photoactivation vs. photoswitching (part 2)

October 9, 2009 at 3:43 pm | | tutorial

In part 1, I discussed the differences between the definitions of photoactivation and photoswitching. Here, I want to talk about some of the applications of the various classes of photochromic dyes.

photochromic-lens

Photochromism typically is used for modulating the absorbance or color of a solution or material. The most obvious example is sunglasses that tint in the sunlight. Photochromism can even be applied to batteries!

Photoactivation is ideal for situations you want an emitter to start in a dark form, be controllably activated to a bright form, emit many photons, then disappear irreversibly. An example application is tracking single molecules when the concentration of molecules is high: you want the probes dark to start to maintain a low background, and you want the probe to turn on irreversibly lest the emitter switches off quickly and you can no longer track it.

Another perfect example is super-resolution imaging (via photoactivation and localization) of relatively static structures: you want to turn on a sparse subset of emitters, localize them once with high precision (requires many photons), then have them disappearĀ permanently. Using a photoswitching probe for imaging static structures isn’t ideal, because relocalizing the same spot over and over wastes time and complicates the subsequent image analysis.

Photoswitching is ideal if you want to relocalize over and over, such as if you are performing super-resolution imaging on a moving or dynamic structure. Photoswitching can also be applied to speckle microscopy, which follows fluctuations in dynamic filaments. Others have used photoswitching to modulate a probe, lock-in to that modulating signal, and filter out the unmodulated background from nonswitching fluorophores (see OLID, figure below).

olid

Photoswitching is also necessary for super-resolution techniques such as STED and structured-illumination, which require many cycles of bright and dark. In fact, both these techniques require photoswitching that is very robust and lasts many many cycles.

Side Note Regarding Photons: It is important to note that a compound being switchable does not necessarily mean that it will ultimately emit more photons. In many cases, cycling emission simply spreads the photons into many bins: the total number of photons emitted over all the cycles sum up to the same number to the situation if the fluorophore had been on continuously. For instance, if the unswitching form of the fluorophore usually emits about a million photons (for the best organic dyes), then you cycle it with each cycle emitting 10,000 photons, the photoswitch will generally last 100 cycles on average before photobleaching.

On the other hand, it is conceivable that a photoswitch could resist photobleaching better than its continuous analog. For instance, if the fluorophore bleaches via producing triplet oxygen that builds up around the dye—eventually colliding and reacting with the compound—then switching to an off state might offer some time for the built up concentration of triplet oxygen to dissipate.

On the third hand, if the switch is capable of bleaching from the dark state, then switching may ultimate reduce the total photon countĀ (e.g. the imaging light may pump the dark form into highly excited triplet or other states, leading the compound to basically explode).

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  1. […] the next installment (part 2), I will describe the situations where photoswitching is preferred over photoactivation and vice […]

    Pingback by Everyday Scientist » photoactivation vs. photoswitching (part 1) — October 9, 2009 #

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