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Index a beam aberrations 332 absorption 14, 16–18, 61, 67, 85–89, beamlets 466 106, 136, 140, 141, 149, 153, 157, beam propagation 466 166, 188, 208, 212–214, 218, 223, beam splitter 311 226, 230, 236, 255, 274, 276, 348, O2 benzylcytosine (BC) 145 389, 394, 397, 408, 410–412, 416, Bessel beam 262 419, 420, 448, 449 Bessel function 38 Abbe’s diffraction limit 368 bioluminescence 95 absorption dipole moment 416 binning 106 acceptor density 426–428 biplane imaging approach 284 acceptor emission filter 431 birefringence 13 acceptor fluorophores 413 bleaching 120 acousto-optical modulator (AOM) 345 blue-shifted dye 334 Airy disk 38, 41–44, 48 Bragg condition 33 Airy disk radius 458 Braun tubes 181 Airy pattern 173, 174 bright field 31–32, 44, 45, 61, 70, 71, angle of incidence 376 73, 74 angleofrefraction 377 brightness 28, 29, 57–62, 67, 74, 76, 80 animalcules 165 astigmatic imaging approach 281–284 astigmatism 21 c ATTO647N 325, 337 camera acquisition software 456 autocollimation telescope 468 carbocyanines 275, 281 autocorrelation function (ACF) 188, cell membrane glycans 283 189, 191, 192, 199 centrosomes 287 autofluorescence 381 charge-coupled device (CCD) 26, 104, Avalanche photodiode (APD) 114 107, 112, 273, 386–387 Avogadro’s number 412 Chinese hamster ovary (CHO) 381 axial resolution 45, 47, 173–179 chromatic reflectors 18–19 chromophore 274 b click chemistry 281–283 back focal plane (BFP) 249, 472–473 cluster size 397 ballistic light 377 coherent beams 295 bandpass filter 324, 326 colliding-pulse mode-locked (CPM) Bayesian statistics 404 218

Fluorescence : From Principles to Biological Applications, Second Edition. Edited by Ulrich Kubitscheck. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA. Published 2017 by Wiley-VCH Verlag GmbH & Co. KGaA. 474 Index

complementary dichroic mirror 96, 97, 102, 375 metal–oxide–semiconductor dichromatic beam splitter 468 (CMOS) 111, 112, 388 dielectric mirrors 16–17 confocal effect 346 differential interference contrast (DIC) confocality 302 13, 23 confocal laser scanning interpretation 81 (CLSM) 342 optical setup 77 confocal scanning microscopy 165, differential interference microscopy 247, 261, 429 77–80 conjugation process, synthetic different lens configurations 259–260 fluorophores 142 diffraction 1, 7, 323, 324, 326, 327 continuous fluorescence coefficients 341, 342, 346 microphotolysis (CFM) 343 limit 269, 270 continuous fluorescence photobleaching pattern 33, 34, 36 343 diffusion-enhanced energy transfer continuous illumination 358 424 continuous wave (CW) 167, 172, 329 diffusion-reaction equation 351, 355 conventional dual-color imaging scheme diffusion time 188 334 digital camera, fluorescence microscopy conventional fluorescence microscopy 113 techniques 264 digitally scanned light sheet microscopy convolution 41 (DSLM) 246, 248 convolution theorem 294, 298 4,4′-diisothiocyanatostilbene-2, copper-catalyzed azide–alkyne 2′-disulfonic (DIDS) 352 cycloaddition 282 dipole–dipole distance 414 Coulombic coupling 411, 414 dipole moments 414, 416 Coulomb’s law 414, 418 Dirac delta distribution 298 coupling FRET 425 discovery, fluorescence of 91–92 critical angle 378 discriminator 172 critical illumination 28–30 dispersion 9 crystallography techniques 422 donor fluorophores 425 cut-off frequency 295, 302 donor emission spectrum 412 cyan fluorescent protein (CFP) 152, donor excitation filter 431 423 donor quantal spectrum 412 cylindrical lens 246, 248 donor quantum yield 412, 417, 431, d 438, 440 DAOSTORM 287 donor quenching (DQ) 428–429, 432 Davson–Danielli–Robertson model double helix PSF 284 341 double-sided illumination 253, 254, defocused imaging 395 258 Deinococcus radiodurans 150 doughnut-shaped depletion beam depth of field (DOF) 49, 81, 392 335 depth of focus 49, 256, 313, 392 dSTORM 275, 282 detection, PSFs 175 dual-color imaging 334 development, dual-view system 375 169 dynamic imaging 373 Index 475 e fluorescence photobleaching techniques electrically tunable lenses (ETLs) 248, CLSMs-assisted photobleaching 256 methods electromagnetic radiation 391, 408 implementation 358–359 electronic delay 332 opportunities 359 electronic filters 103 concepts and procedures electron-multiplying charge-coupled complexity from bottom up device (EM-CCD) 109, 387 346–347 emission dipole 416, 417 principle and several modes emission spectrum 90, 99, 411 340–345 epifluorescence 98 set up 345–346 epi-illumination 31–32 continuous fluorescence equilibrium properties, fluorescence microphotolysis (CFM) emission 88–89 background and data evaluation excitation, PSFs 175 354–357 excitation filter 101 combination of 357 excitation light distribution 311–313 variants 357–358 excited state 325, 327 fluorescence recovery after exciting light intensity 99, 122–123 photobleaching (FRAP) exciting photon 89 binding 350–351 diffusion measurements f 347–350 Fermi’s golden rule 414, 416 membrane transport 351–354 field of view (FOV) 247, 248 fluorescence photon 88 finite optics setup 25 fluorescence quantum yield 136, 147 FlAsH 148 fluorescence recovery after fluid mosaic model 341 photobleaching (FRAP) 160, fluorescein 410 341 fluorescence confinement 326 fluorescent labels fluorescence correlation spectroscopy characteristics 134 (FCS) 172, 354 properties 133, 135, 142 fluorescence cross-correlation sensors in cell 160 spectroscopy (FCCS) 190 fluorescent protein (FPs) 152, 156, fluorescence depletion 326 324, 329, 337 fluorescence emission 272, 300 fluorophore 270, 407 depolarization of 428 classes of 337 fluorescence excitation 293 emission spectrum 326, 336 fluorescence filter 246 excitation 326 fluorescence intensity, measurements of kinetics 328 119–121 photoactivatable/photoconvertible fluorescence labeling 368 272 fluorescence lifetime changes 437–441 photobleaching 270, 355 fluorescence markers 157 photon number 280 fluorescence microscopy 86, 90–95, photoswitchable 273 299 property of 342 fluorescence photobleaching recovery reaction-induced photoswitching (FPR) 341 273 476 Index

fluorophore (contd.) green fluorescent protein (GFP) 150, rhodamine 275 281, 368, 410 ROP 354 grid projection 295 for STED microscopy 337 stimulated emission depletion 324 h flux equation 356 hetero-FRET 413 focal lengths 251 high-pressure mercury vapor focal plane 250, 260, 471–472 arc-discharge lamps (HBO) 99 Förster distance 413, 420 burners 100 Förster equation 414–418 high-resolution information 300–301 Förster radius 399, 400 homo-FRET 414 Förster resonance energy transfer Huygens–Fresnel integral equation 35 (FRET) 155, 191, 369 Huygens wavelet 37 Fourier frequency 36 i Fourier theory 41 illumination intensity (I) 381 Fourier-transformed illumination illumination microscopy 296 pattern 299 illumination pattern 298–301, 4Pi microscopy 357 303–307, 313 Fraunhofer diffraction pattern 38 illumination photons 324 frequency-domain FLIM 443–444 illumination time 381, 382 frequency spectra 415 image brightness 57 fringe distance 315 image contrast 90–93 fringe period 313 image deconvolution 184, 462 fringe projection, see two-beam image integration time 109 interference image processing operations full quantitation 433–434 focal plane 37 full width at half-maximum (FWHM) pixel groups 461–462 175, 372 single pixels 460–461 functional magnetic resonance imaging imaging artifacts 278 (fMRI) 237 imaging contrast, measures improving 285 g immersion media 62–65 Gaussian beam 250–252 immunofluorescence 281 Gaussian filters 462 immunoglobulin G antibodies 147, Gaussian function 272, 394 282 Gaussian intensity profile 348 induced acceptor emission 429–432 Gaussian photon distribution 372 infinity space 24 Gaussian-shaped signal distribution instrument response function (IRF) 390 172 geometric-optical term 176 integrator 172 global fitting approaches 446 intensified charge-coupled device gratings 34, 37, 38, 57, 330 (ICCD) 104, 107, 109 constant 8, 33, 35 intensity-based measurements, FRET distance 299, 312 432–433 structure 34 intensity distribution 173 gray values 189, 317, 455 intensity histograms 288, 459–460 Index 477 intensity zero 332 dark-field 68 interference effects DIC 77 light rays 11 interference contrast 74 pattern 304 optical contrast methods 68 interference microscopy 77 phase contrast, see phase contrast interferometer 303, 304, 309 phase objects 67 plane 306 entrance pupil 50 setup 308 exit pupil 50, 51 interferometric imaging 284–285 field-of-view 28 intermediate image 25 high resolution objectives 65 intrinsically photoswitchable probes illumination system 28 274 beam path 30 inverted 32, 473 image path 24 in vivo/in vitro measurements, FRET magnifications 26 401 CCD 26 IrisFP 154 focal length 27 isoSTED configuration 336 magnifying glass 27 multiview imaging 257–259 j numerical aperture 27 Jablonski diagram 92, 325 telecentricity 51 wave optics and resolution 32 k light propagation 377 Köhler illumination 28–30, 379, 383, light sheet fluorescence microscopy 384 (LSFM) 246 kinetic measurements, FRET 401 light sheet illumination 254, 259, 263 kinetics, fluorescence emission 88–89 light sheet microscopy construction and working 248–249 l photobleaching and toxicity 247 Lambert radiator 58 polarization 253 Lampyris noctiluca 93 principle of 246–247 laser scanning microscopes (LSMs) 3D imaging 255–357 180, 362 water-dipping illumination lens 250 label size vs. structural resolution light sources, STED 332–333 280–282 light waves 2 lateral resolution 41, 175 circular polarized 4 coherent light sources 43 on interference 2 incoherent light sources 41 plane wave 7 optical units 47 transverse wave 3 lens alignment 468 description 3 lens classes 61–62 linear 299 lens design 53–57 polarization 378 light-emitting diodes (LEDs) 100, 101 lipofuscin 316, 318 light microscopy 372 live-cell dynamics 160 angular aperture 27 live-cell labeling 282 apertures 50 LivePalm 286 components 23 localization errors 280 contrast 67 localization precision (LP) 280, 374 478 Index

localization-based super-resolution n microscopy nanocrystals 140 experimental setup for 275–277 nanometers 377 fluorescence labeling 280–285 near-field scanning optical microscopy imaging contrast, measures for (NSOM) 383 improving 285 near-infrared (NIR) region 86 intrinsically photoswitchable probes negative sign, image formation 35 274 N-hydroxysuccinimide (NHS) esters optical resolution and imaging 142, 143, 146–149 artifacts 278–280 Nipkow disk 181, 183 organic fluorophores, photoswitching nitrilotriacetic acid 144 of 275 NMR 422 photoactivatable and noise detector, EM-CCD 389 photoconvertible probes 274 noninvasive measurements 402 single-molecule localization nonlinear 203, 239 microscopy (SMLM) nonlinear optics 206 quantification of 288–289 non-natural amino acids 146 reference structures for nonscanning applications 186 287–288 normalization factor 178 software 285–287 number and brightness analysis (N&B lock-in techniques 384 analysis) 195 Lorentzian model function 327 numerical aperture (NA) 165, 247, 372 low-pass filters 462 Nyquist–Shannon sampling theorem 278 m Nyquist theorem 384, 458 magnification, optical system 48–49 maleimides 143 o mean-square displacement (MSD) objective lens 326 400 objective-type TIR 378 metal halide lamps 99 object size estimation 313 Michaelis–Menten equation 353 occupancy errors 434 microchannel plate 107 oblique angles 466 Micrographia 165 optical aberrations 20 microtubulin 277, 287, 288 optical components, light microscopes mirror alignment 467–468 26 modulation contrast 314, 315 optical delay 332 Moiré effect 300, 301 optical elements 13–19 molecular brightness 136, 196 optical filters 17–18 molecular complexes, stoichiometry of optical lenses 13–15 371 optical metallic mirrors 15–16 Monte Carlo simulations 425 optical parametric oscillator (OPO) multicolor imaging 373 217 multidirectional SPIM (mSPIM) 255 optical path length difference 37 multiparameter fluorescence detection optical projection tomography (OPT) (MFD) 193–195 262 multiple acceptors 424–425 optical resolution 126, 278 multiple donor–acceptor pairs 424 optical sub-diffraction techniques 422 Index 479 optical transfer function (OTF) 40–41, photomultiplier tube (PMT) 113–114, 294, 298 169, 342, 432 organic dyes 138 photon antibunching 397 organic fluorophores, photoswitching of photon noise/shot noise 117 275 photoswitchable fluorophores 273 orthogonal-plane fluorescence optical phototoxicity 125, 137 sectioning (OPFOS) 245 phycobiliproteins 149 oscillation probability 418 physical grating 306 oscillator strength function 416 picture elements, camera 105 oversampling 50 pinhole 169 pivoting light sheet 254 p pixel PA-green fluorescent protein (PA-GFP) oversampling 384 234 size 384 Palm3d 286 undersampling 384 paraffin 316 pixelation noise 390 particle 1, 2, 6 planar distribution 425 parallel data acquisition 455 Planck constant 414, 448 patterned techniques, application of plane of incidence 11 315 plano-convex lenses 466 Perrin formula 448 pointillism 271 Perrin’s equation 414, 415 point spread function (PSF) 38, 40–42, Perrin’s estimation 411 48–50, 65, 67, 294, 297, 314, 323, phase contrast 69 357 conjugate planes 70 Poissonian signal fluctuations 390 focal plane 73 polarization microscopy 395 phasor diagrams 72 polarization vector 305 phase ring 73 precise measurements 399–400 properties 74 primary image plane 25 Zernike’s experiments 70 principles, confocal microscopy of 166 phase fluorimetry 443 prism-type TIR 378 phase modulation technique 443 protein–lipid ratio 341 phasor plot 447 pulsed interleaved excitation (PIE) 191 phosphorescence 88 pulsed irradiation 358 photoactivatable fluorescent proteins pulsed lasers 328 (PA-FPs) 277 diodes 332 photoactivatable (PA) 151, 274, 369 laser systems 332 photobleaching 124, 138, 329, 337 pulse synchronization 332 CLSMs 358 curve 397 q effects 299 Quantenspektrum 416 localization microscopy 374 quantification methods 425 quantum efficiency 342 quantitative measurements 401 technique 435 quantum efficiency 89 photoconvertible probes 274 quantum yields 399, 412, 413, 415, 417 photodetectors 104, 346 QuickPALM 284 480 Index

r sensitive detection 94 radial resolution 173–179 selective plane illumination microscopy radiative lifetime 440 (SPIM) 206, 246 radical anion 275 sensitized emission 429 read noise 118 sensors rapid diffusion limit 424 CCD 107 rapid lifetime determination 445 FRET 431 rapidSTORM 286 sequential data acquisition 457–458 raster image correlation spectroscopy Shannon/Nyquist sampling frequency (RICS) 198 333 rate of transfer 413, 414 shear plate 465 ratiometric measurements (pH) 121 signal fluctuation 389 Rayleigh distance 294 signal-to-noise ratio Rayleigh length 251 detector noise 389 reaction-induced photoswitching 273 signal fluctuation 389 reactive oxygen species (ROS) 137 silicon–rhodamine (SiR) dyes 145, 337 ReAsH 148 sine condition 59–60 redox chemistry 275 single-molecule emitter 397 red-shifted dye 334 single-molecule fluorescence resonance reducing and oxidizing system (ROXS) energy transfer 400 337 single-molecule localization microscopy reflected light image 97 (SMLM) 324 reflection 10–11 principle of 271, 272 refraction 9–10 quantification of 288 refractive index 9 reference structures for 287 region of photolysis (ROP) 342, 343, software 285 346 single-molecule methods, relaxation 325 mobility/anisotropy 371 resolution limit, microscope 35 single-molecule microscopy reversible photoswitching 274 collection efficiency 377 reversible saturable optical fluorescence dual-view system 375 transitions (RESOLFT) 161 dynamic imaging 373 Rhodococcus rhodochrous 145 magnification/resolution 376 rigorous approach, optical resolution multicolor imaging 373 33 photoactivation/photoswitching 373 s photobleaching localization saturated structured illumination microscopy 374 microscopy (SSIM) 161 single-molecule/-particle tracking saturation effects 299, 372 374 scanning confocal imaging 179, 195 super-resolution imaging 372 scanning tunneling microscopy (STM) single-molecule signals 269 brightness 394 scientific complementary color 395 metal-oxide-semiconductor defocused imaging 395 (sCMOS) 247, 277 intensity pattern 393 secondary image plane 25 orientation 394 Index 481

point spread function 391 interference pattern 304 polarization microscopy 394 optical sectioning 301 single-molecule tracking 260 super-resolution microscopy single-photon avalanche diode (SPAD) resolution limit 294 401 spatially modulated illumination single-sided illumination 254, 258 (SMI) solvent relaxation 88 excitation light distribution 311 small objects, misrepresentation object size estimation 313 127–128 overview 309 Snell’s law 377 setup 311 spatial frequency 36, 294 superman cape function 419 spatial resolution 270 synchronization, STED 332 fluorescence microscope 280 synthetic grating 306 spatially modulated illumination excitation light distribution 311 t object size estimation 313 tetracysteine 144 overview 309 thiols 275 setup 311 transfection 159 specificity, fluorescence labeling three-beam interference 295, 302 93–94 three-dimensional SMLM 283 spectral emission bands 431 three-dimensions, fluorescence spectrofluorometry 428 microscopy 121–122 spinning disk confocal microscope time-correlated single-photon counting (SDCM) 181, 184 444–445 stage scanning 179–180 time-domain FLIM 442, 444 standard deviation 372 time-of-flight camera 444 stimulated emission depletion (STED) total internal reflection (TIR) 378 155, 324, 357 total internal reflection fluorescence applications (TIRF) microscopy 354 fluorophore, choice of 336 collimation 384 labeling strategies 337 illumination time (till) 382 experimental setup intensity 380 axial resolution, improving 335 polarization 382 multicolor imaging 334 uniformity 379 scanning and speed 333 wavelength 383 fundamental concepts 324 two-beam illumination 302 key parameters in 328 two-beam interference 295, 297, 298, stochastic optical reconstruction 301 microscopy (STORM) 161, two-dimensional (2D) grating 301 275, 422 two-photon excitation microscope stochastic switching processes 324 203, 329 Stokes’ shift 409–411, 420, 448 absorption 212 structured illumination microscopy advantages 220, 238 (SIM) caged compounds 233 high-resolution information 300 continuous wave 208 illumination pattern 303 depth of imaging 230 image generation in 297 description 205 482 Index

two-photon excitation microscope u (contd.) undersampling 50 detection strategies 219 focal volume 210 v history and theory 207 Venus-YFP (yellow fluorescent protein) in vivo image 229 423 lasers 216 von Bieren condition 33, 35, 60 CPM 218 OPO 217 w pulse widths 218 water-dipping lenses 259 limitations 222 wave 1–11, 13–18, 20, 23, 32–50, 57, diphenylhexatriene 224 67, 70–80 heating, laser powers 223 wavelength effects 330 hyperspectral image 229 wavelength fluorophores 419 photobleaching 227 widefield fluorescence microscope 271 spatial resolution 223 widened parallel laser beam 465–467 PA-GFP 234 single lens 468–470 photochemistry 231 Wollaston prism 375 photobleaching 211 quantum dots 239 x scattering 213 X-ray crystallography 372 UV excitation 231 UV fluorophores 231 z Tyndall effect 85 zero-order diffraction 307