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Manual: TIRF Microscope

Ee-1454

Martijn de Gruiter

Erasmus OIC

Source: NIKON

Total Internal Reflection Fluorescence microscope

Nikon Eclipse T

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

Be-343

tel. 31105

•

Gert-Jan Kremers

Be-346

tel. 43578

Summary of Contents for Eclipse Ti TIFT 1454

Page 1: ...tally 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 Be 343 tel 31105 G...

Page 2: ...Manual TIRF Microscope Ee 1454 Martijn de Gruiter Erasmus OIC 2 ...

Page 3: ...ells uses water o fill reservoir with demineralized water up to level of CO2 tube Allow the system to warm up for 15 30 minutes Settings for the heating unit are paper on the wall choose your settings o General settings for cells on a coverslip o top heater 40 5 C o bath heater 38 5 C o stage heater 38 5 C o lens heater 37 C Ensure that the cells are grown on a glass coverslip Place the slide in t...

Page 4: ... be done with the mercury lamp o Choose the illumination settings for your fluorophore in the Taskbar in Metamorph o Increase or decrease brightness by changing the ND filters o ND 4 8 10 will block intensity the can be combined use minimal intensity to reduce bleaching of your fluorophore When your sample is in focus you can focus the beam of the laser if necessary for detailed explanation ask a ...

Page 5: ...ra o right side mercury lamp or transmission to eyepiece Open FRAP or laser console Metamorph Acquire window Sidebar left Exposure time o Minimum interval time at full chip is 90ms quad chip is 50ms o Exposure time can be lower than interval time Full Chip Quad Chip Use Active Region o Use full chip middle quadrant or region selected by ROI Live bin bin o Combine pixels to increase readout speed o...

Page 6: ...he Illumination choice to Current shutter Tab Special Gain options 10 MHz EM gain gain3 3x is standard setting Control gain between 0 and 1000 Use Frames To Avg option to average images or use longer exposure time Live image window Magnifying glass Display image in other LUT o Grayscale Color by wavelength Histogram of intensities o Manual scaling can be done with the orange arrows Auto scaling op...

Page 7: ...mes to skip during acquisition o Don t show recording faster when exposure time minimal interval time Camera readout default options should be o Camera state HALT o Shutter mode OPEN PRE SEQUENCE o Clear mode CLEAR PRE SEQUENCE Multi Dimensional Acquisition MDA Combine multiple tasks z stack time lapse streaming multiple positions multiple wavelengths use of journals Open the MDA window using Task...

Page 8: ...per o Hold lens paper tight between your fingers and wipe over objective o Clean lens with 2 propanol on your lens paper Cover stage with lens paper box Save data to OIC network storage O drive o To collect the data from your own pc connect to network storage drive via the address oic station oic user guest password guest o When the hard disk of the microscopes pc gets full data will be deleted o ...

Page 9: ...r and or exposure time o view is rotated so move laser in other direction o keep in mind to move red cross into left and right corners of the calibration screen Calibrate with button i To check if your calibration was successful open on the fly tab Change time to 3000 Increase the power of your laser and press Show Live Click in the image and check if laser pulse in at the position of your mouse p...

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