Protocol for Using ISS Alba FCS at Beckman Institute UIUC

Updated 30 December 2010

Protocol for using ISS Alba FCS at Beckman Institute UIUC

Specifications

Located in Beckman Institute, room B606

Single laser excitation wavelength of 469 nm

Start up Instrument

Switch to on: APD 1, APD 2, laserbox, both stage controllers

Log onto computer and launch VistaVision 4.0.112 from Desktop. Use All Users\Braun

Under Excitation Unit, click the filter wheel and select position #1 then position #3 to home it. Do the same for the filter wheel under Detection Unit.

Wait 20 minutes for the laser to warm up.

Sample Preparation

Remove black objective cover plate and place a few drops of immersion water on objective.

Turn on focus lamp.

Clip the pre-prepared standard 8-well plate kept on storage shelf into place on the microscope.

Position the sample holder near standard of interest, but initially along the glue edge.

Turn knob to Eye.

Turning focusing knob clockwise to lower and counterclockwise to raise objective, focus the microscope using the glue on the sample holder.

Make sure objective immersed in immersion water, and after focusing, turn fine focus knob one full turn counterclockwise (raise objective up) in order to make sure past inflection points and in sample.

Turn knob from Eye to L.

Turn off focus lamp and replace black cover plate.

Click the green button to see if the counts are in a good range. If the counts are too high or too low adjust the power appropriately.

Alignment

In the software click Motor Alignment to adjust the pinhole and lens.

For minor adjustments: Leave both channels at pinhole of 50. Under Auto, select 16 Steps and Binary then select Start. Repeat for Lens. Repeat this process until the counts are as high as they will go. Repeat this procedure for channel 2.

For major adjustments: Change the pinhole for both channels to 1000. Under Auto select 32 Steps and Cross then Start. For the Lens select 16 Steps and Binary and Start. Repeat this until you can’t get the counts any higher. Then repeat the procedure for channel 2. Then you may do the minor adjustments.

Measurements

Select Data Acquisition at the bottom left of the software. Press Start to make a measurement. You may have to adjust the acquisition time to get a smoother curve. An intensity trace and autocorrelation plot will be available.

To image your sample, focus deeper, and select Imaging next to Measurement: at the top of the Data Acquisition panel. Then hit start.

Save data by going to File and Save as.

Fit your data in the software. The software can be installed to your home computer. It can be found at the ITG website in the FCS equipment page to download.