How to determine FQY for photoswitchable compounds?
I found most related publications didnt mention, or just said that they used "Absolute quantum yield spectrometer."
I think such technique applied integrating sphere with CCD detector to record emission spectrum to calculate FQY.
My questions:
1.While exciting the photoswitchable compounds to obtain fluorescence, do some of them being photo-converted during measurement, and thus results in underestimated FQY? Or the ~ms irradiation is neglectable?
2.Is it ok to measure FQY of photoswitchable compounds by comparing the slopes of relationship between FL vs. Abs. to reference dye? (figure 6 of "https://www.perkinelmer.com/lab-solutions/resources/docs/APP_Determination_of_Relative_FluorescenceQuantum_Yields_using_FL6500_Fluorescence_Spect.pdf")
Do you know the average amount of time your dye spends in the on vs off state when excited with your chosen wavelength? If so, I would use that information as an estimate of the fraction of molecules in the on state during a steady-state measurement. I would perform the QY experiment (either absolute or relative method) in the standard way and correct the determined QY by this fraction to get the QY of the on state.
Jeremy Pronchik, thanks for replying. I used mild power laser to switch on and off my dyes, half-convert time is about 3-5 seconds. But steady-state FL measurement was from Xe lamp with quite narrow slits (guess the power is < than 1/10 times of my laser), and was measured in 300 ms. Guess the correction term was almost neglectable, but I will take deeper estimation of that part. Thanks.
Bryan Po-Wen Chen , what is the wavelength of the mild laser and what is the wavelength of excitation for your QY measurement? Are they the same?
Laser is 405 nm. For FL, I tuned the Xe light source to 405 nm (with +/- 2 nm slits), and the absorption at 405 nm for both my dye and reference dye were adjusted to the same for relative FQY determination. Sounds reasonable to you?
Hi Bryan Po-Wen Chen ,
Sorry I disappeared. I normally get emails when answers are added to a thread I contributed to...
Ok, I just wanted to make sure the laser that turns the lamp on and off was the same wavelength as excitation, and it sounds like it is. I think I've offered all I can. I don't know the relationship between incident power and on/off rates. I still think that's the key to estimating this. Maybe there are single-molecule experiments that have found rates and the power-rate relationship for your dye? Good luck!
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