1. Microscopes Moisey (2009), Optical Microscope [photograph]. Retrieved from https://commons.wikimedia.org/wiki/File:Optical_microscope_nikon_alphaphot_%2B.jpg

2. Microscopes: Microscopes are some of the most commonly used pieces of lab equipment Seldom maintained Medical uses;  Histology  Pathology  Hematology

3. Types of Microscopes: Compound light Inverted Compound light Phase shift Fluorescence Digital Electron Arranged in order of commonality

4. Types of Microscopes Light microscopes (magnification ~ 2000X) – Compound Staining needed –Stereo For dissection, etc. (low magnification) –Phase contrast Upright or inverted (for cell culture) No staining needed, can see live cells –Fluorescence (usually with phase contrast) (your limit!!- don’t touch) –Confocal Produces 3-D images (Z-stack) Electron microscope (magnification 500,000 X – 5000,000X) –Scanning, Transmission, Cryo Live samples cannot be viewed because of vacuum Sample preparation is laborious (except for cryo) Cannot transport, dedicated facility needed

5. Compound Light Microscopes: Monocular/Binocul ar Inverted Light or Reflector Filtered Aperature Slide clips 3-5 Objectives Stage XYZ Locks

6. Optical Path in a Typical Microscope Wikipedia. “Optical Microscope.” Wikipedia. Retrieved from: https://en.wikipedia.org/wiki/Optical_microscope

7. Components of a Microscope Light source Condenser Specimen stage Fine and coarse focus knobs Objective (s) Eyepiece (s) Camera

8. Basic Light Microscope Components Wikipedia. “Optical Microscope.” Wikipedia. Retrieved from: https://en.wikipedia.org/wiki/Optical_microscope

9. Microscope Components Condenser A condenser is designed to collect, control and concentrate light from the lamp onto the specimen. Iris controls the amount light Egmason (2011), Condensor specimen [drawing]. Retrieved from https://commons.wikimedia.org/wiki/File:Condenser_diagram.svg

10. Microscope Component Specimen Stage Anchors the specimen on the slide, dish, etc. and allows scanning he specimen in X-Y direction using micrometer screws Stage Moisey (2009), Optical Microscope [photograph]. Retrieved from https://commons.wikimedia.org/wiki/File:Optical_microscope_nikon_alphaphot_% 2B.jpg

11. Microscope Component Focus Knob(s) Moves the stage or the objective tube up and down to achieve focus. Uses fine micrometer screws. All microscopes have a ‘coarse’ and ‘fine’ focus knobs. Modern microscopes have them as one piece as shown

12. Microscope Component Objective Heart of the microscope, determines the magnification of the image and resolution. Objectives are interchangeable easily to change the magnification. WHO. “Learning Unit 6: The Microscope.” From the Publication: Basic Malaria Microscopy: part 1. A Learner’s Guide. WHO: Switzerland (2010), p. 37-44. Retrieved from: http://apps.who.int/iris/bitstream/10665/44208/1/9789241547826_eng.pdfhttp://apps.who.int/iris/bitstream/10665/44208/1/9789241547826_eng.pdf

13. Microscope Component Objective- Properties Magnification –How big the image looks –As magnification increases, area seen decreases –Magnification does not always mean better resolution Numerical aperture –How much light is collected from specimen from the objective –Numerical aperture determines resolution Immersion medium –At high magnification, more light can be captured if immersion medium such as oil is used Working distance –How close the objective has to be for optimal function Depth of focus/field –The range of distance of the objective from the specimen within which the image will stay in focus

14. Objective Properties Magnification vs. Resolution High magnification without resolution is not useful; this is known as ‘empty magnification’

15. Objective Properties Oil Immersion Immersion in media with higher refractive index than air (such as oil) increase the amount of light collected, hence creates higher numerical aperture and greater resolution User Gcg (2009), Immersion Microscopy [photograph]. Retrieved from https://commons.wikimedia.org/wiki/File:Immersion_microscopy.jpg

16. Objective Properties Depth of Focus/Field The range of distance of the objective from the specimen within which the image will stay in focus Depth of focus decreases with higher numerical aperture The image will go out of focus with small movements of the objective when depth of focus is small Like f-stop in a camera

17. Microscope Component Eyepiece Eyepiece is the viewing unit of a microscope Can be mono ocular (one eye: old microscopes) or binocular, where both eyes are used Eyepieces add magnification (usually 10X- 20X) to the image produced by the objective Jastrow (2007), Clave 25mm Eyepiece [photograph]. Retrieved from https://commons.wikimedia.org/wiki/File:Clave_25mm_eyepiece.jpg

18. Common Problems: Dirty Lenses Dirty Light path Stiff stage mechanism Broken or missing parts

19. Cleaning method: Clean with 99% alcohol or lens cleaner ONLY! Use a chamois or microfibre cloth;  Cotton batten may be used with care  Light pressure If light path is open take care not to contaminate Cover microscopes when not in use

20. Servicing Microscopes: Clean with 99% alcohol or lens cleaner ONLY! Check light path for contamination Test light range Check all magnifications for focus and tracking Stage must move freely-never force stage movement with fine/coarse controls Check locks & slide clamp Cover when servicing is complete

21. Questions? ?