photoactivation vs. photoswitching (part 1)

September 3, 2009 at 10:18 am | | tutorial

With the development of new super-resolution imaging techniques, photoswitching probes have become very important. Here, I explain the differences in photophysics and applications of photoactivating and photoswitching fluorophores. (These are not hard-and-fast rules, just my definitions of the terms.)

Azobenzene

In general, photochromism refers to the reversable light-induced transformation of a compound between two forms that absorb different energies or amounts of light. By switching between the forms, the color of a solution of the chromophore changes. One or both forms may be fluorescent, but it is possible that neither form fluoresces significantly. Frisbees or beads or shirts that change color in sunlight probably contain some photochromic molecules that are switched using UV irradiation. Common photochromic compounds include azobenzenes (structure above), diarylethenes, and spyropyrans.

Photoswitching refers to the reversable light-induced switching of fluorescence (color or intensity), and is often a type of photochromism. (In general, there is really no reason that the term must refer only to fluorescence, but in the context of imaging it is more helpful.) For instance, photoswitching rhodamines cycle between nonfluorescent and fluorescent forms by closing and opening a lactam ring. When the ring is closed (the thermally stable form), the absorbance is in the UV and the compound is nonfluorescent. Upon irradiation with UV light, the lactam can open; this forms a metastable fluorescent compound that absorbs in the green. Eventually, the lactam ring reforms (either via visible-light irradiation or thermally), and the cycle can repeat. Eventually, some photochemical reaction with change the compound (e.g. photo-oxidation), and the cycling will end (see cycling below). This is called photobleaching. Another example of cycling photoswitching includes Cy5 (here and here), which can be attacked by a thiol and rendered nonfluorescent; by irradiating with green light, the thiol pops back off and the Cy5 becomes fluorescent again. Here is a plot of the cycling fluorescence of Cy5:

photoswitching

Photoactivation refers to the irreversable light-induced conversion of a fluorophore from a dark form to a fluorescent form (or from one emission color to a significantly shifted color, e.g. blue to red). Typically, a chemical reaction transforms the compound, thus making the photoactivation irreversable for all practical purposes. For this reason, a photoactivatable fluorophore is sometimes referred to as being photocaged. There are several photoactivatable fluorescent proteins, such as PA-GFP. Another example close to my heart is the azido DCDHF fluorophore: upon irradiation with blue light, an azide photoreacts into an amine (with the loss of N2) and converts a nonfluorescnt compound to a bright emitter.

azido-DCDHF

In the next installment (part 2), I will describe the situations where photoswitching is preferred over photoactivation and vice versa.

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  1. […] part 1, I discussed the differences between the definitions of photoactivation and photoswitching. Here, I […]

    Pingback by Everyday Scientist » photoactivation vs. photoswitching (part 2) — October 9, 2009 #

  2. […] think this paper is a nice characterization of the photochemistry involved in Cy5 photoswitching. | No Comments […]

    Pingback by Everyday Scientist » how Cy5 switches — December 7, 2009 #

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