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Total Internal Reflection Fluorescence microscope
Nikon Eclipse Ti
Principal:
• Laser is pointed at an angle at specimen
• Due to this angle the laser will totally reflect
on glass/medium interface and creates an evanescent wave above the glass
• Thickness of wave is around 200nm, but decays exponentially
• Possible to only excite fluorophores in membrane, without background signal of the cytoplasm
Benefits:
• Extreme low background signal
• Perfect for membrane studies
• CCD Camera attached will provide in fast imaging
Contact
• Martijn de Gruiter
• Gert-Jan Kremers
Martijn de Gruiter
Source: NIKON
Be-343
tel. 31105
Be-346
tel. 43578
Manual: TIRF Microscope
Erasmus OIC
Ee-1454
1
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Summary of Contents for Nikon Eclipse Ti TIFT 1454
- Page 1 Ee-1454 Manual: TIRF Microscope Total Internal Reflection Fluorescence microscope Nikon Eclipse Ti Principal: • Laser is pointed at an angle at specimen • Due to this angle the laser will totally reflect on glass/medium interface and creates an evanescent wave above the glass •...
- Page 2 Ee-1454 Manual: TIRF Microscope Erasmus OIC Martijn de Gruiter...
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Page 3: Start System
Ee-1454 Manual: TIRF Microscope Start system • Turn on the power by the power socket (on the floor under the table) • Switch the mercury lamp on and then ignite it. o If not needed don’t turn the mercury lamp on o The unit on the shelf closest to the door •... - Page 4 Ee-1454 Manual: TIRF Microscope Finding focus • Put the objective in the lowest position using button A o Lowest position is 500 um • Choose the objective with the right magnification • Put a little drop emersion oil on the lens o Only if it’s an oil emersion objective •...
- Page 5 Ee-1454 Manual: TIRF Microscope Metamorph - Taskbar • On startup your personal taskbar is loaded • Metamorph functions can be accessed easily from a Taskbar and an Acquire window • You can restore illumination settings with the button: "Restore OIC settings" •...
- Page 6 Ee-1454 Manual: TIRF Microscope Tab: Display: • Auto scale option o Enable Auto scale or change custom auto scale o Full bit-range is saved to file, this is only for visualization in the software • Visualize saturated pixels Tab Acquire: •...
- Page 7 Ee-1454 Manual: TIRF Microscope Streaming imaging Open the Stream Acquisition window with the button in the taskbar • Start recording with Acquire button • Check if chosen configuration is ok Tab Acquire: • Acquisition modes: o RAM save later to hard disk o HD choose name and location to save Save as on file or separate TIF files...
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Page 8: Shut Down System
Ee-1454 Manual: TIRF Microscope MDA: Time lapse imaging • Activate the time-lapse option in MDA’s main window • Set the desired interval and time • Choose the desired illumination settings in the wavelength tab o Or multiple when the option multiple wavelengths are selected •... - Page 9 Ee-1454 Manual: TIRF Microscope FRAP on the Fly Calibration Before you can use FRAP the laser must be calibrated • Find an empty piece of glass without cells/fluorescence • Set the exposure time around 100 - 200ms • Open ILAS2 with icon •...
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