1. FNI 2B OM 1 Optical Microscopes

2. FNI 2B OM2 Outline Justification History Components of the Optical Microscope Theory of operation  Basic Microscope Magnification  Refraction of light and Snell’s Law  The thin lens equation Imaging and resolution Confocal Microscopy Vendors

3. FNI 2B OM3 Why Optical Microscopes? Still one of the most commonly used methods of inspection due to ease of use and sample preparation. Often used to prepare samples for electron microscopy or scanning probe microscopy

4. FNI 2B OM4 Labomed Stereo Zoom Scope Top Illumination Power Eye piece Zoom Focus Top Lamp Main power Bottom Lamp With removable glass cover Bottom Illumination Power

5. FNI 2B OM5 Nikon Optiphot Lamp Future camera Coarse focus Fine focus Base Eyepiece Stage Objective Lenses Nosepiece Use this to change the objectives never touch the actual objective lenses Bright field Dark field

6. FNI 2B OM6 Nikon Optiphot Beamsplitter Apertures Stage X, Y Coarse Focus Fine Focus Lamp Intensity Control

7. FNI 2B OM7 Zeiss Axiotron Eyepieces you look through these Brightfield Normal lighting Darkfield Indirect lighting Camera Used to capture images Coarse Focus Fine Focus Light Source Be sure to turn this off when you are finished using it Fiber optics Used to quickly lower the stage so the objectives do not become damaged Objective lenses Used to change magnification Never touch these

8. FNI 2B OM8 Zeiss Axiotron Stage Rotation Stage X and Y Stage handle Vacuum Chuck Not connected to vacuum Beam splitter For camera Motorized objective Always use these buttons to change objectives Always use the front button to increase the magnification and the back button to decrease magnification. Otherwise you may damage the objective lens

9. FNI 2B OM9 Zeiss Axiotron Optional filters Not used Aperture Adjust so it is just beyond the field of view Filters Yellow filter should be used for viewing photoresist The other filter can be used to enhance contrast Polarizer Can be used to enhance contrast

10. FNI 2B OM10 Magnification Objective lenses up to 100x or 150x Eyepieces 10x – 15x Magnification = OL x EP The limit of optical magnification is about 2000x.

11. FNI 2B OM11 Refractive Index Waves do not have the same speed in all materials. The speed of light in vacuum is c=2.998 x 10 8 m/s. The speed of light in glass is slower, only about v=2.000 x 10 8 m/s The refractive index is the ratio of these two speeds This part slows down first θiθi θtθt

12. FNI 2B OM12 Refraction and Snell’s Law http://hyperphysics.phy-astr.gsu.edu/hbase/geoopt/refr.html θiθi θtθt cv Snell’s law describes refraction of sound ( a change in direction as it passes through a boundary) If the incident angle is not zero and the velocities are not equal. This part slows down first θiθi θtθt

13. FNI 2B OM13 Snell’s Law A light wave traveling through a air with propagation speed 2.998 x 10 8 m/s passes into water with propagation speed 2.308 x 10 8 m/s with an incident angle of 50˚. What is the angle of transmittance? θiθi θtθt cv

14. FNI 2B OM14 A Convex Lens The focal point of a lens

15. FNI 2B OM15 Theory of Operation f is focal point distance o is object distance i is image distance M is magnification o f f i

16. FNI 2B OM16 Thin Lens Calculations If a lens has a 4.5 cm focal length and a specimen is 5.3 cm from it where will the image appear to be and what will be the magnification?

17. FNI 2B OM17 Imaging Light field Dark field Resolution Depth of Field Polarizing filters, Nomarski Yellow Filters Apertures Beam splitter Digital image capture Limits of resolution

18. FNI 2B OM18 Sample Preparation Biological sample may be placed on a glass slide with a cover slip. Various stains can be used to highlight different parts of the sample. When gold particles are small enough they no longer appear as yellow shiny metal but can have a variety of colors which depends on their size. These are called quantum dots. These can be combined with antibodies and used to stain different specific proteins different vibrant colors. http://www.invitrogen.com/site/us/en/home/Products-and- Services/Applications/Cell-and-Tissue-Analysis/qdots-microspheres- nanospheres.html

19. FNI 2B OM19 Inspection The optical microscope is the most common method of inspection because they are relatively easy to use and easy to prepare samples for. They are also commonly used when preparing samples for the electron and scanning probe microscopes.

20. FNI 2B OM20 Confocal Microscope

21. FNI 2B OM21 Optical Microscope Vendors Olympus www.olympus.comwww.olympus.com Zeiss www.zeiss.com/microwww.zeiss.com/micro Nikon www.nikonusa.comwww.nikonusa.com Leica www.leica-microsystems.comwww.leica-microsystems.com Microscope U http://www.microscopyu.com/ http://www.microscopyu.com/