anatomy/condensers.html

  13. The condenser: a combination of lenses • Will possess specific • characteristics (correction, • numerical aperture and • others) depending on • manufacturer specifications http://www.zeiss.com/C1256B5E0047FF3F?Open

  14. The lenses: three types in your microscope • The condenser • The objective • The eye-piece or ocular

  15. The objective: a combination of lenses http://micro.magnet.fsu.edu/primer/

  16. The objective: a combination of lenses Motic Zeiss http://micro.magnet.fsu.edu/primer/

  17. The objective: a combination of lenses Motic Zeiss Plan CP-Achromat http://micro.magnet.fsu.edu/primer/

  18. Achromat: Good color correction – exactly for two wavelengths. Field flatness in the image center, refocusing also covers the peripheral areas. Designed for fields of view up to 18 mm diameter. Versions for phase contrast. Budget-priced objectives. Names: CP-Achromat (CP: Clinical Plan) and Achrostigmatism. http://www.zeiss.com/C1256B5E0047FF3F?Open

  19. The effect of chromatic aberration Rays of longer λ focus further away that those of shorter λ. http://micro.magnet.fsu.edu/primer/

  20. The effect of chromatic aberration Rays of longer λ focus further away that those of shorter λ. Rays of longer λ focus further away that those of shorter λ. http://micro.magnet.fsu.edu/primer/ http://micro.magnet.fsu.edu/primer/

  21. Motic Plan and Epiplan:Improved Achromat objectives with good image flatness for fields of view with dia. 20 or even 23 mm. Therefore ideal for photomicrography. Zeiss Achromat: Good color correction – exactly for two wavelengths. Field flatness in the image center, refocusing also covers the peripheral areas. Designed for fields of view up to 18 mm diameter. Versions for phase contrast. Budget-priced objectives. Names: CP-Achromat (CP: Clinical Plan) and Achrostigmatism. http://www.zeiss.com/C1256B5E0047FF3F?Open

  22. Field curvature: the sharpest focus of a lens is on a curved surface rather than on a flat plane. Plant microtechnique and microscopy. E. Ruzin

  23. http://micro.magnet.fsu.edu/primer/java/aberrations/curvatureoffieldhttp://micro.magnet.fsu.edu/primer/java/aberrations/curvatureoffield

  24. http://micro.magnet.fsu.edu/primer/java/aberrations/curvatureoffieldhttp://micro.magnet.fsu.edu/primer/java/aberrations/curvatureoffield

  25. The objective: a combination of lenses Motic Zeiss Plan CP-Achromat 4x / 0.10 10x / 0.25 40x / 0.65 Magnification / Numerical Aperture http://micro.magnet.fsu.edu/primer/

  26. The objective: a combination of lenses Motic Zeiss Plan CP-Achromat 5x / 0.12 10x / 0.25 40x / 0.65 100x / 1.25 oil 4x / 0.10 10x / 0.25 40x / 0.65 Magnification / Numerical Aperture http://micro.magnet.fsu.edu/primer/

  27. The objective: a combination of lenses Motic Zeiss Plan CP-Achromat 5x / 0.12 10x / 0.25 40x / 0.65 100x / 1.25 oil 4x / 0.10 10x / 0.25 40x / 0.65 http://micro.magnet.fsu.edu/primer/

  28. The objective: a combination of lenses Motic Zeiss Plan CP-Achromat 5x / 0.12 10x / 0.25 40x / 0.65 100x / 1.25 oil 4x / 0.10 10x / 0.25 40x / 0.65 ∞ / 0.17 ∞ / - ∞ / 0.17 http://micro.magnet.fsu.edu/primer/

  29. All the objectives mentioned here are members of the family of ICS-Optics (ICS: Infinity Color-corrected System). These objectives project their images to “infinity” first. Only the tube lens produces an intermediate image – to be more precise, at a distance of approx. 164.5 mm behind the tube lens. This distance was chosen to comply with the classical tube length.

  30. The objective: a combination of lenses Motic Zeiss Plan CP-Achromat 5x / 0.12 10x / 0.25 40x / 0.65 100x / 1.25 oil 4x / 0.10 10x / 0.25 40x / 0.65 ∞ / 0.17 ∞ / - ∞ / 0.17 http://micro.magnet.fsu.edu/primer/

  31. A coverslip is • part of the image-forming system, • a lens element, • its power has been taken into account by the manufacturer, • its thickness and the making of the glass will affect the • deviation of the light.

  32. Klosevych, 1989 The thickness of the coverslip and the refractive index of the glass will have an effect of the light path 1 thickness= 0.13 to 0.17 mm Satisfactory for NA ≤ 0.4.

  33. The objective: a combination of lenses Motic Zeiss Plan CP-Achromat 5x / 0.12 10x / 0.25 40x / 0.65 100x / 1.25 oil 4x / 0.10 10x / 0.25 40x / 0.65 ∞ / 0.17 ∞ / - ∞ / 0.17 Any questions? http://micro.magnet.fsu.edu/primer/

  34. The lenses: three types in your microscope • The condenser • The objective • The eye-piece or ocular

  35. http://micro.magnet.fsu.edu/primer/anatomy/oculars.html Eyepieces are not just simple lenses, but are corrected optical systems consisting of several lenses.

  36. http://www.zeiss.com/C1256B5E0047FF3F?Open 1. Position of the intermediate image (also for the reticle) 2. Limit of the field of view (black edge of image) 3. Eye-piece optics (Ramsden ocular) 4. Position of the eyepiece pupil (pupil of the observer’s eye) 5. Focusing ring for the diopter compensation Eyepieces: magnifiers to view the intermediate Image produced by the objective and the tube lens.

  37. The lenses: three types in your microscope • The condenser • The objective • The eye-piece or ocular There will be specifications on these lenses WF for Wide Field of view PL to match the objective correction Magnification 10X Field number which refers to the diameter (in mm) of the fixed diaphragm in the eyepiece. Motic WFPL 10x / 20 Glasses symbol

  38. The lenses: three types in your microscope • The condenser • The objective • The eye-piece or ocular There will be specifications on these lenses Zeiss PL 10x / 18 Glasses symbol Designed for eyeglass wearers. The exit pupil is at a considerable distance from the eyepiece.

  39. Final image Intermediate image Specimen

  40. What is the main purpose of the microscope? • The condenser • The objective • The eye-piece or ocular

  41. What is the main purpose of the microscope? • The condenser • The objective • The eye-piece or ocular • To condense and focus light onto the specimen

  42. What is the main purpose of the microscope? • The condenser • The objective • The eye-piece or ocular • To condense and focus light onto the specimen • To form a clear intermediate image

  43. What is the main purpose of the microscope? • The condenser • The objective • The eye-piece or ocular • To condense and focus light onto the specimen • To form an intermediate image • To form the final image

  44. Final image Intermediate image Specimen Any questions?

  45. What is the main purpose of the microscope? • Magnification: apparent increase in size • Resolution: the minimum distance between • 2 dots that can be discerned

  46. Airy disc: defined as the region enclosed by the first minimum of the Airy pattern and contains 84 % of the luminous energy. http://micro.magnet.fsu.edu/primer/lightandcolor

  47. Resolution: the minimum distance between 2 dots that can be discerned http://micro.magnet.fsu.edu/primer/

  48. The smaller the diameter of the Airy disc produced by a lens, the higher is the resolving power of that lens, the better you can separate two distinct points. http://micro.magnet.fsu.edu/primer/

  49. Resolution = (0.61 λ) / Numerical Aperture The larger the numerical aperture of a lens, The smaller the Airy disc, The smaller and better the resolution. http://micro.magnet.fsu.edu/primer/

  50. Airy disk sizes vary with changes in objective numerical aperture and illumination wavelength. Resolution = (0.61 λ) / Numerical Aperture Resolution = (0.61 λ) / (n x sin θ) λ: wavelength of light n: refractive index of the medium in the object space Θ: angular aperture

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