1. Light Microscopy Uses Uses Before you go to the EM Before you go to the EM Quick check Quick check Maybe all you need Maybe all you need Hydrated and living materials Hydrated and living materials

2. Light Microscopy Systems Systems Parts Parts Illumination Illumination Sample stage Sample stage Imaging Imaging

3. Light Microscopy Parts of the light microscope Parts of the light microscope Stand Stand Illuminator Illuminator Stage Stage Objective(s) Objective(s) Tube Tube Eyepiece(s) Eyepiece(s) Eye, camera Eye, camera

4. Light Microscopy Illumination Illumination Through the sample Through the sample Incident on surface of sample Incident on surface of sample

5. Light Microscopy Through sample illumination Through sample illumination Kohler illumination Kohler illumination Open field and condenser diaphragm Open field and condenser diaphragm Focus sample Focus sample Close field diaphragm and focus with condenser focus control. Use centering controls to center image. Close field diaphragm and focus with condenser focus control. Use centering controls to center image. Open field diaphragm to fill image Open field diaphragm to fill image Take out eyepiece and adjust condenser aperture to ¾ fill BFP of objective Take out eyepiece and adjust condenser aperture to ¾ fill BFP of objective

6. Light Microscopy Incident Light illumination Incident Light illumination Use special objectives Use special objectives Almost setup for Kohler illumination Almost setup for Kohler illumination Objectives serve as condenser too Objectives serve as condenser too

7. Light Microscopy Objectives Objectives Achromats- RB chromatic and G spherical corrected Achromats- RB chromatic and G spherical corrected Semi-apochromats- RBG chromatic + BG spherical Semi-apochromats- RBG chromatic + BG spherical Apochromats-RBBG chromatic + BG spherical Apochromats-RBBG chromatic + BG spherical “Plan” objectives correct for field curvature “Plan” objectives correct for field curvature

8. Light Microscopy Special Techniques Special Techniques Darkfield (hollow cone illumination) Darkfield (hollow cone illumination)

9. Light Microscopy Special Techniques Special Techniques Phase contrast (blocking phase object interactions with illumination) Phase contrast (blocking phase object interactions with illumination)

10. Light Microscopy Special Techniques Special Techniques Polarized light (polarized light interaction with birefringent materials) Polarized light (polarized light interaction with birefringent materials)

11. Light Microscopy Special Techniques Special Techniques DIC (Nomarski) (shearing interferometry) DIC (Nomarski) (shearing interferometry)