Also you already set your acquisition mode to kinetic, triggering internal, readout mode image.

For smFRET: 500 or even better 1000 images (kinetic series length), no prescans, 1 accumulation, exposure 100 ms.

For super-resolution: 4000 images, no prescans, 1 accumulation, exposure 100 ms.

Run the camera in preview mode with take video, and if you are ready for acquiring data for storage use take signal.

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 (500 frames).

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.

Storing the data should be done automatically for taking signal, and it should be placed in the folder D:\data. The next time it will be overwritten, so make sure you make a new folder and move your data there. Use the right save settings (can be found in acquisition setup> auto-save>TIFF options): 16-bit gray tiff format, 0-65535 data range (otherwise your data gets re-scaled resulting in unrecognisable spots in your image).

Do not forget to analyze the files that are created by this program. You can do this with the given files in the data analysis part of this practicum page.