1 / 9

Different Ksv-s

Different Ksv-s. The dynamic range of the probe in the Seahorse seems to be larger (~4.2) compared to the measurements with the confocal microscope (~3.3) Is there a gain / offset problem? (How are the power of LEDs and the gain and zero of the photodiodes determined? Linearity?).

aristotle-olson
Download Presentation

Different Ksv-s

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  1. Different Ksv-s The dynamic range of the probe in the Seahorse seems to be larger (~4.2) compared to the measurements with the confocal microscope (~3.3) Is there a gain / offset problem? (How are the power of LEDs and the gain and zero of the photodiodes determined? Linearity?)

  2. Ksv and F0 are mutually inter-related!! Given by Seahorse By measurement of F0 by dithionite By Ksv measuremt on the Pascal Fixing gain (and zero) to match the F0 measured in dithionite to the one calculated from the measurement of Ksv on the Pascal.

  3. Synaptosome (2008-06-11-SYNAPTO-36(1)) different calibrations of S2 O2 5 ug 10 ug Dithionite Pascal Seahorse S-V Curve fit 15 ug

  4. Linear back-diffusion correction Fick’s law, here C is function of x and t <> Means spatially averaged C for the assay volume , so C is only the function of t l is the distance between the bottom and the probe, But it is built in the Dapp apparent diffusion constant C0 is the ambient, dissolved O2, best if measured in the temperature control wells. OCR measured Real consumption Rate of back-diffusion Differentiation is very sensitive for noise, so let’s integrate both sides of the equation: The virtual[O2] tells that how much O2 would be there without back-diffusion. This can be normally a negative number, therefore called virtual. Or call it O2 credits??

  5. First calculating the mean of F0 for Stern-Volmer calibration (from the maxal F of each ditihionite trace) Changing slope linearized Then analyzing the slope By finding the optimal Dapp, The corrected trace is closest to linear (Pearsons correlation is closest to 1)

  6. 3. S-V 3. S-V linearized virtual The virtual [O2] is zero when the cells consumed that much oxigen since the start of the measurement as much oxygen was there. Of course, it was meanwhile replenished by back-diffusion, so the actual [O2] is not zero at this point.

  7. saturation + offset (y-axis) nonlinearity - offset (y-axis)

  8. Back diffusion theoretical curve

More Related