Stem Cell Instrumentation Foundry (SCIF) Orientation: Microscopy

1. Stem Cell Instrumentation Foundry (SCIF) Orientation: Microscopy Venu Polineni Research Associate, SCIF University of California, Merced Phone: (559) 253-3068

2. Outline • SCIF Scheduler • Microscopy basics • Upright microscope at SCIF • Confocal Microscopy: Basics • Confocal Microscopy at SCIF • SOP for Microscopy room at SCIF • Applications

4. Ocular Separation and Diopter Adjustment You can adjust binocular microscope to fit the distance between you eyes and to correct for some prescription lenses you may have. To adjust the oculars to your eye-separation simple push the oculars , while looking through them, closer together or farther apart until you see a single image of the object. For Diopter adjustments follow the steps below: Step 1: Focus your sample in bright field. Step 2: While looking through your right eye (cover your other eye or simply close it) focus on a single point in your sample using the fine focusing knob and remember its location. Step 3: Now, while looking through your left eye (cover your other eye or simply close it) focus on the same point as in step 2 using the Ocular Diopter Knob. Ocular Diopter Knob Step 4: Open both eyes and now you should be able to see a clearer image. You do not need your prescription glasses to continue observing though the microscope. Fine Focusing Knob

5. Köhler Illumination Illumination of the specimen is the most important variable in achieving high-quality images in microscopy and critical photomicrography. Köhler Illumination is as a method of providing the optimum specimen illumination. This method guarantees that the condenser is providing the best amount of light possible for the combination of lenses. Step 1: Focus your sample in bright field. Step 2: Close the field diaphragm. Step 3: Focus the edge of the diaphragm by adjusting the condenser height. Step 4: Center the image using the two centering screws. Step 5: Open the field diaphragm until it is at the edge of the field view.

6. Camera Stage controller Temperature control chamber Smart shutter Upright Microscope

9. Raster scanning • Detector : Photomultiplier tube (PMT)

10. Confocal Microscopy at SCIF • Nikon Ti Eclipse Microscope • C1 Series; Optical sectioning • Nikon Elements software for post image processing • DIC, Phase Contrast, Fluorescence • Lasers: 408nm; 488nm; 560nm • Objectives: 10x; 20x; 40x

11. Camera Stage controller Lasers Microscope Log book Floating Table Confocal Microscope

12. Prior Stage Ctrl Shutter Ctrl Microscope Ctrl Halogen Lamp Ctrl Laser Ctrl UV Source

13. Microscope turn on sequence 1. Release compressed air to the floating table (50Psi) 2. UV light source 3.Lasers 4. Halogen lamp 5.Shutter control unit 6. Stage control unit 7. Microscope controlling unit 8. Camera 9. Microscope 10. Computer

14. SOP for the Microscopy room • Gloves necessary when handling slides containing a potential bio-hazard. • Please make sure to bring fully prepared slides/specimen. Any additional sample preparation time at SCIF will automatically be added to your registered time. • Any user not trained and certified is not supposed to accompany the registered user

15. Slides/cover slips/ Petri dishes/gloves are not provided by the SCIF • Cancellations are to be notified 24 hours in advance. Last minute cancellations or a no show will be charged nonetheless • Assistance on the weekends must be notified 3 days in advance • Unless prior approval from SCIF staff no user is allowed to work alone during after hours and weekends

16. Applications • Live cell imaging • Optical sectioning • Phase contrast • Temperature and growth condition control • Fluorescence, DIC, Phase contrast • 3D volume view; XYZ; Timelapse imaging

17. Thank you.