Nature Methods | Methagora

Increasing your fluorescence signal using pulsed excitation

Stefan Hell and colleagues propose that the use of pulsed excitation with a delay of at least one microsecond between pulses can dramatically reduce photobleaching and increase the effective fluorescence signal in both single and multi-photon fluorescence microscopy. Is this something you would consider trying in your lab? Do you think the described benefits are worth the necessary equipment changes?

Read the paper here and then add your opinion.

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    Taekjip Ha said:

    This approach sounds potentially very interesting. Ultimately, information content in single molecule fluorescence imaging will be limited by the excited state photophysical processes if one wants to push the sensitivity and time resolution to the extreme. I am curious as to what I need to do to implement this approach for example on the 532 nm CW laser we are using currently in our group. It will certainly be interesting to find out if the approach boosts the photobleaching lifetimes of Cy dyes when their properties are already optimized using soluble additives (oxygen scavenger system, trolox, etc).

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    Daniel Evanko said:

    Taekjip Ha raises two interesting points:

    1) The use of this technique for single molecule imaging, and

    2) Can it be implemented using a continuous wave laser?

    This paper was not evaluated from the standpoint of single molecule work but we did think people doing such work might be intrigued by the technique since bleaching is very problematic when imaging single fluorophores.

    We assumed that implementation would require the use of special pulsed lasers. This is a potential roadblock in adoption of the method. However, it might be possible to modulate the output of a CW laser to create pulses. Is there anyone who can comment on this? Even if such modulation is possible it could be cheaper and easier in the long run to install a second (pulsed) laser on the system.

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    Dave Piston said:

    I was also thinking about the point that Taekjip Ha raises. However, it looks like the power levels where this paper shows the improvement are well above the onset of saturation, and thus we would not expect any enhancement for regular confocal imaging, where excitations are much less than 1 mW at the sample.

    The case of single molecule imaging is interesting. In our lab, we use the lowest possible laser intensities for single molecule measurements, but perhaps those trying to take data with a faster time line could see some improvement by going to a pulsed source. In terms of reduced photobleaching, though, this would assume that the molecules are spending time in a dark state (triplet?) where furhter excitation can lead to increased bleaching. If there isn’t a non-linear bleaching change, then it seems that the pulsed excitation could only reduce the total fluorescence rate for a single molecule. While the amount of fluorescence per input intensity might increase with pulsed excitation, the absolute fluorescence intensity per molecule can only decrease with pulsed excitation. In any case, however, it probably isn’t practical to start with most CW lasers because the low duty cycle would not allow sufficient power to reach the sample.