Transcript Stem Cell Instrumentation Foundry (SCIF) Orientation

Stem Cell Instrumentation Foundry
(SCIF) Orientation: Microscopy
Venu Polineni
Research Associate, SCIF
University of California, Merced
Phone: (559) 253-3068
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Outline
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SCIF Scheduler
Microscopy basics
Upright microscope at SCIF
Confocal Microscopy: Basics
Confocal Microscopy at SCIF
SOP for Microscopy room at SCIF
Applications
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.
Ocular Diopter
Knob
Fine Focusing
Knob
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.
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.
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.
Camera
Stage controller
Temperature control
chamber
Smart shutter
Upright Microscope
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• Raster scanning
• Detector : Photomultiplier tube (PMT)
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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
Camera
Stage controller
Lasers
Log book
Microscope
Floating Table
Confocal Microscope
Shutter
Ctrl
Prior
Stage
Ctrl
Halogen
Lamp
Ctrl
Microscope
Ctrl
Laser
Ctrl
UV Source
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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
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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
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• 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
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Applications
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Live cell imaging
Optical sectioning
Phase contrast
Temperature and growth condition control
Fluorescence, DIC, Phase contrast
3D volume view; XYZ; Timelapse imaging
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Thank you.