The setup is ready for measurement when
**Checklist**
- The laser has become stable.
- The camera has a stable temperature (about -85C)
- The right filter and lens are selected in the microscope.
- The slide is in place.
- There is total internal reflection.
Before opening the camera shutter, make sure all the lights are switched off. The normal light can damage the CCD of the camera.
First open the camera shutter. Then open the laser shutter. With caution, use the microscope to focus the image.
Make sure you switch off the ‘auto’-settings for the CCD gain control. The values for normal conditions are:
Gain 200
Background subtraction 420
Data scalar 2
Exposure Time 0.1
After entering these values click on the set-gain button. Feel free to experiment with these values to achieve a better image.
These are two examples of what the image might look like.
Figure 1: These are examples of what you will see while preparing for a measurement. The left is a very poor quality. Although the image is quite focused, there are very few molecules in the image. This is not good enough for a measurement. The right image has multiple molucules that are clearly visible in both sides of the image. Also the focus is quite good. If the picture remains focused, this is a very good image for one measurement.
When the molecules are clearly visible, it is time to start the measurement. The ideal time for measurement is somewhat one or two minutes.
While measuring the image can lose focus. This has to do with the optics. This problem can be solved by first focus the image, close the laser shutter for a minute and finally focus the image again.
Do not forget to analyze the files that are created by this program. You can do this with “Analyse_all” this wil analyse all files in the measurement folder. After you analyzed the files you can remove the movie (large file) and place the remaining files in another folder.