As a calibatin method for the new TimeHarp 300, is required to determine very accuratelly the focal spot size.
They recomend to do this using FCS measurements of Rodhamine 6G, as standard
As a comment about our new Pico Harp 300 , is that the focal spot size is a value completely arbitrary in the Symphotime software. I dont ike that , cause is very easy to make a mistake or even make wrong use of it , in order to achieve better resolution in the microscope and so on
So if anyone has a very acurate or another ways to determine the focal volume or focal spot , I will appreciate your coments
Best wishes to all scientists !!!
You can use the calibration script offered in the symphotime software. http://www.picoquant.com/images/uploads/downloads/calibrate_the_confocal_volume_using_the_fcs-calibration-script_step_by_step.pdf
But I image gold nanorod (non bleaching luminiscent object with size less than diffration limit, fluorescence beads another option) in XY, XZ, and YZ plane. From the images you can get the FWHM in all dimensions and use for focal volume calculation.
http://www.picoquant.com/images/uploads/page/files/7351/appnote_quantfcs.pdf
I am already rewading the second file, which I did not have .
Thanks Biswajit !!!
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