Turn on Input/Output Controller. This controls camera-software timing. Turn on Definite Focus Controller. This allows you to keep focus while moving stage. Turn on TIRF Controller. This controls the laser power. (Optional) Turn on EXPO mercury lamp. This provides white light. Sample loading procedure: On Microscope Control Box, do the following: Home Load position Set work position. This lowers the objective lens. Control Reflector 74 HE GF/mRF Control Objectives 100x Oil. On the 100x objective, drop immersion oil (Immersol 518F) onto lens without letting the dropper touch the lens. Load your sample onto the stage. Note: Wipe the glass slide before loading. Make sure the sample holder is securely fitted inside the stage. Sometimes the holder pops out. Use the coarse focus adjuster knob to bring the oil on the lens in contact with sample. Stop immediately when oil spreads. Then use finer adjuster knob to spread the oil evenly. Use joystick to move lens to the correct Note: If the stage moves too fast/slow, push the joystick button to change the speed. Place the laser safety cover on top of the stage. Data acquisition procedure: the AxioVision software on the computer. Go to Microscope Microscope Under Reflected tab, select GFP/mRFP”. “74HE Under Laser tab, set laser power based on how bright your samples are. Set angle using this button and set it 70 degrees. WARNING: Do not use , otherwise laser will point towards you and could hurt your eyes! Turn on laser and make sure the lasers are visible on the stage. WARNING: Do not look at lasers closely when checking! It should be visible through the laser safety cover. Go to Hardware Change camera exposure time. (50 - 100 ms is okay.) Enable EM-CCD. This enhances signal. Note: If camera is oversaturated, it will turn off and make an alarm noise. Uncheck and Check the EM-CCD checkbox to turn off the alarm noise. Go to Live View. Select the image contrast option. Min/Max or Best Fit. Find focus using Live View. WARNING: Do not over focus! Can cause damages to lens. Visually check the lens frequently when focusing for the first time. Go back to Hardware. Go to Definite tab and set it to ON. This keeps the sample in focus. Confirm that autofocus is working through Live View. When exit Live View.
Turn on Input/Output Controller. This controls camera-software timing. Turn on Definite Focus Controller. This allows you to keep focus while moving stage. Turn on TIRF Controller. This controls the laser power. (Optional) Turn on EXPO mercury lamp. This provides white light. Sample loading procedure: On Microscope Control Box, do the following: Home Load position Set work position. This lowers the objective lens. Control Reflector 74 HE GF/mRF Control Objectives 100x Oil. On the 100x objective, drop immersion oil (Immersol 518F) onto lens without letting the dropper touch the lens. Load your sample onto the stage. Note: Wipe the glass slide before loading. Make sure the sample holder is securely fitted inside the stage. Sometimes the holder pops out. Use the coarse focus adjuster knob to bring the oil on the lens in contact with sample. Stop immediately when oil spreads. Then use finer adjuster knob to spread the oil evenly. Use joystick to move lens to the correct Note: If the stage moves too fast/slow, push the joystick button to change the speed. Place the laser safety cover on top of the stage. Data acquisition procedure: the AxioVision software on the computer. Go to Microscope Microscope Under Reflected tab, select “74HE GFP/mRFP”. Under Laser tab, set laser power based on how bright your samples are. Set angle using this button and set it 70 degrees. WARNING: Do not use , otherwise laser will point towards you and could hurt your eyes! Turn on laser and make sure the lasers are visible on the stage. WARNING: Do not look at lasers closely when checking! It should be visible through the laser safety cover. Go to Hardware Change camera exposure time. (50 - 100 ms is okay.) Enable EM-CCD. This enhances signal. Note: If camera is oversaturated, it will turn off and make an alarm noise. Uncheck and Check the EM-CCD checkbox to turn off the alarm noise. Go to Live View. Select the image contrast option. Min/Max or Best Fit. Find focus using Live View. WARNING: Do not over focus! Can cause damages to lens. Visually check the lens frequently when focusing for the first time. Go back to Hardware. Go to Definite tab and set it to ON. This keeps the sample in focus. Confirm that autofocus is working through Live View. When exit Live View.
Standard%20Operating%20Procedure%20-%20TIRF.docx