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220 Acousto-Op 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 microscope (CLSM) avalanche photodiodes (APDs) 113, 217 123–124, 204 confocal microscopy 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 confocal microscopy (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 microscopes (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´
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