Microscope efficiency report for Example Widefield (Sedat)
This page calculates excitation efficiency and collection efficiency for every probe in the database with each optical configuration saved on this microscope. This info is combined with probe brightness to calculate a "predicted" brightness metric for each fluorophore/optical configuration pair (indicated by the size of the points on the chart). The table below the chart can be used to explore the data more selectively. For more information on how each efficiency metric is calculated, see the documentation.
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This report only includes probes in the database for which spectra are available. Effective brightess is only calculated for probes whose extinction coefficients and quantum yields are known. "Brightess" on this page refers to the product of excitation efficiency, collection efficiency, extinction coefficient, and quantum yield.
In addition to the efficiency calculation notes in the documentation, here are some things to keep in mind when evaluating this information.
Emission efficiency is often more important than excitation efficiency. Fluorophores emit a limited number of photons before bleaching, so it's important to collect as many as possible. While we can sometimes compensate for poor excitation efficiency simply by increasing the illumination light power (at the potential cost of phototoxicity), the same cannot be said for poor collection efficiency: those lost emission photons are gone forever. Therefore, if the "brightest" probe for a given channel has much lower collection efficiency ("em eff") than other probes, you may have better practical success with the other probes.
Do not forget about expression level, and FP maturation.. These numbers are theoretical! "Brightness" here only considers the relative compatibility of a dye with an optical configuration, and "molecular brightness" of the probe (EC * QY). However, a rapidly maturing (or over-expressing) FP may be practically brighter than a poorly maturing FP, even if the rapidly maturing FP was predicted to be dimmer on this page.
However, for single molecule imaging... Poor FP maturation will manifest not as decreased intensity, but rather as decreased "labeling efficiency". For single molecule experiments, you may wish to maximize collection efficiency and quantum yield.
Consider bleedthrough. If you are designing a multi-color experiment, note the predicted brightness for your candidate probes in all of optical configurations you will be using. For example, if your brightest "green" probe has a much higher predicted brightness in your "red" channel than a slightly less-bright alternative green probe, you may experience more bleedthrough (cross-talk) with the brightest green probe than the alternative; and it might be worth trying the slightly dimmer one as well.
These numbers are only as accurate as the underlying spectral data.. If the spectral data for one of the probes is inaccurate, the resulting efficiency calculation will also be inaccurate. For instance, if the spectrum for a given probe is heavily truncated, then it will (falsely) appear to have a very narrow excitation/emission spectrum and may yield an artificially high or low efficiency prediction.