Hein, B.

Hell, S.

Jakobs, S.

Kellner, R.

Medda, R.

Willig, K.

English

MPG.PuRe

Nanoscale resolution inGFP-based microscopyKatrin I Willig, Robert R Kellner, Rebecca Medda,Birka Hein, Stefan Jakobs & Stefan W HellWe report attainment of subdiffraction resolution usingstimulated emission depletion (STED) microscopy with GFP-labeled samples. The B70 nm lateral resolution attained in thisstudy is demonstrated by imaging GFP-labeled viruses and theendoplasmic reticulum (ER) of a mammalian cell. Our resultsmark the advent of nanoscale biological microscopy withgenetically encoded markers.The resolution of a light-focusing microscope is usually limited toDr¼ l / 2NA E 200 nm, with NA denoting the numerical apertureof the lens and l the wavelength of light1. Because fluorescencemicroscopy is the most popular imaging mode for subcellularresearch, the diffraction resolution limit severely hampers lifescience studies. Image deconvolution may increase the resolutionby up to a factor of two, but further improvement using mathe-matics is viable only by using a priori knowledge of the sample2.STED microscopy3,4 is distinct from commonly applied fluor-escence microscopy modes because its resolution is no longerstrictly limited by l. Rather it follows that:Dr ffi l=ð2NAffiffiffiffiffiffiffiffiffiffi1 + zpÞ;with z describing the magnitude of STED5,6. Hence, the maximumresolution attainable using this microscope is given by the largestpractical value of z, whereas the conceptual limit is about the size ofthe label.STED microscopy has proven to be decisive in solving keyproblems in biology. For example, it was used to establish thatthe synaptic vesicle protein synaptotagmin I forms clusters on thepresynaptic membrane7, a fact which has crucial implications forsynaptic protein recycling. It also revealed that the Drosophila sp.protein bruchpilot forms ring-like structures that define the synap-tic active zones in the Drosophila sp. neuromuscular junction8.Furthermore, it proved that the SNARE protein syntaxin remainsaggregated in the plasma membrane even upon massive over-expression9. All imaging applications using STED so far have reliedon organic dyes that stained organelles either directly4 or throughantibody labeling10. Here we reveal that nanoscale resolution canalso be realized with fluorescent proteins.A STED microscope design capable of imaging GFP is shown inFigure 1 (see Supplementary Methods online for details). Theprotein is excited at l¼ 490 nm using a laser diode emitting 100-pspulses (PicoTA, Picoquant) operating at 80 MHz. The resultingfocal excitation spot with a full-width-half-maximum (FWHM) of240 nm represents the resolution obtained by conventional micro-scopy, that is, the Airy disk of the 1.4 NA lens used (Planapo oil,100; Leica).STEDExcitationFluorescence Excitation490 nmSTED575 nmPhase-retardation0 2πExcitation/emissionWavelength (nm)a bcSTEDExcitationEGFP350 400 450 500 550 600 65000.51.0DetectionDetectorDC1DC2MLensExcitation STEDPAL-SLMFigure 1 | STED microscope for GFP imaging. (a) Blue (490 nm) excitationlight forms a regular Airy disk in the lens focal plane and is overlapped witha yellow (575 nm) doughnut spot for STED. The doughnut is produced byapplying a helical phase delay on the STED beam wavefront, exp(if), with0 r f r 2p, using a programmable wavefront modulator (PAL-SLM,Hamamatsu Photonics). The linear helical progression of the phase from0 to 2p ensures that the light field from opposite parts of the entrance pupilof the lens interferes destructively on the optic axis. The focal intensitythus assumes a cylindrical shape centered around the dark optic axis.(b,c) Excitation, fluorescence and stimulated emission in the energydiagram (b) in relation to the excitation and emission spectrum of GFP (c).STED is performed at the red tail of GFP emission.RECEIVED 5 JUNE; ACCEPTED 17 JULY; PUBLISHED ONLINE 9 AUGUST 2006; DOI:10.1038/NMETH922Max Planck Institute for Biophysical Chemistry, Department of NanoBiophotonics, Am Fassberg 11, 37077 Göttingen, Germany. Correspondence should be addressed toS.W.H. (shell@gwdg.de).NATURE METHODS | VOL.3 NO.9 | SEPTEMBER 2006 | 721BRIEF COMMUNICATIONS©2006 Nature Publishing Group  http://www.nature.com/naturemethodsTo overcome this limit, the excitation spot is overlaid with adoughnut-shaped counterpart, the STED beam, which provides200-ps pulses of lower-energy photons for molecular de-excitation,synchronized at 80 MHz. The fluorescent state depopulates asexp(–z)  exp(–sF), with F denoting the number of stimulatingphotons per pulse and per area. With a typical cross-section forstimulated emission, s E 1017 cm2, we require F 4 1017photons/cm2 to obtain z 4 1 and hence subdiffraction values Dr.As the doughnut area is B109 cm2, the STED pulse shouldcontain at least 108 photons, corresponding to a STED beamaverage power on the order of P E 3 mW.The value of P to be actually applied depends inversely on theprecise value of s, which in turn scales with the emission spectrumof the dye11. Tuning the STED pulse to the peak emission (510 nm),however, is not advisable for imaging GFP, because of the residualexcitation at this wavelength (Fig. 1c). Screening revealed that forimaging GFP, STED prevails from 570 to 600 nm. Therefore, weoptimized our emission filter to detect fluorescence in the 510–560nm range. We focused the fluorescence onto a detector openingthat was equivalent to common pinhole settings used in confocalmicroscopy1. Confocalization is not required, but it convenientlyprovides confocal axial resolution and, by blocking the STED beam,direct comparison with this standard technique. We acquired theimages by scanning the sample with a piezo stage.To explore the resolution, we tuned to575 nm and imaged rotavirus-derived parti-cles12. The rotavirus is trilayered, containingthe VP2 proteins in the innermost layer, VP6in the medial layer, and VP4 and VP7 in itsoutermost coat. Coexpression of VP6 and GFP-tagged VP2 in abaculovirus expression system yields double-layered, GFP-taggedvirus-like particles containing GFP-VP2 molecules in their 40-nmdiameter inner capsids12.Comparison of confocal and STED recording at 7.2 mW(excitation power 70 nW) reveals the improved resolution of thelatter technique (Fig. 2). The virus-like particles are individuallyresolved in areas where confocal microscopy only renders blurredpatches, and this finding is substantiated by the raw data profiles(Fig. 2e). Featuring an FWHM of 190 nm, the confocal profile isbasically the point-spread function (PSF) of a confocal micro-scope1. The profile in the STED images displays an FWHM of76 nm. Considering the 40-nm capsid, it follows that the PSF of theSTED imaging mode features an FWHM of B70 nm.To label the ER of fixed mammalian cells, we expressed GFPfused to an ER-targeting sequence in rat kangaroo kidney epithelial(PtK2) cells (Fig. 3). Comparison between the confocal and theSTED recordings reveals the improved resolution of the latter.Linear deconvolution of raw data increases the resolution further.The addition of the STED doughnut reduced the spot area byninefold, but, owing to its cylindrical shape, did not squeeze thespot axially. Therefore, in a convoluted organelle like the ER, theimproved lateral resolution alone cannot unfold the method’s fullpower. Remedies to this problem that leave the cell intact are anac dbe f200160120804000FWHM (nm)3001000–1003001000–100–300x (nm)x (nm)10203040OccurenceSTED76nm190nmConfocal00.51.0Intensity (a.u.)STEDConfocalxyConfocal STED1880 Counts / 0.3 ms 760 Counts / 0.3 msConfocal STED60Counts / 0.3 ms0190Counts / 0.3 ms0STEDlin decSTEDSTEDConfocallin decConfocalConfocala b c de fFigure 3 | Subdiffraction imaging of GFP-labeledER in PtK2 cell (a–d) Confocal (a) and STED (b)images. The improved resolution in b is alsoevident in c and d displaying magnified views ofthe boxed regions in a and b, respectively; datais smoothed by a Gaussian filter over 1.5 pixels.(e,f) Images after a regularized lineardeconvolution using the PSF obtained fromFigure 2. The arrows indicate details indeconvolved STED data (f) that are notobtainable via deconvolution of confocal data (e).Scale bars, 1 mm.Figure 2 | STED overcomes the diffractionresolution barrier in GFP-based microscopy.(a,b) GFP-labeled rotavirus-derived particlesimaged confocally (a) and with STED (b). Thethreefold increase in lateral resolution in bseparates many more of the 40-nm sized particles.(c,d) Magnified view of the marked areademonstrates blurred blobs in the confocal image(c), whereas most of the particles are resolved bySTED (d). (e) Raw data profile through an overlayof ten individual particles displays a FWHM of 190nm for the confocal and 76 nm for STED mode.(f) Histograms of the FWHM detail the decreaseof spot size. Scale bars, 500 nm.722 | VOL.3 NO.9 | SEPTEMBER 2006 | NATURE METHODSBRIEF COMMUNICATIONS©2006 Nature Publishing Group  http://www.nature.com/naturemethodsadditional STED beam with specific peaks above and below thefocal plane, or the combination of STED with 4Pi microscopy10.Keeping the intensity at the doughnut center close to zero isimperative in STED microscopy, because a finite STED beamintensity at the minimum reduces the flux of the fluorescencephotons from the central spot to be recorded. If the centralminimum amounts to b percent of the doughnut crest, the peakflux is approximately exp(–0.01bz) times its value without STED,meaning that b should not exceed B5%. Therefore, the applicationof STED microscopy in scattering media, such as GFP-labeledtissue, depends on how well the ‘zero’ can be kept free of scatteredSTED light. Our preliminary results with organic dyes displaysubdiffraction resolution several cell layers deep. Several optionscan readily be envisaged to keep b low, such as the application ofactive optical elements and of annihilating light fields.We are confident that the resolution can be further augmented byrefining the pulse duration and the timing. Live-cell GFP imagingshould be facilitated by implementing rapid image acquisitionthrough beam scanning. The most straightforward option is tomerge the excitation and the phase-modified STED beam in acommon path and subject both beams to fast galvanometerscanning. As any increase in resolution calls for finer imagepixelation, it will be preferable to reduce the field of view by thesame factor by which the spot area has been decreased. Because thespot size can be tuned by adjusting z, one can balance between thedegree of resolution on the one hand, and the signal strength, theimaging speed and the field of view on the other hand. In thefuture, this zooming should be possible while scanning.We also obtained B70-nm resolution with the enhanced yellowfluorescent protein (EYFP) by applying STED at 595 nm. Theyellow protein mCitrine13 can also be depleted at this wavelength.The red fluorescent protein mRFP14 exhibits STED at 710 nm.Screening of recent additions to the fluorescent protein family15 isunraveling further options; for example, mStrawberry lends itself todepletion at 666 nm.De-excitation by stimulated emission is as fundamental a mole-cular process as excitation. STED is therefore prevalent in allfluorophores, reaching its maximum efficiency at predefined wave-lengths and pulse durations. In fact, screening of organic dyes hasso far shown that STED can be applied to any dye with a fluorescentlifetime 40.8 ns. Therefore, STED microscopy is compatible withmulticolor labeling. A possible combination of functionalizedorganic dyes is Atto532 (AttoTech), Dy-510XL (Dyomics) andAtto647N (AttoTech), featuring green (550 nm), yellow (590 nm)and red (670 nm) emission peaks, respectively. We also expectfluorescent protein–based multicolor STED microscopy to beviable, for example with the set of proteins listed above. For severaldye pairs, for example GFP and mCitrine-EYFP, the same excitationor STED wavelength can be applied, so that the set of requiredwavelengths can be conveniently reduced. Notwithstanding this,multicolor STED microscopy should be greatly facilitated byemerging technologies. Compact ‘white light’ sources such asdiode-pumped photonic crystal fibers provide spatially cleanwhole-spectrum (500–900 nm) subnanosecond pulses that shouldbe ideal for STED.In conclusion, nanoscale fluorescent protein microscopy shouldopen up a new avenue for answering many key questions in biology.Note: Supplementary information is available on the Nature Methods website.ACKNOWLEDGMENTSPurified GFP-VLPs were provided by J. Cohen and the plasmid eGFP-ER by P. Lipp.We thank S. Verrier, T. Rosenmund, A.C. Schauss, J.J. Sieber and T. Müller forproviding samples, A. Schönle for help with the software ImSpector, V. Westphaland B. Harke for valuable discussions, as well as J. Keller and B. Rankin for criticalreading. We thank R.Y. Tsien for providing the plasmids coding for mCitrine,mStrawbery and mRFP.COMPETING INTERESTS STATEMENTThe authors declare that they have no competing financial interests.Published online at http://www.nature.com/naturemethods/Reprints and permissions information is available online athttp://npg.nature.com/reprintsandpermissions/1. Pawley, J.B. (ed.) Handbook of biological confocal microscopy (Springer, New York,2006).2. Conchello, J-A. & Lichtman, J.W. Nat. Methods 2, 920–931 (2005).3. Hell, S.W. & Wichmann, J. Opt. Lett. 19, 780–782 (1994).4. Klar, T.A., Jakobs, S., Dyba, M., Egner, A. & Hell, S.W. Proc. Natl. Acad. Sci. USA 97,8206–8210 (2000).5. Hell, S.W. Nat. Biotechnol. 21, 1347–1355 (2003).6. Westphal, V. & Hell, S.W. Phys. Rev. Lett. 94, 143903 (2005).7. Willig, K.I., Rizzoli, S.O., Westphal, V., Jahn, R. & Hell, S.W. Nature 440, 935–939(2006).8. Kittel, R.J. et al. Science 312, 1051–1054 (2006).9. Sieber, J.J., Willig, K.I., Heintzmann, R., Hell, S.W. & Lang, T. Biophys. J. 90,2843–2851 (2006).10. Dyba, M., Jakobs, S. & Hell, S.W. Nat. Biotechnol. 21, 1303–1304 (2003).11. Schäfer, F.P. Dye Lasers (Springer, Berlin, 1973).12. Charpilienne, A. J. Biol. Chem. 276, 29361–29367 (2001).13. Griesbeck, O., Baird, G.S., Campbell, R.E., Zacharias, D.A. & Tsien, R.Y. J. Biol.Chem. 276, 29188–29194 (2001).14. Campbell, R.E. et al. Proc. Natl. Acad. Sci. USA 99, 7877–7882 (2002).15. Shaner, N.C., Steinbach, P.A. & Tsien, R.Y. Nat. Methods 2, 905–909(2005).NATURE METHODS | VOL.3 NO.9 | SEPTEMBER 2006 | 723BRIEF COMMUNICATIONS©2006 Nature Publishing Group  http://www.nature.com/naturemethods

Nanoscale resolution in GFP-based microscopy.

https://pure.mpg.de/pubman/item/item_596155_4/component/file_596154/287629.pdf

Nanoscale resolution in GFP-based microscopy.

Abstract

Similar works

Full text

Available Versions

MPG.PuRe