Choosing a confocal for live imaging / how resonant scanning actually works?

Hi @djc,

Hot/somewhat conterversial topic we’re wading into here, but in my opinion, a reduction in photobleaching resulting from resonant scanning has not been rigorously/quantitatively demonstrated.

Most arguments I’ve heard point to a couple sources:

The one that I see most often referenced, and which appears to have the most direct comment is this letter in MRT from many years ago (I’m not sure if it’s peer reviewed):

This is a purely theoretical paper that argues that the pulsed illumination in resonant scanners may reduce photobleaching by reducing the accumulation of fluorophores in the excited triplet state (T2 below; which is said to lead to photobleaching), by giving fluorophores that have entered a longer-lived triplet state (T1) time to undergo relaxation to the ground state (S0) before getting immediately re-excited.

Obviously this game hinges entirely on these rate constants (k1-k7) and the actual duty cycle of excitation in a resonant scanner. That paper simply makes up rate constants that support the argument being made, with no references to back them up. Furthermore, there are no time units in the graphs there, and in simulations I’ve made to try to reproduce the curves they show, I have to pick pretty outlandish parameters to come up with anything approaching the benefit shown in figure 4. Nonetheless, that paper frequently gets used as rationale for the “relaxation to the ground state”.

Another, much more rigorous/quantitative paper exploring the effect of pulsed illumination on photon yield is Donnert et al 2007

There, they adsorbed various FPs and dyes to glass, then irradiated them with a (stationary) pulsed beam and measured rate of bleaching and total photon yield of the spot as the varied the repetition rate of the laser. They found that inter-pulse intervals on the order of 0.5-2µs increased the total photon yield:

unfortunately, when people reference that paper in support of “pulsed illumination is better”, they tend to miss the very important detail in that paper that the effect depends a lot on the irradiance, as shown in figure 2b. Specifically, the integral under that curve in figure one only starts to get a lot better with larger inter-pulse intervals once we start to get into the range of many MW/cm2

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Those are irradiation levels that are not uncommon in STED, but are very uncommon even in point scanning confocal (and unheard of for spinning disc and widefield techniques). Note: tens of MW/cm2 is at least an order of magnitude larger than a “saturating” light dose (i.e. approaching ground state depletion) for EGFP. God help you if you’re putting that much light on your live sample :wink:

There are many other papers that have tried to explore this relationship between temporal light dose and photobleaching (non exhaustive list below) … but properly normalizing absolute total irradiance while varying illumination protocol is a famously hard thing to do… and i think that all of these leave at least some room for debate (or at the very least, are hard to generalize beyond the specifics of the experimental setup… and are of questionable application to resonant scanning in a point scanning confocal)

To be clear, I’m not trying to argue that resonant scanners have no benefit over conventional point scanners… but in my experience, this argument has tended more towards qualitative anecdotal reports. (Though always happy to hear some counter opinions here!)

I’ll leave you with one additional back of the envelope calculation. consider that end the end of the day we’re limited by the fluorophore:

  • the lifetime of an EGFP molecule is ~3ns and the quantum yield is ~60% (meaning it can give you no more than 0.2 photons / ns at saturating light intensities
  • the dwell time of 12KHz resonant scanner is ~80ns for a 1k x 1k field of view.

even with a high NA lens, a high QE detector and a perfectly aligned microscope, you’re looking at a best case scenario of ~.6 photons per EGFP per 80ns dwell… (assuming you’re dumping upwards of .6MW/cm2 of light onto the specimen)

That’s not a lot of SNR, so very often you’ll find yourself averaging frames/lines to recover that signal.
There’s no free lunch.