403

Index

a bioluminescence 103 aberrations, in imaging 65 BODIPY dyes 150 aberrations, optical 23–24 Bragg condition 43 absorption filters 19 Braun tubes 191 acousto-optical modulator (AOM) 220 Brewster angle 13 acousto-optic tunable filters (AOTFs) 112–113 c actual focus point (AFP) 79 cage fluorophores 238 adaptive optics 23 carbon dots 151 advanced correlation techniques carrier proteins (CPs) 155 – burst analysis with multiparameter charge-coupled device (CCD) 113–117 fluorescence detection 202–205 – features 122 – fluorescence cross-correlation spectroscopy charge-coupled device (CCD) cameras 200–201 311–312 – pulsed interleaved excitation 201–202 chemical toxicity 147 Airy function 47–51, 56 chromatic aberrations 24, 65 Airy pattern 74 chromatic reflectors 20 AlexaFluor dyes 149 circular polarization 12 amplitude 5 CLIP tag 154 angular aperture 38, 175 Col-F 101–102 animalcules 175 coma 24 antibunching 31 complementary-metal-oxide semiconductor astigmatism 24 (CMOS) 120–121 autocollimation telescope 397 – detectors 313 autocorrelation function (ACF) 198–200, 202 – features 122 autofluorescence 309 confocal laser scanning (CLSM) avalanche photodiodes (APDs) 113, 217 123–124, 204 confocal axial resolution 56–59 – applications 196 – – advanced correlation techniques b 200–205 back focal plane, of objective lens 40, 44ff, – – beyond imaging 205–210 401–402 – – nonscanning 196–200 back-illuminated charge-coupled device – conventional widefield microscopy evolution (BI CCD) cameras 114, 117 and limits 175–177 beam walk 395–396 – history and development 177–180 Bertrand lens 85 – theory binning 116 – – confocal deconvolution 194–195

Fluorescence Microscopy: From Principles to Biological Applications, First Edition. Edited by Ulrich Kubitscheck. © 2013 Wiley-VCH Verlag GmbH & Co. KGaA. Published 2013 by Wiley-VCH Verlag GmbH & Co. KGaA. 404 Index

(contd.) digital camera 122 – – principle 180–182 direct stochastical optical reconstruction – – radial and axial resolution and pinhole microscopy (dSTORM) 331 size impact 182–189 dispersion 10 – – scanning 189–194 distance of closest approach 263 confocal photobleaching 233, 234–235 donor photobleaching 273–275 – arbitrarily shaped regions 236 donor quenching (DQ) 270 – artifacts and remedies 237–238 DRAQ5 101–102 – data evaluation improvement 236 Dronpa 239 – high time resolution 236–237 dye classes comparison 165 – laser scanning (LSMs) in dynamic imaging 299 experiments 233–234 dynamic range, of detector 125 – three-dimensional analysis 237 continuous fluorescence microphotolysis e (CFM) 217–219 electric telescope 191 – combination with other techniques 241 electron multiplied charge-coupled device – theoretical background and data evaluation (EMCCD) cameras 113 229–231 electron-multiplying charge-coupled device – variants 232–233 (EMCCD) 118–120, 312–313 continuous wave (CW) lasers 182, 381, 385 electronic filters 112–113 contrast 78–80 elliptic polarization 12 – dark field 80–81 enhanced green fluorescent protein (eGFP) – differential interference contrast (DIC) 136 89–94 epifluorescence 104 – interference contrast 86–89 epi-illumination 64 – phase contrast 81–86 – and bright field microscope 41–42 convolution theorem 346, 350 epipolic dispersion 99 corpuscular theory of light 1 excitation filters 111 coumarins 149 critical illumination 39–40, 305 f critical molecule distance 251 far field 20 cross-correlation 200 Fermi’s golden rule 28 cross-correlation function (CCF) 201 filters 19 curvature of field 24 finite optics setup 39 flavins 309 d fluorescence microcopy 97–98 dark current 126–127 – avalanche photodiode (APD) 123–124 – nonuniformity 128 – charge-coupled device (CCD) 114–117 dark field 80–81, 97 – – features 122 decay kinetics 272 – complementary-metal-oxide semiconductor – fluorescence lifetime changes 276–278 (CMOS) 120–121 – photobleaching rate 272–275 – – features 122 destructive interference 2 – current avenues of development 140 dichroic mirrors 20 – digital camera 122 dichromatic beam splitter 396 – electronic filters 112–113 dielectric mirror 18 – electron-multiplying charge-coupled device differential interference contrast (DIC) 14, (EMCCD) 118–120 89 – exciting light sources 108–110 – comparison with phase contrast 93–94 – features – image interpretation 93 – – image contrast 98–101 – optical setup of microscope 89–93 – – sensitivity of detection 102–103 diffraction 1, 7–9, 43–52, 293, 294, 298, 299, – – specificity of fluorescence labeling 309, 310, 324, 329, 330 100–101 diffusion-enhanced energy transfer 262 – intensified CCD (iCCD) 117–118 Index 405

– limitations 134 fluorescence photoactivation localization – – optical resolution 136–138 microscopy (fPALM) 329 – – photobleaching 134–135 fluorescence photobleaching recovery (FPR) – – phototoxicity 136 215 – – reversible photobleaching under oxidizing fluorescence recovery after photobleaching and reducing conditions 135–136 (FRAP) 168, 215, 217, 221–222 – – small objects misrepresentation – binding 225 138–139 – diffusion measurements evaluation – noise types in digital microscopy image 222–224 124–128 – membrane transport 226–228 – operation principle 103–106 fluorescent proteins (FPs) 146, 147, 158, – optical filters 111–112 160–161, 163, 167, 389–390 – photodetectors 113 – with modified chromophores 161–162 – photomultiplier tube (PMT) 122–123 fluorophores, as sensors inside cell 167 – quantitative fluorescence microscopy focal depth 188 – – dimension measurement in 3D focal plane of lens 34, 399–400 fluorescence microscopy 132 Forster¨ resonance energy transfer (FRET) – – exciting light intensity measurements 103, 126, 246 133 – analysis and pitfalls, 250–286 – – fluorescence intensity measurements – – average lifetime and multiple lifetime and labeled target concentration fitting, 285–286 128–131 – fluorescence lifetime imaging microscopy – – ratiometric measurements 131–132 (FLIM), 280–282 – – technical tips 133–134 – – frequency-domain, 282 – scientific CMOS (sCMOS) 121–122 – – time-domain, 283–285 fluorescence correlation spectroscopy (FCS) – historical development, 246–254 168 – measurements, 265 fluorescence labeling 143 – – decay kinetics, 272–278 – genetically encoded labels – – spectral changes, 266–272 – – GFP-like proteins 158–163 – and molecular conformations – – phycobiliproteins 158 determination, 325–329 – key properties 144–148 – as molecular ruler, 258–261 – principles 143–144 – to monitor intramolecular conformational – selection, for particular applications 163 dynamics, 163–167 – – fluorophores as sensors inside cell 167 – requirements, 254–258 – – FRET to monitor intramolecular – special conditions, 262–265 conformational dynamics 163–167 Fourier frequency 46–47 – – live-cell dynamics 168 frame-transfer CCD 118 – – protein expression in cells 167 Franck Condon principle 27 – synthetic fluorophores Frits Zernike’s experiments 82–85 – – conjugation strategies 152–155 front-illuminated charge-coupled device – – fluorescent nanoparticles 150–152 (FI CCD) cameras 114, 116–117 – – fluorophore and target 156–157 full width at half maximum (FWHM) 53, – – nonnatural amino acids 155–156 183, 187–188, 354 – – organic dyes 149–150 fusion proteins 154–155 fluorescence lifetime imaging microscopy (FLIM) 245, 277, 280–282 – frequency-domain g – – operational principle and technical Gaussian function 184–185 aspects 282 genetically encoded labels – time-domain 283 – GFP-like proteins 159–163 – – time-correlated single-photon counting – phycobiliproteins 158 283 global fitting approaches 286 – – time gating 284–285 gold nanoparticles (AuNPs) 151 406 Index

green fluorescent protein (GFP) 159–163, – – illumination beam path 39–42 208, 307 ––imagingpath 34–36 – – magnification 36–37 h – contrast 78–80 HaloTag 154 ––darkfield 80–81 high-pressure mercury vapor arc-discharge – – differential interference contrast (DIC) lamps (HBO) 108–110 86–94 Huygens’ principle 7–8, 17, 47 – – phase contrast 81–86 Huygens–Fresnel integral equation 45–46 – microscope objectives 64 Huygens wavelets 43, 46, 48, 55 – – immersion media 73–77 – – light collection efficiency and image i brightness 68–72 image contrast 80–93, 98–101 – – objective lens classes 73 imaging optical path 41 – – objective lens design 64–68 immersion media 73–77 – – specific applications 77 impact ionization 119 – wave optics and resolution 42–43 infinity space 34 – – Airy function 47–50 instrument response function (IRF) 182 – – axial resolution 56–59 intensified CCD (iCCD) 117–118 – – depth of field and focus 60–61 intensified charge-coupled device (iCCD) – – imaging process description 43–47 113 – – lateral resolution of coherent light sources interference 54–56 – diffraction and refraction 7–14 – – lateral resolution using incoherent light – light as wave 2–7 sources 52–54 interference contrast 86–89 – – magnification and resolution 59–60 interferometer of Mach–Zehnder type 3 – – over and under sampling 61 internal conversion (IC) 144 – – point spread function and optical transfer inverted microscopes 42 function 50–52 lipofuscin 309, 369 j live-cell dynamics 168 Jablonski diagram 26, 29, 100, 144 localized precision (LP) 299, 300, 302, 310, – simplified 377 311, 316–317, 330 long-distance objectives 77 k Kohler¨ illumination 40–41, 63, 305, 308 m Kohler¨ principle 82 mean square displacement 333 mercury arc lamps 108 l metal halide lamps 108 Lambert radiator 69 metallic mirror 17–18 laser scanning 190 microchannel plate (MCP) 117, 282, 285 laser scanning confocal microscope 113 mirror alignment 395–396 laser scanning cytometer (LSC) 130 Moire´ effect 352 laser scanning microscope (LSM) 375, 384 molecular brightness 147 lenses 14–17 molecular physiology 245 – alignment 396 monochromatic aberrations 65 lensmaker’s equation 15 multicolor imaging 299 light-emitting diodes (LEDs) 110 light microscopy 33 n – apertures, pupils, and telecentricity near field 20 61–64 N-hydroxysuccinimide (NHS) esters – construction 152–153 – – angular and numerical aperture 38 Nipkow disk 191–192 – – components 33–34 noise types, in digital microscopy image – – field of view 38–39 124–128 Index 407

Nomarski prisms 93 p nominal focus point (NFP) 79 paraxial theory 56–57 nonscanning applications patterned techniques, application of – fluorescence correlation spectroscopy (FCS) 368–372 196–200 Pendry’s near-field lens 23 number and brightness analysis Perrin equation 247 205–208 phase contrast 81–82 numerical aperture (NA) 38, 298, 302–305, – Frits Zernike’s experiments 82–85 317, 322, 355 – images properties of, 85–86 – microscope setup 85 Nyquist theorem 310 phase front 4, 8 phase mask (PM) 378, 383 phase ring 85 o phasor diagrams and complex wave 5–7 objective lens classes 73 phasor plot 287 on-chip multiplication 118, 120 photoactivatable green fluorescent protein optical alignment 393 (PA-GFP) 329 – autocollimation telescope 397 photoactivated localization microscopy – focal plane of lens 399–400 (PALM) 329 – lens alignment 396 photoactivation and dissipation – mirror alignment 395–396 – basic aspects 238–239 – objective lens back focal plane – reversible flux measurements 239–241 401–402 – theory and instrumentation 239–239 – single lens alignment using laser beam photobleaching 134–135, 148 397–399 – approaching complexity from bottom up – widened parallel laser beam 393–395 220–221 optical contrast methods 80 – confocal 233–235 optical filters 111–112 – – arbitrarily shaped regions 236 optical path length differences (OPDs) – – artifacts and remedies 237–238 48, 68 – – data evaluation improvement 236 optical reciprocity 16 – – high time resolution 236–237 – – laser scanning microscopes (LSMs) in optical resolution 136–138 experiments 233–234 optical transfer function (OTF) – – three-dimensional analysis 237 52, 346–347, 354, 362 – continuous fluorescence microphotolysis optics and photophysics 1–2 (CFM) 228 – diffraction and refraction 7–14 – – combination with other techniques – far-field, near-field, and evanescent waves 231–232 20–23 – – theoretical background and data – light as wave 2–7 evaluation 229–231 – optical aberrations 23–24 – – variants 232–233 – optical elements 14 – fluorescence recovery after photobleaching – – chromatic reflectors 20 (FRAP) 168, 215, 217, 221–222 – – dielectric mirror 18 – – binding 225 ––filters 19 – – diffusion measurements evaluation ––lenses 14–17 222–224 ––metallicmirror 17–18 – – membrane transport 226–228 ––pinholes 18–19 – FRAP/CFM instrument 219–221 – photons, Poisson statistics, and – localization microscopy 299–300 anti-bunching 30–31 – rate 272–275 – physical background 24–30 – reversible, under oxidizing and reducing organic dyes 149–150 conditions 135–136 oscillator strength function – working 216–219 253–254 photodetectors 113 408 Index

photoinduced blinking 307 refraction 9–14 photomultiplier 123 refractive index 10, 38 photomultiplier tube (PMT) 122–123 region of photolysis (ROP) 217–218, 220, photon bunching 109 234 photon noise 127, 128 resolution limit 43, 177 photons 1, 30–31 retinal pigment epithelium (RPE) 368–369 photoresponse nonuniformity 128 reversible photobleaching, under oxidizing photoswitching 238 and reducing conditions 135–136 phototoxicity 136, 148 reversible saturable optical fluorescence phycobiliproteins 158 transitions (RESOLFT) 376 pinholes 18–19 rhodamines 149 pinhole-shifting FLIM 284 Richardson–Lucy algorithm 195 231, 232 root-mean-square-optical path length plane wave 4, 48 difference (rms-OPD) 68 point spread function (PSF) 48, 50–52, 176, 177, 183–184, 187, 195, 346, 349 s Poisson’s spot 1 scamper 234 Poisson distribution 31 scanning near-field optical microscopy propagation waves 20 (SNOM) 22–23 pulsed lasers 182, 380–382 scientific CMOS (sCMOS) 121–122 secondary electrons 119–120 q sensitized emission (SE) 266, 268, 270–271 quantitative fluorescence microscopy shear plate 393–395 – dimension measurement in 3D shot noise. See photon noise fluorescence microscopy 132 signal and noise 315 – exciting light intensity sine condition 70–71 measurements 133 single lens alignment, using laser beam – fluorescence intensity measurements 397–399 and labeled target concentration single-molecule/-particle tracking 299 128–131 single-molecule microscopy 163, 293–295 – ratiometric measurements 131–132 – analyzing 316 – technical tips 133–134 – – brightness 320–322 quantum dots (QDs) 146–148, 150–151, – – defocused imaging 322–322 163, 168 – – intensity patterns along optical quantum efficiency, of CCD 116–117 axis 320 – of photobleaching 216 – – localizing in two dimensions quantum electrodynamics 25 316–318 quantum optics 2 – – multicolor microscopy 322 – – point-spread function shape analysis r 318–319 radial and axial resolution and pinhole size – – polarization microscopy and orientation impact 182–189 321–322 radiative lifetime 278 – building 301 rapid lifetime determination (RLD) – – charge-coupled device (CCD) cameras 284–285 311–312 Raster image correlation spectroscopy (RICS) – – CMOS detectors 313 208–210 – – collimation 309 Rayleigh criterion 45, 54, 56, 176 – – dual view 301–302 ray tracing 66 – – electron-multiplying CCD cameras reactive oxygen species (ROS) 148, 163, 167 312–313 read noise 128 – – excitation intensity 305–308 reducing and oxidizing systems – – illumination time 308 (ROXs) 389 – – light polarization 308–309 reflected light imaging 106 – – light wavelength 309 Index 409

– – objective 302–304 – – wavelength effects 382 – – pixel size 310 – stimulated emission depletion 376–378 – – uniformity in illumination, 304–305 – switching between optical states 376 – molecular association determination stochastic optical reconstruction microscopy 323–325 (STORM) 329–331 – molecular conformations determination via Stokes shift 26, 100, 144, 247, 251 FRET 325–329 structured illumination microscopy (SIM) – superresolution 329–331 347–349 – tracking 332 – high-resolution information extraction – transitions detection 332–334 352–353 – unique information 295 – illumination pattern generation 355 – – bioanalysis 300–301 – image generation 349–352 – – full probability distribution – interference pattern mathematical measurement 296–297 derivation 355–358 – – resolving of kinetics 295 – optical sectioning 353–355 – – structures and functional – setups examples 358–362 states 297–298 super-resolution microscopy interference – – structures and superresolution and pattern techniques 345–346 imaging 298–300 – patterned techniques application 368–372 single-pair Forster¨ resonance energy transfer – resolution limit 346–347 (spFRET) 203 – spatially modulated illumination (SMI) single-photon avalanche diodes microscopy 362–363 (SPADs) 328 – – optical path 364–366 small-molecule organic dyes 389 – – setup 363–364 Snell’s law 11, 14, 21 – – size estimation 366–368 Sparrow criterion 177 – structured illumination microscopy (SIM) spatial light modulator (SLM) 359–360 347–349 spatially modulated illumination (SMI) – – high-resolution information extraction microscopy 362–363 352–353 – optical path 364–366 – – illumination pattern generation 355 – setup 363–364 – – image generation 349–352 – size estimation 366–368 – – interference pattern mathematical spherical aberration 23–24, 79 derivation 355–358 spherical wave 26, 47 – – optical sectioning 353–355 spinning disk confocal microscope (SDCM) – – setups examples 358–362 179, 190–194 surface normal 11 – comparison with laser scanning confocal synthetic fluorophores microscopy 194 – conjugation strategies 152–155 stage scanning 189–190 – fluorescent nanoparticles 150–152 stimulated emission depletion (STED) – fluorophore and target 156–157 microscopy 375–380 – nonnatural amino acids 155–156 – applications 388 – organic dyes 149–150 – – fluorophore choice 388–389 – – labeling strategies 389–390 t – experimental setup 384 tetracysteine (TC) tag 154 – – axial resolution improvement 388 time-correlated single-photon counting 283 – – light sources and synchronization time-domain FLIM 280 384–385 time gating 284–285 – – multicolor imaging 386–387 total internal reflection fluorescence – – scanning and speed 385–386 microscopy (TIRF) 232, 303–304, 308 – key parameters 380 transistor–transistor logic (TTL) 182 – – PSF shape and quality 382–384 transition dipole movement 11 – – pulsed lasers and fluorophore kinetics transition dipole orientation 256 380–382 transverse wave 4 410 Index

tunneling 22 – lateral resolution using incoherent light Twyman–Green interferometer 360 sources 52–54 Tyndall effect 97, 177 – magnification and resolution 59–60 – over and under sampling 61 v – point spread function and optical transfer von Bieren condition 46, 71 function 50–52 Weyl expansion 20 w Wollaston prism 89–91 wave 1, 5–7 wave optics and resolution 42–43 x – Airy function 47–51, 56 xenon arc lamps 108 – axial resolution 56–59 – depth of field and focus 60–61 z – imaging process description 43–47 Z-scanning 189 – lateral resolution of coherent light sources Zernike modes 23 54–56