Microscopy beyond imaging: space-resolved photochemistry and ...

This article is published as part of a themed issue ofPhotochemical & Photobiological Sciences onMicroscopy beyond imaging: space-resolvedphotochemistry and photobiologyEdited by Frans de Schryver and Santi NonellPublished in issue 4, 2009EditorialMicroscopy beyond imaging: space-resolved photochemistry and photobiologyF. de Schryver and S. Nonell, Photochem. Photobiol. Sci., 2009, 8, 441PerspectivePhotosensitized production of singlet oxygen: spatially-resolved optical studies in singlecellsT. Breitenbach, M. K. Kuimova, P. Gbur, S. Hatz, N. B. Schack, B. W. Pedersen, J. D. C. Lambert, L.Poulsen and P. R. Ogilby, Photochem. Photobiol. Sci., 2009, 8, 442CommunicationTowards direct monitoring of discrete events in a catalytic cycle at the single molecule levelR. Ameloot, M. Roeffaers, M. Baruah, G. De Cremer, B. Sels, D. De Vos and J. Hofkens, Photochem.Photobiol. Sci., 2009, 8, 453PapersSpatial and temporal dynamics of in vitro photodynamic cell killing: extracellular hydrogenperoxide mediates neighbouring cell deathN. Rubio, S. P. Fleury and R. W. Redmond, Photochem. Photobiol. Sci., 2009, 8, 457Multicolor photoswitching microscopy for subdiffraction-resolution fluorescence imagingS. van de Linde, U. Endesfelder, A. Mukherjee, M. Schüttpelz, G. Wiebusch, S. Wolter, M. Heilemann and M.Sauer, Photochem. Photobiol. Sci., 2009, 8, 465Multiparameter fluorescence image spectroscopy to study molecular interactionsS. Weidtkamp-Peters, S. Felekyan, A. Bleckmann, R. Simon, W. Becker, R. Kühnemuth and C. A. M. Seidel,Photochem. Photobiol. Sci., 2009, 8, 470Triplet-relaxation microscopy with bunched pulsed excitationG. Donnert, C. Eggeling and S. W. Hell, Photochem. Photobiol. Sci., 2009, 8, 481Single-molecule photophysics of oxazines on DNA and its application in a FRET switchJ. Vogelsang, T. Cordes and P. Tinnefeld, Photochem. Photobiol. Sci., 2009, 8, 486

Fig. 3 Comparison of pulsed fluorescence excitation in bunches and atconstant repetition on a dye layer of Atto532. Various trains consisting ofbunches of 10 and 5 excitation pulses, arriving in time intervals of 2.5 mswere compared with a continuous 40 MHz pulse train. The timelines ofthe different pulse trains are schematically plotted in (A). The 10 and5 pulses were either applied with a repetition rate of 40 MHz with darkperiods filling up the remainder of the 2.5 ms, or evenly distributed intime with repetition rates of 4 and 2 MHz, respectively. Illumination witha total number of 5 ¥ 10 5 pulses and thus with total acquisition timesof 250 ms (5-bunch or 2 MHz) and 125 ms (10-bunch or 4 MHz)leaves differently photobleached dark spots (B) with levels of remainingfluorescence (inset B) and integrated fluorescence signal (C) depending onthe pulse intensity I p and the pulse timing of illumination. The levels ofremaining fluorescence (profile across the photobleached dye layer) andintegrated fluorescence are plotted for I p = 2.9 MW cm -2 (arrow). Underthese conditions, the same number of pulses leads to the same levels ofphotobleaching and to a similar amount of integrated fluorescence signalat different I p (C), which changes upon addition of a triplet quencher(inset C), favoring excitation with a uniform repetition of pulses (circle).The lines in (C) are drawn for guidance.during illumination between the bunched and non-bunched modes(Fig. 3C).The time distance between succeeding pulses of 0.5 and 0.25 msof the continuous 2 and 4 MHz illumination mode, respectively,is smaller than the triplet lifetime of ~1 ms in the samplesinvestigated. 4 Only dark periods exceeding the average time neededfor triplet relaxation render ideal T-Rex illumination. 4 In our case,nearly ideal T-Rex illumination is attained only with interpulsetime differences of >2.5 ms, i.e. pulse repetition rates 400 kHz, i.e. to~1–2 MHz. The inset to Fig. 3C shows the fluorescence signal ofthe Atto532 layer in the presence of the triplet quencher againobtained for a total of 5 ¥ 10 5 pulses, for 5 pulses either bunchedor evenly distributed over the duty cycle of 2.5 ms. As before for the400 kHz T-Rex mode (Fig. 2), the (in this case ideal) 2 MHz T-Rexillumination is now favored over pulse bunching, resulting in about10% less signal of the bunched illumination as compared to itscontinuous counterpart. This difference can also be theoreticallyexplained for well determined kinetic parameters of rhodaminedyes, 18 specifically by a ~10-fold shorter triplet lifetime.This observation also highlights the dependence of our approachon environmental conditions. Since the lifetime of thetriplet state may change with experimental conditions such asadditives, concentration of molecular oxygen, or temperature,adjusting the timing of the experiment is required. Yet, the tripletlifetime is usually prolonged to >1 ms in biological samples orother mounting media, thus shifting the ideal T-Rex illuminationregime to repetition rates

ReferencesFig. 4 Balancing between large fluorescence signal and short acquisitiontime for fluorescence data recording applying the different modes ofexcitation: continuous (fast 40–80 MHz pulsed or CW excitation),excitation in bunches with an increasing number of pulses within a bunchand excitation with continuous low (400 kHz) repetition rate. Experimentaldata recorded for the GFP dye layer are given as black stars. CW or 40 MHzpulsed excitation realizes short acquisition times but also low levels ofsignal due to pronounced dark state populations (grey area). Excitationwith low repetition rates renders large signal levels at the expense of longacquisition times (orange area). This gap can be bridged by applyingbunched excitation (blue area) providing a good compromise betweenfluorescence signal and acquisition time.variable can be optimized for choosing the suitable excitationscheme.ConclusionOur study demonstrates that within a certain range fast scanningor bunched excitation provides an effective way of improvingthe photostability and fluorescence signal brought about by theT-Rex excitation scheme. Limiting the excitation to repetitive shortperiods of ~100–200 ns followed by breaks that are long enoughfor dark (triplet) state relaxation reduces the recording time whilestill operating close to T-Rex conditions. This observation holdstrue both for Atto532, a prominent member of the rhodaminedye family, and GFP, the archetype of fluorescent proteins. Theverification of this concept with these fluorophores indicates thegenerality of the concept. It is extendable to both CW andmulti-photon excitation, as well as to STED imaging. As aresult, the bunched excitation scheme provides new degrees offreedom for balancing between signal maximization and imagingspeed, thus complementing the existing modes of fluorescenceexcitation.AcknowledgementsWe acknowledge V. Westphal for helping with the electronics andS. Jakobs for providing purified GFP. We thank S. Bretschneiderand A. Giske for help with the experimental setup and A. Schönlefor support with the IMSPECTOR software.1 R. Y. Tsien, Imagining imaging’s future, Nat.CellBiol., 2003, SS16–SS21.2 J. B. Pawley, (ed.), Handbook of biological confocal microscopy,Springer, New York, 2nd edn., 2006.3 G. Donnert, J. Keller, R. Medda, M. A. Andrei, S. O. Rizzoli, R.Lührmann, R. Jahn, C. Eggeling and S. W. Hell, Macromolecular-scaleresolution in biological fluorescence microscopy, Proc. Natl. Acad. Sci.U. S. A., 2006, 103, 11440–11445.4 G. Donnert, C. Eggeling and S. W. Hell, Major signal increase influrorescence microscopy through dark-state relaxation, Nat. 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