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Transcript of Index [] Index A AAQ (acryl-azobenzene-quaternary ammonium), 38 AAV. SeeAdeno-associated virus...
1061
Index
AAAQ (acryl-azobenzene-quaternary
ammonium), 38AAV. See Adeno-associated virusAAV-G-CaMP3, 68AAV plasmid preparation (recipe), 604–605Ablation, for in situ histology of brain tissue
with femtosecond laser pulses,437–445
Acepromazine, 515, 526Acetoxymethyl (AM)-conjugated indicators
dextran-conjugated indicators compared,131–132, 132f, 137
limitations of, 131loading cerebellar granule cell axons in
brain slices (protocol), 123–129experimental method, 124–125imaging calcium transients in parallel
fiber presynaptic terminals, 125imaging setup, 123loading granule cell presynaptic terminals
in rodent transverse cerebellarbrain slices, 124–125, 125f
materials, 123–124preparation of AM-conjugated calcium
indicator loading solution, 124recipes, 129troubleshooting, 126
presynaptic calcium measurements usingbulk loading, 121–129
loading conditions, determiningappropriate, 128, 128f
overview, 121–122protocol, 123–126selection of indicator, 127–128, 127f
Acetoxymethyl (AM) ester calcium indicatorsimaging action potentials with, 369–374loading of Bergmann glia with (protocol),
716–717Acousto-optical deflector (AOD)-based
patterned UV neurotransmitteruncaging, 255–270
application, 258–260, 259fglutamate, patterned uncaging of,
258–260, 259f
implementation of AOD-based system,258
comparison with other beam-steeringapproaches, 258
comparison with two-photonuncaging system, 258
implementing beam steering by softwarecontrol of AOD, 267
analog outputs, 267daily recalibration, 267parking the beam, 267stimulus design, 267
overview, 255–257acousto-optic deflectors, 256–257characteristics of caged compounds,
256one- and two-photon imaging, 256spatial resolution, 257
protocols, 261–270alignment and implementation of
uncaging, 264–267aligning uncaged and imaging
beams, 266course alignment, 265discussion, 266–267experimental method, 264–265implementing beam steering by
software control of AOD, 267materials, 264preparation of caged fluorescein
dried sample, 264troubleshooting, 265–266
integrating AOD-based patterneduncaging with UV light into atwo-photon microscope,261–263
assembling UV beam path andincorporating the AODs, 263
basic control of AODs, 262imaging setup, 262overview of UV beam path,
261–262, 261fpreparing and handling caged
compound solutions, 268–270application of caged compounds,
269
bath application, 268caged glutamate, preparation of,
268capillary application, 269discussion, 269–270, 269fexperimental method, 268–269focal application through capillary
tubing, 269–270, 269fmaterials, 268
Acousto-optical deflector (AOD)-based two-photon microscopy for high-speed calcium imaging ofneuronal population activity,543–555
overview, 543–544random-access pattern scanning
(protocol), 545–555application example, 554discussion, 554–555experimental method, 548–552
alignment and optimizationprocedures, 549–550
animal preparation, 550microscope setup, 548–549random access pattern scanning,
550–552, 551fimaging setup, 545–547, 547fmaterials, 547–548troubleshooting, 552–553
Acousto-optical deflectors (AODs), 256–257application to astrocyte imaging, 6953D laser scanning and, 539limitations of, 544patterned UV neurotransmitter uncaging
and, 255–270Acousto-optical tunable filter (AOTF), for
confocal spot detection, 142Acryl-azobenzene-quaternary ammonium
(AAQ), 38ACSF. See Artificial cerebrospinal fluidAction potentials
Aplysia abdominal ganglion, 472, 473f,476–477
back-propagated action potential (bAP),293
calcium dynamics and, 63, 66, 70, 71
Page references followed by f denote figures; page references followed by t denote tables.
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Action potentials (Continued)imaging neuronal population activity,
839–849action potential detection, 845–847application example, 847, 848fdata analysis, 843–847, 844fexperimental procedures, 840–843future prospects, 848–849
imaging with calcium indicators, 369–374advantages, 374application example, 373, 373finferring spikes, 373, 374flimitations, 374protocol
experimental method, 370–372imaging setup, 370loading embryonic and neonatal
acute cortical slices, 371loading juvenile and adult acute
cortical slices, 371materials, 370pressure injection, 371–372
interferometric detection of, 195–199advantages and limitations, 199application example, 198–199, 198fprinciples of interferometric detection,
195–198secretion-coupled light scattering and,
161–168AdEasy-1, 718, 719Adeno-associated virus (AAV)
in Alzheimer’s disease imaging, 991f, 992,993, 994f
for opsin expression in mammalian brain,867, 869, 871–872
recombinant (rAAV), 588–590, 588f–589fAdenosine triphosphate (ATP), as
gliotransmitter, 639Adenovirus, recombinant for gene transfer
into astrocytes, 707, 708f,710–712, 718
Adenylyl cyclases, photoactivated, 867ADVASEP-7, 179, 188Aldehyde dehydrogenase (Aldh1L1) promoter,
687Alexa dyes
Alexa-488 dextran, 138Alexa-594 dextran, 138Alexa Fluor 488
two-photon calcium imaging ofdendritic spines, 275, 276f
Alexa Fluor 594for detection of astrocyte vacuole-like
vesicles, 644–645, 645ffor imaging neuronal population
activity, 842for two-photon calcium imaging of
dendritic spines, 274–276, 275fAlexa Fluor 568 hydrazide, 132f
Algebraic reconstruction technique, 1028Alkaline trapping, 668, 669fAllen Brain Atlas, 377Allen Institute for Brain Science, 116
All-optical in situ histology of brain tissue,437–445
advantages and limitations, 443–444application example, 443, 444flaser alignment, 442–443overview, 437–438, 440fprotocol, 438–445
blocking and staining, 442discussion, 442–444experimental method, 441–442imaging setup, 439, 440fiterative imaging and ablation, 442,
443tmapping region of interest, 441–442materials, 439, 441perfusing with fluorescent gel, 442recipes, 445subblocking and mounting, 442
All-trans retinal, 864αCaMKII promoter, 585, 586fα-chloralose, 931Alzheimer’s disease
imaging neural networks in mouse model(protocol), 1000–1009
discussion, 1007–1008, 1008fexperimental method, 1001–1005
multicolor imaging, 1004f, 1005plaque-staining procedure, 1002,
1003fstaining neurons and glia with
calcium indicator dyes,1002–1003
surgical procedure, 1001–1002imaging setup, 1000materials, 1000–1001recipes, 1008–1009troubleshooting, 1005–1006
imaging of structure and function in,989–995
mouse models of, 990, 1000–1009near-infrared fluorescence (NIRF)
tomography, 989, 990overview, 989, 999postmortem diagnosis, 989two-photon imaging, 990–995, 1000–1009
amyloid plaques, 991–992calcium imaging, 993–994, 994ffunctional in vivo imaging, 993–995,
994f, 995fneurofibrillary tangles, 992–993oxidative stress, 994, 995fstructural in vivo imaging, 991–993
AMPA receptor (AMPAR), 7, 8fimaging with two-photon uncaging
microscopy, 251–252, 251fmapping distribution using two-photon
photostimulation, 429Amplex Red, 994, 995FAmyloid β, 989–995, 999. See also Alzheimer’s
diseaseAmyloid plaques, labeling of, 1002, 1003f, 1007Amyloid precursor protein (APP), 989, 992,
999
Anesthesiacat, 514–515, 526Drosophila, 561mouse, 308, 322, 324, 492, 504, 534, 550,
569, 595, 602–603, 615, 688,711, 714, 724, 726, 728, 871,952, 965, 985, 1001, 1015, 1016,1019, 1026
rat, 534, 871, 931rodent, 514, 526zebrafish, 569
Anesthetic for cats (recipe), 526Anesthetic for rodents (recipe), 526AngioSense, 1025Antigen presenting cells (APCs), preparation
of, 984Antioxidants, to decrease photodynamic
damage, 337AODs. See Acousto-optical deflectors (AODs)AOTF (acousto-optical tunable filter), for
confocal spot detection, 142APCs (antigen presenting cells), preparation
of, 984Aplysia abdominal ganglion action potentials,
472, 473f, 476–477APP (amyloid precursor protein), 989, 992,
999Arc lamp, 1037–1038Artificial cerebrospinal fluid (ACSF) (recipe),
129, 244, 315, 415, 424,486–487, 527, 652, 681–682,696, 705, 733, 760, 976, 988
HEPES-buffered, 719high-divalent, 425holding, 486–487modified, 487modified, free of carbonate and
phosphate, 936Ascorbate, 337ASLV (avian sarcoma and leucosis virus), 85,
96Astrocytes, 521
current status of in vivo imaging of, 695imaging astrocyte calcium and vacuole-
like vesicles, 639–653detection of calcium signals by two-
photon microscopy, 642–644,643f
detection from astrocytes infusedwith membrane-impermeablecalcium indicators, 643–644,643f
detection from multiple astrocytesin slices, 642, 643f
detection from photolysis of cagedcompound in single astrocyte,644, 644f
detection of vacuole-like vesicles bytwo-photon microscopy,644–647, 645f
using Fluo-4 AM, 646using Fluo-4 potassium and Alexa
Fluor 594, 644–645, 645f
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Index / 1063
using FM1-43, 646–647, 647fidentification of astrocytes and
neurons in hippocampal slices,640–642, 641f
by biocytin staining, 642by electrophysiological properties,
642by GRAP staining, 642morphology confirmation, 640,
641f, 642imaging setup, 640protocols, 648–653
biocytin staining procedure, 648recipes, 652–653two-photon imaging of acute brain
slices preincubated with Fluo-4AM, 649
two-photon imaging of astrocytesfilled with photolabile cagedcalcium compound, 651
monitoring exocytosis in, 655–670overview, 655–656protocols
identification and staining ofdense-core granules (DCGs),659–660
identification and staining oflysosomes, 661
identification and staining ofsynaptic-like microvesicles(SLMVs), 657–658
imaging exocytosis and recycling atthe whole-cell level, 667–669
imaging exocytosis at the singlevesicle level with EWi, 663–666,665f
transfection of, 658, 659–660two-photon imaging astrocytic excitation
in cerebellar cortex of awakemobile mice, 745–760
in vivo calcium imaging of cerebellarcerebellar craniotomy for in vivo
imaging (protocol), 713–715injection of recombinant adenovirus
for gene transfer of FCIP G-CaMP2 into astrocytes ofcerebellar cortex (protocol),710–712
experimental method, 711–712materials, 710–711troubleshooting, 712
overview, 707–709, 708fpreferential loading of Bergmann glia
with synthetic acetoxymethylcalcium dyes (protocol),716–717
in vivo imaging of structural andfunctional properties, 685–696
future developments in imaging, 695overview, 685–687protocols
imaging of calcium dynamics incortical astrocytes: loading with
calcium indicator dyes, 690imaging of calcium dynamics in
cortical astrocytes: loading withphotolabile caged calciumcompounds, 691
in vivo imaging of astrocytesstained with SR101, 688–689
in vivo imaging of redox stateusing NADH intrinsicfluorescence with cellularresolution, 692–694, 693f
in vivo labeling of cortical astrocytes withsulforhodamine 101, 673–682,674f
application example, 680, 689foverview, 673–674, 674fprotocol, 675–682
ATP (adenosine triphosphate),gliotransmitter, 639
Atropine, 514, 515, 526Autofluorescence, of flavoproteins, 919Automated imaging and analysis of behavior
in Caenorhabditis elegans,763–775
Avertin, 1015, 1016, 1019Avian sarcoma and leucosis virus (ASLV), 85,
96Awake animals, imaging in
fiber-optic calcium monitoring ofdendritic activity, 903, 904f
imaging neuronal population activity,839–849
application example, 847, 848fdata analysis, 843–847, 844f
action potential detection, 845–847correction for movement artifacts,
845estimation of noise levels and
neuropil contamination, 845normalization of fluorescence
values, 843–844experimental procedures, 840–843
cell-attached electrophysiology,842–843, 844f
dye preparation for injection,842–843
habituation and training for awakeexperiments, 840
head plate design, 840–841, 841fhead plate implantation, 841–842surgical considerations, 842targeting sensory areas, 842
future prospects, 848–849miniaturization of two-photon
microscopy for imaging infreely moving animals, 851–860
optical imaging based on intrinsic signals,912
optogenetics in freely moving mammals,877–885
overview, 851–862two-photon imaging/microscopy of
astrocytic and neuronal
excitation in cerebellar cortexof awake mobile mice, 745–760
two-photon imaging of neural activity inawake mobile mice, 827–836
data acquisition and processing,831–834, 833f
examples of imaging in different brainregions, 834, 835f
experimental apparatus, 828–829, 828ffuture outlook, 836surgical procedures and dye loading,
829–831, 830fvoltage-sensitive dye imaging of cortical
spatiotemporal dynamics inawake behaving mice, 817–824
voltage-sensitive dye imaging ofneocortical activity of corticaldynamics in behaving monkeys,810–811
Axonscorrelated light and electron microscopy
of green fluorescent protein–labeled, 227–236
interferometric detection of actionpotentials, 195–199
loading cerebellar granule cell axons inbrain slices with acetoxymethyl(AM)-conjugated calciumindicators, 123–129
sodium imaging in, 201–206Azobenzene, 34f, 35–38
BBAC (bacterial artificial chromosome)
advantages for labeling genetically definedcell types, 116
Cre mice, 116, 118–119transgenic mice, 113–119
application to neuroimaging studies,116–118, 117f, 118f
generation of, 114–115, 115fGENSAT Project database, 118–119ordering mouse lines, 119
Back-propagated action potential (bAP), 293Bacteriorhodopsin
light-induced dipole switching, 43foverview, 42f, 866photocycle, 43f, 44–45, 48structural and functional homologies with
channelrhodopsin, 46f, 47Bafilomycin A1, 668, 669fBallistic delivery of dyes, 447–456
advantages of, 455application examples, 454, 454fdisadvantages of, 455–456overview, 447–448protocol, 449–456
discussion, 454–456experimental method, 450–452imaging setup, 449labeling cells with water-soluble dyes,
452materials, 449–450
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Ballistic delivery of dyes (Continued)preparation and delivery of lipophilic
dyes, 450–452bullet preparation, 450–451coating tungsten beads, 450dye diffusion, tissue mounting, and
imaging, 451–452dye preparation, 450particle delivery, 451, 451f
troubleshooting, 452–453BAP (back-propagated action potential), 293BBS (BES-buffered saline 2x) (recipe), 605Beam splitter, dichroic, 1033f, 1034Behavior
automated imaging and analysis ofbehavior in Caenorhabditiselegans, 763–775
fiber-optic dendritic calcium monitoringduring, 897–905
imaging motor behavior in zebrafish,783–789
optogenetic modulation of dopamineneurons, 877–886, 879f
functional integration during opticalcontrol, 884–885, 885f
integrating behavioral readouts withreward circuit modulation,880–884
conditioned place preference,881–883, 882f
operant conditioning, 883–884two-photon imaging of astrocytic and
neuronal excitation incerebellar cortex of awakemobile mice, 745–760
habituation of mice (protocol), 750–751optical window preparation and tissue
(protocol), 752–754surgical implantation of a head plate
(protocol), 747–749, 748ftwo-photon imaging, 755–760
discussion, 759–760example data, 758fexperimental method, 756–757imaging setup, 755materials, 756recipe, 760troubleshooting, 757
two-photon imaging of neural activity inawake mobile mice, 827–836
voltage-sensitive dye imaging of corticalspatiotemporal dynamics inawake behaving mice, 817–824
voltage-sensitive dye imaging ofneocortical activity, long-termin behaving monkeys, 810–811
Benzonase, 600, 602Bepanthen, 724Bergmann glia
imaging calcium waves in, 699–705, 704fpreferential loading with synthetic
acetoxymethyl calcium dyes(protocol), 716–717
in vivo calcium imaging with syntheticand genetic indicators,707–719, 708f
Biocytin-filled cells, histologicalreconstruction of, 381
Biocytin staining of astrocytes, 641f, 642, 648Bioluminescence imaging, for monitoring
doxycycline (Dox)-controlledgene expression, 585, 586f, 592,595
Bipolar cells, loading and TIRF imaging ofdissociated, 187–188
experimental method, 187–188materials, 187troubleshooting, 188
Birefringent plate, in interferometry, 196Bleaching
G-CaMP and, 558, 558fin stimulated emission depletion (STED),
242voltage-sensitive dyes, 480
Blocking solution (recipe), 445Blood–brain barrier, 990, 1011–1012, 1021Blood flow
optical imaging based on intrinsic signals,913–917
optically induced occlusion of single bloodvessels in neocortex, 939–947
application example, 947histology, 945–946, 945flimitations of, 947overview, 939–940protocol, 941–946
two-photon imaging of blood flow incortex, 927–936, 930f, 933f
two-photon imaging of neuronalstructural plasticity in miceduring and after ischemia,949–957
overview, 949protocol, 950–957
discussion, 957experimental method, 951–955,
951f, 953fimaging setup, 950materials, 950–951troubleshooting, 955–956
Blowfly, in vivo dendritic calcium imaging invisual system, 777–780
[Ca2+] measurements, 779–780, 780fcell labeling, 778imaging, 779imaging setup, 778–779, 778fwhole-mount preparation, 777–778
Blue dyes, in vivo voltage-sensitive dyeimaging of mammalian cortexusing, 481–483
BOLD (blood-oxygenation-level-dependent)functional magnetic resonanceimaging (fMRI), 915, 916
Bovine chromaffin cells, transfection andTIRF imaging of cultured,189–190
experimental method, 189–190materials, 189troubleshooting, 190
BPB (bromophenol blue), 180, 180fBrainbow
advantages, 109color-assisted circuit tracing, 102, 103colors, 101–102generation and imaging of Brainbow mice
(protocol), 105–110discussion, 109experimental method, 106–108imaging setup, 105materials, 106recipe, 109–110Reconstruct software, 105, 107–108troubleshooting, 108
GRE mice, 104limitations, 109overview of approach, 99–100, 100fstrategies, 100–101, 102ttransgene generation, 103–104
elements for optimized expression, 104fluorescent proteins, 103FRT site, 104lox sites, 103promoter, 103subcellular localization signals, 103–104
transgenic micegeneration and imaging of, 105–110lines, 104
Brain slicescircuit mapping by ultraviolet uncaging of
glutamate, 417–425imaging astrocyte calcium and vacuole-
like vesicles, 639–653imaging microglia in, 735–742infrared-guided laser stimulation of
neurons in, 388–390loading AM-conjugated indicators in
cerebellar granule cell axons,123–129
maintaining live slices, 331–338mapping connections in, 432–434, 433f,
434foptical measurement of dopamine nerve
terminals in, 884–885voltage-sensitive dye imaging of
population signals in, 478–480Brain tumor imaging, 1011–1021
fluorescence molecular tomography,1023–1029
imaging brain metastasis using a brain-metastasizing breastadenocarcinoma (protocol),1018–1021
discussion, 1021experimental method, 1019–1020
analysis of tumor metastasis byfluorescence imaging,1019–1020, 1020f
preparation of cells for injection,1019
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Index / 1065
tumor cell injection by intracardiacinjection, 1019
imaging setup, 1018materials, 1018–1019
live imaging of glioma by two-photonmicroscopy (protocol),1013–1017
discussion, 1017experimental method, 1014–1016
cranial window preparation, 1015postimaging analysis, 1016preparation of cells for injection,
1014tumor cell injection into brain,
1015two-photon imaging, 1016, 1016f
imaging setup, 1013materials, 1013–1014troubleshooting, 1017
Breast cancer, metastatic, 1012, 1018–1021,1020f
Breathing artifacts, 10066-bromo-7-hydroxycoumarin-4-ylmethoxy-
carbonyl-glutamate (Bhc-glu),246, 246f
Bromophenol blue (BPB), 180, 180fBungarotoxin (BTX), 221–222Buprenorphine, 324, 712, 872, 902, 1015,
1019
CCAA (cerebral amyloid angiopathy), 992, 994,
995fCaenorhabditis elegans, automated imaging
and analysis of behavior in,763–775
Caffeine, dorsal root ganglion (DRG) neuronresponse to, 343, 343f
Ca2+-free Ringer’s solution (recipe), 509Caged calcium chelators, 393–401
chemical properties of, 393–397, 394f,394t, 395f, 396f
detection from photolysis of cagedcompound in single astrocyte,644, 644f, 651
illumination systems, 399–401light sources, 399–400measuring effectiveness of photolysis,
401setup and accessories, 400
overview, 393practical challenges to use in neurons,
397–399cell loading, 397–398estimating the efficiency of photolysis,
398quantification of ∆[Ca2+]i, 398–399,
399fCaged compounds, characteristics of, 256Caged glutamate stock solution (recipe), 425Calcium (Ca2+)
confocal spot detection of Ca2+ domains,141–149
application to study of presynapticCa2+ microdomains, 147–149,148f
hardware overview, 142–143, 143foptical conditions for, 143–147
Ca2+ indicator choice forappropriate brightness andsensitivity, 144–146
characterization of confocalvolume, 144
point spread function (PSF), 144rapidly responding indicator,
146–147shortcomings of, 147signal-to-noise ratio (SNR), 147–149,
148feffects on light scattering associated with
secretion from peptidergicnerve terminals, 163–164, 163f
fiber-optic monitoring of dendriticactivity in vivo, 897–905
single compartment model of calciumdynamics, 355–366
uncaging calcium in neurons, 393–401uncaging in nerve terminals, 151–159
examining transmitter release at calyxof Held synapse, 158–159, 158f
overview, 151–152three-point calibration procedure
(protocol), 153–159calculation of Keff, 157determination of post-flash
calibration constants, 157experimental method, 154–157imaging setup, 153in-cell calibration, 155–157, 156fmaterials, 153, 154tratiometric measurements of
[Ca2+]i, 154–155recipes, 159in vitro calibration, 155, 156f
in vivo recordings and channelrhodopsin-2 activation through an opticalfiber, 889–895
application examples, 895overview, 889protocol, 890–894, 893f
anesthesia, 892experimental method, 892–893, 893fimaging setup, 890, 891fimplantation and fixation of
optical fiber, 892–893materials, 890–891skull preparation, 892troubleshooting, 894
wide-field charge coupled device (CCD)camera based imaging ofcalcium waves and sparks incentral neurons
loading cells and detecting [Ca2+]ichanges (protocol), 282–285
application examples, 284–285,284f, 285f
discussion, 284–285experimental method, 283materials, 282–283
Calcium chelators. See Caged calciumchelators
Calcium dynamicsimaging in cortical astrocytes
loading with calcium indicator dyes,690
loading with photolabile caged calciumcompounds, 691
simultaneous recording of tectal Ca2+
dynamics and motor behaviorin zebrafish, 785
single compartment model of, 355–366application examples, 363dynamics of single [Ca2+]i transients,
360extensions to model, 364–366
buffered calcium diffusion, 364deviations from linear behavior,
364–365, 365fmeasurement of calcium-driven
reactions, 365–366saturation of buffers and pumps,
364slow buffers, 365
materials, 356model application, 361–363
estimates of amplitude and totalcalcium charge, 362–363
estimates of endogenous Ca2+-binding ratio and clearancerate, 361–362, 362f
estimates of unperturbed calciumdynamics, 363
model assumptions and parameters,356–360, 358t–359t
buffering, 358clearance, 360influx, 357–358
overview, 355–356schematic of model, 357fsummation of [Ca2+]i transients during
repetitive calcium influx, 360–361Calcium Green-1 (CG-1)
two-photon calcium imaging of dendriticspines, 278
for two-photon imaging of neural activityin awake mobile mice, 831
Calcium Green-1 (CG-1) dextran, 132fballistic delivery of, 452, 454, 454fcalcium imaging in olfactory neurons, 566,
566fimaging neural activity in zebrafish larvae,
793, 796, 796fCalcium imaging
acousto-optical deflector (AOD)-basedtwo-photon microscopy forhigh-speed imaging of neuronalpopulation activity, 543–555
in Alzheimer’s disease, 993–994, 994f,1002–1005
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Calcium imaging (Continued)confocal imaging of neuronal activity in
larval zebrafish, 791–797in Drosophila olfactory system with a
genetic indicator, 557–563functional neuron-specific expression of
genetically encoded fluorescentcalcium indicator proteins inliving mice, 583–606
imaging astrocyte calcium and vacuole-like vesicles, 639–653
imaging calcium waves and sparks incentral neurons, 281–285
in intact olfactory system of zebrafish andmouse, 565–571
NADH imaging in combination with, 694of neuronal activity and motor behavior
in zebrafish, 783–789in neuronal endoplasmic reticulum,
339–344application example, 343, 343foverview, 339, 340fprotocol, 341–344
advantages and limitations,343–344
discussion, 343–344imaging setup, 341intracellular calibration of
MagFura-2 signals, 342materials, 341real-time video imaging, 342recipes, 344staining neurons with MagFura-2
AM and Fluo-3, 342of neuronal population activity in awake
and anesthetized rodents,839–849
of neurons in visual cortex using troponinC–based indicator, 611–620,612f
in populations of olfactory neurons byplanar illumination microscopy,573–580
of two-photon of dendritic spines,273–278
overview, 273, 278protocol, 274–278
estimating changes in calciumconcentration from ∆F/F, 277
experimental method, 274–277imaging calcium transients in
dendritic spines, 276–277loading neurons with calcium
indicators and Alexa dyes,274–276, 275f, 276f
materials, 274recipe, 278
in vivo dendritic imaging in fly visualsystem, 777–780
in vivo local dye electroporation, 501–509in vivo of cerebellar astrocytes, 707–719in vivo two-photon imaging in visual
system, 511–527
in vivo two-photon using multicell bolusloading of fluorescentindicators, 491–499
Calcium indicatorsconfocal spot detection of presynaptic
Ca2+ domains, 141–149detection of calcium signals from
astrocytes by two-photonmicroscopy, 643–644, 643f
imaging action potentials with, 369–374imaging neuronal activity with genetically
encoded calcium indicators(GECIs), 63–73
imaging of calcium dynamics in corticalastrocytes, 690
with photolabile caged calciumcompounds, 691
imaging presynaptic calcium transientsusing dextran-conjugatedindicators, 131–138
influence of rate constant on speed ofcalcium-dependentfluorescence response, 146
loading into olfactory sensory neurons,566–567
bolus loading of neurons downstreamfrom glomeruli, 567
dextran-coupled (protocol), 568–571genetically encoded indicators, 567selective loading of neurons projecting
to glomeruli, 566multicell bolus loading, 491–499presynaptic calcium measurements using
bulk loading of acetoxymethylindicators, 121–129
signal-to-noise ratio, 64, 145–146single compartment model of calcium
dynamics, 356two-photon calcium imaging of dendritic
spines, 273–278in vivo calcium imaging of cerebellar
astrocytes, 707–719, 708fCalcium phosphate transfection, 597, 599–600Calcium waves
imaging in central neurons, 281–285imaging in cerebellar Bergmann glia,
699–705, 707, 708f, 709application example, 704, 704foverview, 699–700protocol, 701–705
discussion, 704experimental method, 701–702imaging setup, 701materials, 701recipes, 705troubleshooting, 702–703
Calibrating solutions (recipe), 344Calliphora vicina, in vivo dendritic calcium
imaging in visual system,777–780
[Ca2+] measurements, 779–780, 780fcell labeling, 778imaging, 779
imaging setup, 778–779, 778fwhole-mount preparation, 777–778
Calmodulin (CaM), 583, 584Cameleon
development of Yellow Cameleons,583–584
neuronal imaging in Caenorhabditiselegans, 764
CameraCCD camera
electron-multiplying (EMCCD), 578imaging calcium waves in Bergmann
glia, 699–705, 704fobjective-coupled planar illumination
(OCPI) microscopy, 578use in dendritic voltage imaging, 288,
292f, 293wide-field charge coupled device
(CCD) camera-based imaging,of calcium waves and sparks incentral neurons, 281–285
choice for voltage-sensitive dye imaging,475
infrared (IR), 378Camgaroo-1, 584Camgaroo-2, 584, 585, 586f–587fCamKIIα promoter, 867Capase indicators, 995Capillary tubing, application of caged
compounds through, 269–270Carbon dioxide, for Drosophila anesthesia, 5614-carboxymethoxy-5,7-dinitroindolinyl-
glutamate (CDNI-glu), 246,246f
Carprofen, 324, 615Cat
anesthesia induction, 514–515surgery, 515
craniotomy, 516durotomy, 517
in vivo two-photon calcium imaging invisual system, 511–527
Cautions, 1051–1058CCD camera
electron-multiplying (EMCCD), 578imaging calcium waves in Bergmann glia,
699–705, 704fobjective-coupled planar illumination
(OCPI) microscopy, 578use in dendritic voltage imaging, 288,
292f, 293wide-field charge coupled device (CCD)
camera-based imaging, ofcalcium waves and sparks incentral neurons, 281–285
CCLp, 584CD63, 661cDNA, recovery of replication-competent
rabies virus from (protocol),87–92
cell culture, 89fixation of cells and direct
immunofluorescence, 90
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materials, 88–89transfection and virus recovery, 89–90virus stock preparation, 92virus titration, 91, 91f
CDNI-glu (4-carboxymethoxy-5,7-dinitro-indolinyl-glutamate), 246, 246f
Cell-attached electrophysiology, combiningimaging with, 842–843, 844f
Cell populationslabeling genetically defined using BAC
transgenic mice, 113–119in vivo calcium imaging using multicell
bolus loading of fluorescentindicators, 491–499
Cerebellar craniotomy, 713–715Cerebral amyloid angiopathy (CAA), 992, 994,
995fCerebral blood flow, two-photon imaging of,
927–936, 930f, 933fCerebrospinal fluid, artificial. See Artificial
cerebrospinal fluidCFP. See Cyan fluorescent proteinCG-1. See Calcium Green-1 (CG-1)Channelrhodopsin (ChR), 864
biophysical properties, 48–49ChR1, 46–47, 48–49ChR2, 46f, 47, 48–49
activation spectrum of, 864activation through an optical fiber,
889–895, 893fdeactivation rate, 866light required for activation, 868
light-gated ion channels, 46–47light-induced dipole switching, 43foverview, 42f, 46–47step-function opsin genes (SFOs), 47structural and functional homologies with
bacteriorhodopsin, 46f, 47Channelrhodopsin-2-assisted circuit mapping
(CRACM), 424Chelators. See Caged calcium chelatorsChemical two-photon uncaging, 25–31
disadvantage of, 30overview, 25–26, 26fprotocol, 27–30
application example, 29, 29fdiscussion, 29–30experimental method, 28imaging setup, 27materials, 27–28troubleshooting, 28
Chlamydomonas reinhardtii, 46–47, 864Chloride imaging using Clomeleon, 75–80
advantages, 79application example, 79, 80flimitations, 79–80mode of action, 75protocol, 77–78
calibration procedure, 78imaging setup, 77materials, 77slice preparation, 77–78
structure of, 75, 76f
Chloride imaging using MQAE, 623–632advantages and limitations, 630application example, 627f, 628f, 630overview, 623–624protocol, 624–631
discussion, 630experimental method, 626–628imaging setup, 625intracellular calibration of MQAE,
627–628materials, 625–626recipes, 630–631staining cells or tissue slices via bath
application, 626, 627fstaining of neurons and glia using
multicell bolus loading, 626,628f
troubleshooting, 629two-photon imaging of stained cells,
626Choleratoxin B-Alexa (CTB-Alexa), 384ChR. See Channelrhodopsin (ChR)Chrysamine-G, 1007Ciona intestinalis, 53, 57Circuit mapping by ultraviolet uncaging of
glutamate, 417–425overview, 417–418protocol, 419–425
analyzing traces, 422discussion, 424experimental method, 420–422, 421fimaging setup, 419mapping a neuron’s excitation profile,
422mapping a neuron’s synaptic inputs,
420–421, 421fmaterials, 420preparation of brain slices, 420recipes, 424–425troubleshooting, 422–423
Circuit tracingcolor-assisted in Brainbow studies, 102,
103in vivo local dye electroporation for,
501–509Circularly permuted fluorescent proteins, 584Classical conditioning, for assessing reward-
related behavior, 881Cleaning optical equipment, 1039–1040Clomeleon, 75–80
chloride imaging, 623–624advantages, 79application example, 79, 80flimitations, 79–80mode of action, 75protocol, 77–78
structure of, 75, 76fCNS-1 cell line, rat, 1011–1017, 1016fCollagenase type IA, 593Common carotid artery occlusion, 951–954,
951fComplementation, rabies virus vectors and,
84–85, 85f
Conditioned place preference, 881–883, 882fConfocal calcium imaging of neuronal activity
in larval zebrafish, 791–797Confocal spot detection of Ca2+ domains,
141–149application to study of presynaptic Ca2+
microdomains, 147–149, 148fhardware overview, 142–143, 143foptical conditions for, 143–147
Ca2+ indicator choice for appropriatebrightness and sensitivity,144–146
characterization of confocal volume,144
point spread function (PSF), 144rapidly responding indicator, 146–147
shortcomings of, 147signal-to-noise ratio (SNR), 147–149, 148f
Congo Red, 1007Contact lens, 516Continuous-wave (CW) lasers
use in dendritic voltage imaging, 289use in stimulated emission depletion
(STED), 243Coomassie Brilliant Blue staining solution
(recipe), 605CoroNa Green, 202, 303Cortex buffer (recipe), 328, 620Cortical blood volume (CBV), activity-
dependent increase in, 914–915Cortical spatiotemporal dynamics, voltage-
sensitive dye (VSD) imaging inawake behaving mice, 817–824
Cortical stress response, 322cpVenus, 584CRACM (channelrhodopsin-2-assisted circuit
mapping), 424Cranial window
imaging through chronic, 319–328fluorophore choice, 320overview, 319–320protocol, 321–328
application example, 325fexperimental method, 322–326image acquisition, 324, 325fimage analysis, 324, 326imaging setup, 321materials, 321–322recipe, 328surgery, 322–324, 323ftroubleshooting, 326
quantification of morphologicalchanges, 327
transcranial imaging compared, 314implantation for calcium imaging using
TN-XXL, 615preparation for glioma imaging, 1015,
1015fpreparation for imaging excitation in
cerebellar cortex, 753–754preparation in rat for two-photon imaging
of blood flow in cortex(protocol), 929–936, 930f, 933f
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Cranial window (Continued)application example, 933–934, 933fexperimental method, 931–932materials, 929–931setup, 930f
for two-photon imaging of microglia, 968Craniotomy
for Alzheimer’s disease imaging, 1002bleeding during, 1005cat, 516cerebellar, 713–715rodent, 516, 516f
Cre/lox recombinationin Brainbow strategies, 99–104CreER/tamoxifen system, 101, 106–107, 108doxycycline (Dox)-controlled gene
expression, 585in Thy1 mice, 211–213
Cre mice, 104, 116, 118–119Cre recombinase
in optogenetics studies, 878, 879fuse in optogenetics, 867–868
Crick, Francis, 886CTB-Alexa (choleratoxin B-Alexa), 384Culture medium (recipe), 192Cutting solution (recipe), 425CW lasers. See Continuous-wave (CW) lasersCyan fluorescent protein (CFP)
in Clomeleon, 75, 76f, 77, 79–80, 80fFRET and, 583, 584in VSFPs (voltage-sensing fluorescent
proteins), 55, 55t, 56f, 57Cysteine-maleimide reactivity, 38Cytomegalovirus (CMV) immediate-early
(IE) promoter, 707, 708f, 718
DDAB (diaminobenzidine), 176–178, 176fDark fringe method, 196Dark noise, in voltage-sensitive dye imaging,
472, 474DCGs. See Dense-core granulesD3cpv, 584, 590, 591fD3cpVenus, 64, 65f, 67, 67tDCU (dispersion compensation unit),
546–547, 547f, 549–550Dendrites
correlated light and electron microscopyof green fluorescentprotein–labeled, 227–236
fiber-optic calcium monitoring ofdendritic activity in vivo,897–905
infrared-guided neurotransmitteruncaging on dendrites, 387–391
single compartment model of calciumdynamics in, 355–366
sodium imaging in, 201–206in vivo calcium imaging in fly visual
system, 777–780Dendritic spines
GFP expression levels required to detect,320
induction of structural plasticity at single,252, 253f
multiphoton stimulation of, 411–415stimulated emission depletion (STED)
imaging of, 237–244two-photon calcium imaging of, 273–278two-photon imaging and uncaging of,
247–250two-photon sodium imaging in, 297–304
Dendritic voltage imaging, 287–294imaging setup, 288–289overview, 287–288staining individual vertebrate neurons
(protocol), 290–294advantages and limitations, 294example, 292f, 293experimental method, 290–291, 291fmaterials, 290recipes, 294troubleshooting, 291
Dense-core granules (DCGs)identification, 659staining (protocol), 659–660
2-deoxyglucose (2-DG), 908Deoxyhemoglobin, 914, 915Destaining solution (recipe), 605Developmental studies, using optical imaging,
912Dexamethasone, 322, 515, 526, 615Dextran-conjugated indicators
acetoxymethyl (AM)-conjugated indicatorscompared, 131–132, 132f, 137
advantages of, 137imaging presynaptic calcium transients
using dextran-conjugatedindicators, 131–138
overview, 131–133presynaptic imaging of projection
fibers by in vivo injection ofdextran-conjugated calciumindicators (protocol), 134–136
experimental method, 135imaging setup, 134loading solution preparation, 135materials, 134–135preparation of brain slices, 135presynaptic imaging, 135–136troubleshooting, 136in vivo injection of dextran
indicators, 135properties of, 137–138red, 138
Diaminobenzidine (DAB), 176–178, 176fDi-8-ANEPEQ dye, 484Diazo-2, 393, 394f, 397Dichroic beam splitter, 1033f, 1034DIC microscopy, for identification of
astrocytes and neurons inhippocampal slices, 640–642,641f
DiD, 450Diffractive optical element (DOE), 430–431Digital micromirror device (DMD), 258
DiI, 450DiO, 450Dispersion compensation unit (DCU),
546–547, 547f, 549–550Dissection medium (recipe), 742DMD (digital micromirror device), 258DMEM3+ (recipe), 98DM-nitrophen (DMNP), 151–153, 154t, 156f,
157, 159, 393, 394f–396f, 394t,395–401
DOE (diffractive optical element), 430–431Dopamine, optogenetic modulation of
neurons, 877–886, 879ffunctional integration during optical
control, 884–885, 885foptical measurement of dopamine
nerve terminals in brain slices,884–885
optical measurement of dopaminerelease in vivo, 885
integrating behavioral readouts withreward circuit modulation,880–884
conditioned place preference, 881–883,882f
operant conditioning, 883–884Doxycycline (Dox)-controlled gene
expression, 585–586, 586f–587f,592, 595–596
DQ gelatin, 995, 995fDrd1, 878Drosophila
anesthesia, 561antennae–brain preparation, 558f, 561calcium imaging in olfactory system,
557–563Drosophila adult hemolymph-like saline
(AHLS) (recipe), 561D-serine, as gliotransmitter, 639, 657DsRed, for astrocyte imaging, 707, 708fDurotomy, 517Dye-containing pipette solution (recipe), 352Dye loading with whole-cell recordings,
347–353dye loading with patch pipettes (protocol),
348–353discussion, 352experimental method, 349–350, 350fimaging setup, 348materials, 348–349recipes, 352–353troubleshooting, 350–351
overview, 347Dye-making recipe, 527Dyes. See also Fluorescent dyes; FM dyes;
Voltage-sensitive dye; specificapplications; specific dyes
ballistic delivery of, 447–456organic, labeling and imaging receptors
using, 2, 4–7, 4tDye solution (recipe), 905Dynamic high bandwidth atomic force
microscopy (HBAFM), 164
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EEar biopsy, 593eGFP. See Enhanced green fluorescent proteinElectromagnetic pulse (EMP), 1038Electromagnetic spectrum, 1031, 1032fElectron microscopy
correlated light and electron microscopyof green fluorescentprotein–labeled axons anddendrites, 227–236
imaging green fluorescentprotein–labeled neurons usinglight and electron microscopy(protocol), 228–236
discussion, 235experimental protocol, 230–234finding imaged neuron in sections,
231–232, 232ffixation and immunochemistry,
230fixation of imaged slices, 230–231imaging dendrites in EM, 234, 235fimaging setup, 228materials, 228–229recipes, 236resin embedding for EM, 231serial sectioning and imaging in
EM, 233–234, 233ftroubleshooting, 234–235
overview, 227FM dyes as endocytic markers for,
176–178, 176fElectron-multiplying CCD (EMCCD) camera,
578Electro-optic modulator, in interferometry,
196, 197fElectroporation
targeted in vivo, 463–464, 464fin utero, 213, 612, 613in vivo local dye electroporation for
calcium imaging and neuronal-circuit tracing, 501–509
overview, 501–502protocol, 503–509
application examples, 504, 506f,507f, 508f
discussion, 509experimental method, 504, 505fimaging setup, 503materials, 503recipe, 509troubleshooting, 506–508
Embryo medium (recipe), 789EMCCD (electron-multiplying CCD) camera,
578Emission filter, 1033–1034, 1033fEmission maxima, for fluorochromes,
1035t–1036tEMP (electromagnetic pulse), 1038Endocytosis and imaging of synaptic vesicle
recycling using FM dyes,171–181
Endoplasmic reticulum, calcium imaging inneuronal, 339–344
Enhanced green fluorescent protein (eGFP)in Alzheimer’s disease imaging, 1005in BAC (bacterial artificial chromosome)
transgenic mice, 113–114,116–119, 117f, 118f
dense-core granule staining, 659–660expression in astrocytes, 687in imaging synaptic-like microvesicles
(SLMVs), 658immune cell expression of, 987, 987fneuropeptide Y–enhanced green
fluorescent protein (NPY-eGFP), 183, 185, 189, 191, 192f
in rabies virus vectors, 84f, 85, 87–96, 95fEnvA, of avian sarcoma and leucosis virus
(ASLV), 85, 96Envelope switching, rabies virus vectors and,
85Ephus, 419Epifluorescence illumination (EPIi), 574, 574f,
662, 667–669, 669fEpifluorescent microscopy
for fiber optic calcium monitoring ofdendritic activity in vivo, 897,899–900, 899f
for voltage-sensitive dye (VSD) imaging ofcortical spatiotemporaldynamics in awake behavingmice, 818–823, 818f
Epigenetic silencing, 595EPSCs (excitatory postsynaptic currents), 259EPSPs (excitatory postsynaptic potentials),
411, 432, 433f, 434fEvanescent field, 183, 184fEvanescent field fluorescence microscopy. See
Total internal reflectionfluorescence microscopy
Evans solution (recipe), 789EWi (evanescent wave illumination), 662–670,
665f, 669fExcitation filter, 1033, 1033fExcitation in mouse cerebellar cortex,
imaging, 745–760Excitation maxima, for fluorochromes,
1035t–1036tExcitation-secretion coupling, peptidergic
nerve terminals and, 161–168Excitatory postsynaptic currents (EPSCs), 259Excitatory postsynaptic potentials (EPSPs),
411, 432, 433f, 434fExocytosis
imaging with total internal reflectionfluorescence microscopy(TIRFM), 183–193, 655–670
kiss and run fusion, 665, 665fmonitoring in astrocytes, 655–670regulated, 655–656
Experimental autoimmune encephalomyelitis,981, 987f
Expression of genetically encoded calciumindicators, 583–606
Expression vector, Thy1, 208–209, 208fExternal solution (recipe), 352–353
FFast Green (1% stock solution) recipe, 129,
138Fast-scan cyclic voltammetry (FSCV), to
measure dopamine release, 884,885f
FCIP. See Fluorescent calcium indicatorprotein
Fentanyl, 615Fiber optics
calcium monitoring of dendritic activityin vivo, 897–905
overview, 897–898protocol, 899–905
bolus loading, 902–903discussion, 905experimental method, 902–903,
904fhead mount, 903imaging setup, 899–900, 899f, 900fmaterials, 901–902recipes, 905troubleshooting, 903–904
periscope, 900, 900f, 905in vivo calcium recordings and
channelrhodopsin-2 activationthrough an optical fiber,889–895
application examples, 895overview, 889protocol, 890–894, 893f
Fiberscope. See Two-photon fiberscopesFilamentTracer, 378Filter culture medium (FCM) (recipe), 742Filters, fluorescence microscopy, 1033–1034,
1033fFirefly luciferase activity, for monitoring
doxycycline (Dox)-controlledgene expression, 585, 586f, 592,595
Fixative (recipe), 652FlaSh (fluorescent Shaker), 54, 55t, 56f, 486Flavoproteins, autofluorescence of, 919Flp recombinase, use in Brainbow studies,
101, 104Fluo-3, use in calcium imaging in neuronal
endoplasmic reticulum,339–343, 340f
Fluo-4for detection of astrocyte vacuole-like
vesicles, 644–645, 645ftwo-photon calcium imaging of dendritic
spines, 275, 275f, 276ftwo-photon imaging of astrocytes filled
with, 650Fluo-4 AM
for detection of astrocyte vacuole-likevesicles, 646
imaging astrocyte calcium activity, 642,643f, 646, 690
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Fluo-4 AM (Continued)for in vivo calcium imaging of cerebellar
astrocytes, 707, 716–717two-photon imaging of acute brain slices
preincubated with, 649Fluo-4 dextran, 138Fluo-5F AM, for in vivo calcium imaging of
cerebellar astrocytes, 707, 708f,716–717
Fluorescein, caged, 264–266Fluorescence, normalization of values,
843–844Fluorescence microscopy. See also specific
applicationsbrain tumor metastasis, 1018–1020, 1020ffilters, 1033–1034, 1033ffluorescence molecular tomography of
brain tumors, 1023–1029illumination methods in, 574fsafe operation of fluoresence microscope,
1037sodium imaging in axons and dendrites,
201–206stimulated emission depletion (STED),
237–244total internal reflection fluorescence
microscopy (TIRFM), imagingexocytosis with, 183–193
Fluorescence molecular tomography of braintumors in mice
experimental method, 1026–1028, 1026finitial image processing, 1027tomographic reconstruction, 1028
imaging setup, 1024–1025, 1025fautomation and data acquisition, 1025illumination, 1024, 1025timaging, 1025mounting, 1025
materials, 1025–1026troubleshooting, 1028
Fluorescent calcium indicator protein (FCIP)calcium indicator protein expression using
mouse transgenesis (protocol),592–596
experimental method, 593–595preparation of mouse ear
fibroblasts and quantitativegene expression assay, 593, 595
screening for transgenic fortransgenic mouse founderswith high FCIP expression, 593
imaging setup, 592materials, 592–593table of transgenic mice, 594t
calcium indicator protein expression usingrAAVs (protocol), 597–603
discussion, 603experimental method, 599–603
calcium phosphate–mediated genetransfection of HEK cells,599–600
purification of rAAV by heparincolumn, 601–602
purification of rAAV by iodixanolgradient, 600–601
stereotactic injection, 602in vivo imaging in olfactory bulb,
603in vivo two-photon imaging, 603
imaging setup, 597materials, 597–599
development of, 583–584functional neuron-specific expression of
proteins in living mice, 583–606future prospects, 604ratiometric, 584recipes, 604–605single GFP-based, 584strategies for expression, 585–591
using mouse transgenesis, 585–587,586f–587f
viral delivery using rAAV, 588–590,588f–589f
in vivo calcium imaging of cerebellarastrocytes, 707–712, 708f, 718
Fluorescent dyes. See also specific applications;specific dyes
ballistic delivery of, 447–456free radical generation by, 176loading via pipettes in whole-cell patch-
clamp method, 347–353for membrane voltage measurement,
53–54sodium-binding benzofuran isophthalase
(SBFI) for sodium imaging,202–206, 205f
Fluorescent gel solution (recipe), 445Fluorescent proteins
Brainbow mice, imaging multicolor,99–109
circularly permuted, 584genetically encoded calcium indicators
(GECIs), 63–71for imaging synaptic-like microvesicles
(SLMVs), 658for labeling and imaging receptors, 2, 4–7,
4tfor membrane voltage measurements,
53–59Fluorescent Shaker (FlaSh), 54, 55t, 56f, 486Fluorochromes
emission maxima, 1035t–1036texcitation maxima, 1035t–1036t
FM1-43, for detection of astrocyte vacuole-like vesicles, 646–647, 647f
FM dyeschemical structure, 172f, 173destaining, 175, 179for imaging synaptic-like microvesicles
(SLMVs), 658imaging synaptic vesicle recycling with,
171–181application examples, 179–181, 180fexocytosis imaging, 179–181, 180foverview, 171–173, 172fprotocols
FM dye photoconversion, 176–178,176f
recipes, 181staining and destaining synaptic
vesicles with FM dyes, 174–175slice preparation, 179
loading of astrocytes with, 661properties and mechanism of action, 172f,
173total internal reflection fluorescence
microscopy (TIRFM) forimaging exocytosis, 183–193,191f
FMRI. See Functional magnetic resonanceimaging
Förster resonance energy transfer. See FRETFree radicals, generation by fluorescent dyes,
176Frequency, 1031FRET
Clomeleon use as chloride indicator, 75,79
described, 583fluorescent calcium indicator protein
(FCIP) development and,583–584
genetically encoded calcium indicators(GECIs), 64, 65f, 67
in membrane voltage measurement, 54neuronal imaging in Caenorhabditis
elegans, 764TN-XXL and, 611, 613, 618
FRT sites, in Brainbow constructs, 101, 104FSCV (fast-scan cyclic voltammetry), to
measure dopamine release, 884,885f
Functional magnetic resonance imaging(fMRI)
BOLD (blood-oxygenation-level-dependent), 915, 916
contrast-agent-based, 917low spatial resolution of, 908perfusion-based, 917
Functional neuron-specific expression ofgenetically encoded fluorescentcalcium indicator proteins inliving mice, 583–606
Fungizone, 593Fura-2
calcium wave imaging in cerebellarBergmann glia, 701–702
detection of changes in amplitude ofpresynaptic calcium influx,127–128, 127f
Fura-2 AM for action potential imaging,370–373, 373f, 374f
Fura-2 dextran, 137Fura-2FF use in Ca2+ uncaging in nerve
terminals, 151, 154–156, 154t,156f, 158
GGABA (γ-aminobutyric acid), 623
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caged use in optical fiber-based uncagingsystem, 405–408
receptors, 389Gal4/UAS gene expression system
calcium imaging in Drosophila olfactorysystem, 557, 558f, 563
Galvanometers, voltage-controlled mirror, 419Galvanometric scanning, 258G-CaMP
benefits of, 584calcium imaging in Drosophila olfactory
system, 557–563G-CaMP2, 68, 69f, 707–712, 708f, 718G-CaMP3, 64, 65f, 67, 67t, 68, 69fneuronal imaging in Caenorhabditis
elegans, 764in vivo calcium imaging of cerebellar
astrocytes, 707–712, 708f, 718GECIs. See Genetically encoded calcium
indicatorsGelfoam, 323Gene gun for ballistic delivery of dyes,
447–456Genetically defined neuronal populations,
structure–function analysis of,377–385
Genetically encoded calcium indicators(GECIs), 63–73
application examples, 64calcium imaging in Drosophila olfactory
system with a genetic indicator,557–563
functional neuron-specific expression ofproteins in living mice, 583–606
future design, 71to measure neural activity, 71optimization, 68–70
G-CaMP3, 68, 69fGECI expression, 68practical improvement, 68subcellular targeting, 70
overview, 63–64properties influencing performance, 64–67
expression level, 66–67fluorescence properties, 67, 67tunderlying calcium dynamics, 64, 66
structure of, 64, 65ftesting standardization, 70TN-XXL, calcium imaging of neurons in
visual cortex using, 611–620,612f
GENSAT (Gene Expression Nervous SystemAtlas) Project, 113–119, 377
GFAP (glial fibrillary acidic protein), 640,641f, 642, 687
GFP. See Green fluorescent proteinGH146-Gal4, 557, 558fGlia. See also Astrocytes
ballistic delivery of dyes, 447–456Bergmann
imaging calcium waves in, 699–705,704f
preferential loading with synthetic
acetoxymethyl calcium dyes(protocol), 716–717
in vivo calcium imaging with syntheticand genetic indicators,707–719, 708f
imaging microglia in brain slices and slicecultures, 735–742
two-photon imaging/microscopy in spinalcord in vivo, 721–733
two-photon imaging of microglia inmouse cortex in vivo, 961–976
Glial fibrillary acidic protein (GFAP), 640,641f, 642, 687
Glial fibrillary acidic protein (GFAP)promoter, 687, 708f, 867
Glioblastoma, 1012, 1017Glioma, 1011–1017, 1016fGliotransmitters, 639–640, 655, 657Glossary, 1041–1050Glutamate
cagedacousto-optical deflector (AOD)-based
patterned UV neurotransmitteruncaging, 255–270
circuit mapping by ultravioletuncaging, 417–425
methoxy-7-nitroindolinyl-glutamate(MNI-glu), 246, 246f, 249–253,251f, 253f, 268, 277, 431–432
optical fiber-based uncaging system,405–408
properties of caged glutamate for two-photon photolysis, 246, 246f
ruthenium-bipyridine-trimethylphosphine-glutamate(RuBi-glutamate), 431–432
two-photon calcium imaging ofdendritic spines, 276f, 277
two-photon mapping of neuralcircuits, 429–434, 430f, 432t
using patterned uncaging to simulatenetwork activity in vitro,258–260
as gliotransmitter, 639, 657Glutamate receptors
light-activated ionotropic glutamatereceptor (LiGluR), 36
spatially resolved flash photolysis viachemical two-photon uncaging,29, 29f
Glutamate transporter 1 (GLT1) promoter, 687Glycine, 623Glycosylphosphatidylinositol (GPI), 208GlyR (glycine receptor) lateral diffusion,
measuring using quantum dots,15, 16f
GMEM4+ (recipe), 98Gold nanoparticles
detection by photothermal heterodyneimaging (PHI), 2
labeling and imaging receptors using, 2,4–7, 4t
tracking using single-nanoparticle
photothermal tracking(SNaPT), 2
G-protein-coupled receptors (GPCRs)in chimeric opsins, 867of type II opsins, 44
Green fluorescent protein (GFP). See alsoEnhanced green fluorescentprotein
in Alzheimer’s disease imaging, 991f, 992correlated light and electron microscopy
of green fluorescent protein–labeled axons and dendrites,227–236
in FlaSh (fluorescent Shaker), 54, 55t, 56flevel required for dendritic spine
detection, 320pHluorins, 19–20SPARC (sodium channel protein-based
activity reporting construct),55, 55t, 56f
HHabituation and training for awake
experiments, 840Halobacterium salinarum, 45Halorhodopsin
light-induced dipole switching, 43fmode of action, 45overview, 42f, 866
Hazardous materials, 1053–1058HBAFM (high bandwidth atomic force
microscopy), dynamic, 164Head plate
for imaging neuronal population activityhead plate design, 840–841, 841fhead plate implantation, 841–842
implantation protocol, 747–749, 748ffor two-photon imaging of neural activity
in awake mobile mice, 828–829,829f
Heat filter, 1034Held synapse, using Ca2+ uncaging to examine
transmitter release at calyx of,158–159, 158f
Hemoglobin, oxygen saturation of, 913–914,915–916
Heparin column, purification of rAAV by,601–602
HEPES-buffered culture medium (HCM)(recipe), 742
HEPES-KRH buffer (recipe), 670Hidden Markov model (HMM), 832, 845High bandwidth atomic force microscopy
(HBAFM), dynamic, 164High-divalent ACSF (recipe), 425High-K+ Ringer’s solution (recipe), 192Histology, in situ of brain tissue with
femtosecond laser pulses,437–445
HIV-1 Env, 85HMM (hidden Markov model), 832, 845Human clinical studies, using optical imaging,
912–913
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IIce cube model, 910, 911fIGOR Pro, 267ImageJ, 736, 1000, 1016, 1020Immune cells, two-photon imaging of,
981–988overview, 981protocol, 982–988
cocultures of acute hippocampal sliceswith immune cells, 984–985
discussion, 987–988, 987fexperimental method, 984–985imaging acquistion and analysis, 985imaging setup, 982, 983fmaterials, 982–984preparation of brain stem of
anesthetized mice for intravitalimaging, 985
troubleshooting, 986Imspector software package, 241Incident light, noise in, 472Infrared (IR) camera, 378Infrared-guided laser stimulation of neurons
in brain slices, 388–390advantages and limitations of photolytic
neurotransmitter activation,390
applications of targeted stimulation, 389,390f
experimental method, 389–390imaging setup, 388materials, 388–389
Infrared-guided neurotransmitter uncagingon dendrites, 387–391
IntegriSense, 1025Interferometric detection of action potentials,
195–199advantages and limitations, 199application example, 198–199, 198fprinciples of interferometric detection,
195–198Internal pipette solution (recipe), 206Internal recording solution (recipe), 278, 467Internal solution (recipe), 192Intracellular saline containing SBFI (recipe),
303Intrapipette solution (recipe), 344, 824Intrinsic imaging
basics of, 634chronic, 634–637, 635fof functional map development in
mammalian visual cortex,633–637, 635f, 636f
overview, 633–634Intrinsic signals
optical imaging based on, 907–919combining with other techniques, 917,
918flimitations of, 919outlook for, 917, 919overview, 908–913
sources of, 913–915
In utero electroporation, 213, 612, 613Inverse pericam, 586f–587f, 587In vitro calibration solutions (recipe), 631Iodixanol gradient, purification of rAAV by,
600–601Ion channels
engineering light-regulated, 33–40modifications to existing light-gated
channels, 37–38photoswitchable tethered ligands
(PTLs), 35practical considerations, 38strategies, 33, 34ftargeted proteins, 36
light-gated, 46–47Ion pumps, light-gated, 44–46Ischemia
optically induced occlusion of single bloodvessels in neocortex, 939–947
two-photon imaging of neuronalstructural plasticity in miceduring and after ischemia,949–957
Isoflurane, 492, 514, 515, 714, 728, 753, 871,892, 902, 931, 952, 965, 985,1001, 1026
Isolectin B4 (IB4), 737–739, 741Isolectin B4 (IB4) derived from Giffonia
simplicifolia seeds (recipe), 742
JJackson Laboratory, 211
KKatushka, 1023Ketamine, 308, 315, 322, 324, 514, 515, 526,
569, 595, 602–603, 688, 711,714, 871, 892, 952
Kiss-and-run fusion, 171, 179–181, 180fKrebs-Ringer solution (recipe), 391Kwik-sil, 830–831KX mixture (recipe), 315
LLabVIEW, 267, 492, 531Lamp1, 661Laser
light sources for photolysis, 399–400mode-locked, 248, 307, 411–412pulsed nitrogen, 400safe operation of, 1037–1038ultraviolet, 258, 261, 262, 399–400
Laser pulses, in situ histology of brain tissuewith, 437–445
advantages and limitations, 443–444application example, 443, 444flaser alignment, 442–443overview, 437–438, 440fprotocol, 438–445
blocking and staining, 442discussion, 442–444experimental method, 441–442imaging setup, 439, 440f
iterative imaging and ablation, 442,443t
mapping region of interest, 441–442materials, 439, 441perfusing with fluorescent gel, 442recipes, 445subblocking and mounting, 442
Laser scanning photostimulation (LSPS)circuit mapping by ultraviolet uncaging of
glutamate, 417–425hardware, 419software, 419
Laser-targeted focal photothrombotic strokemodel, 954–955, 955f
Lead citrate (recipe), 236Lentivirus, for opsin expression in
mammalian brain, 867, 869,871–872
Lidocaine, 492, 603, 902, 931, 952, 965Light-activated ionotropic glutamate receptor
(LiGluR), 36Light-gated ion channels, 46–47Light-gated ion pumps, 44–46Light-regulated ion channels, engineering,
33–38modifications to existing light-gated
channels, 37–38engineering ion channel targets, 37photochromic ligands, 37–38photoswitch tuning, 37
photoswitchable tethered ligands (PTLs),35
ligand, 35photoswitch, 35reactive moiety, 35
practical considerations, 38cysteine-maleimide reactivity, 38light, 38
strategies, 33, 34ftargeted proteins, 36
ionotropic glutamate receptor, 36nicotinic acetylcholine receptor, 36voltage-gated potassium channel, 36
Light scatteringcalcium effects on, 163–164, 163fsecretion-coupled from peptidergic nerve
terminals, 161–168effects of Ca2+ on intrinsic optical
signal components, 163–164,163f
measuring intrinsic optical signalsfrom mammalian nerveterminals (protocol), 166–168
overview, 161–163, 162funderlying mechanisms, 164–165, 164f
Light-sheet microscopy, 573–575Line averaging, 336–337Lipofectamine 2000, 595Lipofuscin, 1008Lipophilic dyes, ballistic delivery of, 447–456Live cells and tissue slices, maintaining,
331–338ill health indicators, 337
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Index / 1073
maintaining in the imaging setup, 332–337collecting images, 336–337gas and pH condition maintenance
after mounting, 334, 334f, 335fmedium considerations, 332mounting specimens for microscopic
observation, 332–335, 333ftemperature considerations, 336
overview, 331–332Lobster, interferometry of walking leg nerve,
198, 198f, 199Local dye electroporation for calcium imaging
and neuronal-circuit tracing,501–509
overview, 501–502protocol, 503–509
application examples, 504, 506f, 507f,508f
discussion, 509experimental method, 504, 505fimaging setup, 503materials, 503recipe, 509troubleshooting, 506–508
Low-Ca+ Ringer’s solution (recipe), 193LSPS. See Laser scanning photostimulationLucas–Kanade algorithm, 845Luciferin, 595LY-411575, 992Lysosome identification and staining in
astrocytes, 661Lysosome marker proteins, 661
MMagFura-2 AM, use in calcium imaging in
neuronal endoplasmicreticulum, 339–344, 340f
MagFura-5 AM, 132f, 137MagIndo-1 AM, 432Magnesium Green, 127–128, 127fMagnetic resonance imaging (MRI). See also
Functional magnetic resonanceimaging
of Alzheimer’s disease, 989, 990functional brain mapping with, 913
Maleimides, cysteine-reactivity of, 38Mannitol, 711Mapping
functional based on intrinsic signals,913–917
single-condition, 916–917two-photon of neural circuits, 429–434
MATLAB, 267, 784, 935Matrix metalloproteinases (MMPs), 995, 995fMauthner cells, 796, 796fMB-231BR mouse cell line, 1012, 1018–1020,
1020fMCBL. See Multicell bolus loading (MCBL) of
fluorescent indicatorsmCherry
in fluorescence molecular tomography,1023, 1028
for imaging synaptic-like microvesicles
(SLMVs), 658synaptic-like microvesicles (SLMVs)
imaging, 663–666, 665fVGLUT1 tagged with, 663, 664–665, 665f
Mechanical drift, in stimulated emissiondepletion (STED), 242
Medetomidine, 615MEM (recipe), 670Membrane voltage
fluorescent proteins for measurement of,53–59
applications, 58, 58fdiscussion of, 59experimental setup, 58FlaSh (fluorescent Shaker), 54, 55t, 56fmechanism of fluorescence change,
56f, 57–58Mermaid, 55t, 57, 58, 58fSPARC (sodium channel protein-based
activity reporting construct),55, 55t, 56f
VSFPs (voltage-sensing fluorescentproteins), 54–55, 55t, 56f, 57
optical approaches to studying, 53–54fluorescent dyes as sensors, 53–54protein-based sensors, 54
MEM medium for quantum dot imaging(recipe), 17
MEMS (microelectromechanical system), 856MEPSCs (miniature excitatory postsynaptic
currents), 251–252, 251fMEQ (6-methoxy-N-ethylquinolinium
chloride), 624Mercury arc lamp, 1037, 1038
as light sources for photolysis, 399–400Mermaid, 55t, 57, 58, 58fMetastasis, 1012, 1018–1021, 1020fMethohexital, 724–725Methoxy-7-nitroindolinyl-glutamate (MNI-
glu), 246, 246f, 249–253, 251f,253f, 268, 277, 431–432
Methoxy-XO4, 990, 991f, 992, 1007Mice
Alzheimer’s disease model, 991anesthesia, 308, 322, 324, 492, 504, 534,
550, 569, 595, 602–603, 615,688, 711, 714, 724, 726, 728,871, 952, 965, 985, 1001, 1015,1016, 1019, 1026
BAC transgenic, 113–119Brainbow, generation and imaging
multicolor, 99–111calcium imaging in intact olfactory system
of, 565–571craniotomy, 516, 516ffluorescence molecular tomography of
brain tumors, 1023–1029fluorescent calcium indicator protein
(FCIP) expression intransgenic, 585–587, 586f–587f,594t
functional neuron-specific expression ofgenetically encoded fluorescent
calcium indicator proteins inliving mice, 583–606
GENSAT (Gene Expression NervousSystem Atlas) Project, 113–119
head plate implantation, 747–749, 748f,963–965, 964f
imaging neocortical neurons through achronic cranial window,319–328
intubation, 725MB-231BR cell ine, 1012, 1018–1020,
1020fmeasuring intrinsic optical signals from
mammalian nerve terminals(protocol), 166–168
Mutant Mouse Regional Resource Center(MMRRC), 119
ordering mouse lines, 119Thy1 lines, 207–225transgenic
BAC, 113–119with constitutive promoters driving
FCIP expression, 594tfluorescent calcium indicator protein
(FCIP) expression in, 585–587,586f–587f, 594t
for targeting reward circuitry, 879ttet-activator (tTA), 594ttet-responder, 594t
two-photon imaging of astrocytic andneuronal excitation incerebellar cortex of awakemobile mice, 745–760
two-photon imaging of microglia inmouse cortex in vivo, 961–976
two-photon imaging of neural activity inawake mobile mice, 827–836
two-photon imaging of neurons and gliain spinal cord in vivo, 721–733
in vivo two-photon calcium imaging invisual system, 511–527
voltage-sensitive dye imaging of corticalspatiotemporal dynamics inawake behaving mice, 817–824
Microbial opsins. See also Opsinsbiophysical properties, 48–49homologies, 44light-gated ion channels, 46–47light-gated ion pumps, 44–46for optical control of neural activity, 41–50overview, 41–42, 42f
Microelectromechanical system (MEMS), 856Microglia
imaging in brain slices and slice cultures,735–742
imaging setup, 735–736migration of microglia, 740fprotocol, 737–742
confocal and two-photon imaging,739
discussion, 740–741, 740f, 741fexperimental method, 737–739labeling with fluorescent lectin, 738
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Microglia (Continued)materials, 737mounting tissue slices for imaging,
738–739recipes, 742tissue slice preparation, 737–738troubleshooting, 739
two-photon imaging in mouse cortex invivo, 961–976
discussion, 975–976optical window preparation (protocol),
966–968materials, 966–967sealed craniotomy method, 968thinned skull method, 967–968troubleshooting, 968
overview, 961–962surgical implantation of head plate in
preparation for imaging(protocol), 963–965
experimental method, 964–965materials, 963–964setup, 964f
two-photon imaging (protocol),969–974
experimental method, 970–972,971f, 973f
imaging setup, 969–970materials, 970troubleshooting, 972–974
Microinjection buffer (recipe), 223Microinjection solution (recipe), 696Microscope objective, 1039–1040
for confocal spot detection, 142selection for total internal reflection
fluorescence microscopy(TIRFM), 185
Microscopy. See also specific applicationsacousto-optical deflector (AOD)-based
two photon microscope,545–547, 547f
objective-coupled planar illumination(OCPI), 573–580, 576f, 578f
photothermal imaging microscope, 3planar illumination microscopy, 573–580single-molecule epifluorescence
microscope, 3stimulated emission depletion (STED),
237–244total internal reflection fluorescence
microscopy (TIRFM), 183–193two-photon uncaging, 245–253
Midazolam, 615Miniature excitatory postsynaptic currents
(mEPSCs), 251–252, 251fMiniaturization of two-photon microscopy
for imaging in freely movinganimals, 851–860
application examples, 857–859, 858ffluorescent dye labeling in vivo, 857imaging setup, 852–857, 853t, 854f
components, 852, 853fdesign considerations, 852–857, 854f
fluorescence detection, 856mechanical attachment to animal, 857miniaturized fiber-scanning devices,
855–856small microscope objectives, 856two-photon excitation through optical
fibers, 853, 855MitoMice, 211fmKOκ (monomeric kusabira orange), 57MLCKp (myosin light chain kinase), 583MMPs (matrix metalloproteinases), 995, 995fMMPSense, 1025MMRRC (Mutant Mouse Regional Resource
Center), 119MNI-glu (methoxy-7-nitroindolinyl-
glutamate), 246, 246f, 249–253,251f, 253f, 268, 277, 431–432
Mode-locked laser, 248, 307, 411–412Modified balanced salt solution (MBSS)
(recipe), 742MOG peptide (myelin oligodendrocyte
glycoprotein peptide), 984MOM (movable objective microscope), 828Monkey, voltage-sensitive dye imaging of
neocortical activity of corticaldynamics in behaving, 810–811
Monomeric kusabira orange (mKOκ), 57Monomeric umi-kinoko green (mUKG), 57Monosynaptic tracing, with recombinant
fluorescent rabies virus vectors,83–98, 85f
Motor behavior, imaging in zebrafish,783–789
Mouse model of Alzheimer’s disease, 999–1009Mouse Ringer’s solution (recipe), 168Movable objective microscope (MOM), 828Movement artifacts, 845, 1006Mowiol 4-88 mounting medium (recipe), 385MPScope, 439MQAE (N-(6-methoxyquinolyl) acetoethyl
ester), chloride imaging using,623–632
advantages and limitations, 630application example, 627f, 628f, 630overview, 623–624protocol, 624–631
discussion, 630experimental method, 626–628imaging setup, 625intracellular calibration of MQAE,
627–628materials, 625–626recipes, 630–631staining cells or tissue slices via bath
application, 626, 627fstaining of neurons and glia using
multicell bolus loading, 626, 628ftroubleshooting, 629two-photon imaging of stained cells,
626MQAE cuvette calibration solutions (recipe),
631mRaspberry, 1023
MRI. See Magnetic resonance imagingMS-222, 569–570mUKG (monomeric umi-kinoko green), 57Multicell bolus loading (MCBL) of fluorescent
indicators, 491–499advantages and limitations, 497–498in Alzheimer’s disease imaging,
1002–1003, 1007–1008application examples, 495–497, 495f, 496foverview, 491protocol for in vivo two-photon calcium
imaging, 492–499experimental method, 493–494imaging setup, 492materials, 492–493troubleshooting, 494
in vivo calcium imaging of cerebellarastrocytes, 707, 708f, 716–717
Multiphoton imaging, in olfactory system ofzebrafish and mouse, 566f, 567,568
Multiphoton stimulation of neurons andspines, 411–415
advantages of, 415application example, 414, 414f, 415fexperimental protocol, 412–413imaging setup, 412limitations of, 414–415materials, 412
Multiple sclerosis, murine models of, 981Multi-worm Tracker, 770–773, 773fMutant Mouse Regional Resource Center
(MMRRC), 119Myelin oligodendrocyte glycoprotein peptide
(MOG peptide), 984Myosin light chain kinase (MLCKp), 583
NNADH intrinsic fluorescence, in vivo imaging
of redox state using, 692–694,693f
Nanolabels, tracking receptors usingadvantages, 7limitations, 7overview, 1–2protocol, 3–7
analysis, 6application example, 7, 8f.experimental method, 4–6image acquisition, 6imaging setup, 3labeling cells, 4–5, 4tmaterials, 4particle tracking, 6recipe, 7signal characterization, 5
National Center for Biotechnology (NCBI),119
Natronomonas pharaonis, 45, 46f, 866NCAM (neuronal cell adhesion molecule), 84Near-infrared fluorescence (NIRF)
tomography, of Alzheimer’sdisease, 989, 990
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Nematodes. SeeWorm trackingNembutal, 504Neocortical activity, voltage-sensitive dye
imaging of, 799–812Neocortical neurons, imaging through a
chronic cranial window,319–328
Nerve terminalsCa2+ uncaging in, 151–159light scattering changes associated with
secretion from peptidergic,161–168
single compartment model of calciumdynamics in, 355–366
Neural circuits, two-photon mapping,429–434
overview, 429–431practical examples, 431–434
choice of caged glutamate, 431–432,432t
mapping connections in brain slices,432–434, 433f, 434f
Neural networkacousto-optical deflector (AOD)-based
two-photon microscopy forhigh-speed calcium imaging ofneuronal population activity,543–555
three-dimensional imaging of activity,529–540
application example, 538, 538fmeasuring neuronal population
activity using 3D laser scanning(protocol), 531–537
animal preparation and 3Dimaging procedure, 534
assigning fluorescence signals tocells within the scan volume,535f
creating 3D scan trajectories,532–533, 532f
discussion, 539experimental method, 532–536imaging setup, 531laser intensity adjustment, 533–534materials, 531–532, 532frecipe, 540signal assignment and 3D
visualization of networkdynamics, 534, 536
troubleshooting, 536–537overview, 529–530principles of 3D laser scanning, 532f
voltage-sensitive dye imaging ofneocortical activity, 799–812
NeuroCCD-SM Imaging System, 288Neurointermediate lobe, isolation of, 166–167Neurolucida, 378Neuromantic, 378Neuromuscular explant, imaging of Thy1 lines
using an acute, 221–222experimental method, 222imaging setup, 221
materials, 221Neuronal activity
confocal calcium imaging in larvalzebrafish, 791–797
imaging activity and motor behavior inzebrafish, 783–789
Neuronal cell adhesion molecule (NCAM), 84Neuronal membrane receptors, labeling single
receptors with quantum dots,13–17
experimental method, 14imaging setup, 13interpretation of imaging data, 15, 16fmaterials, 13measuring GlyR (glycine receptor) lateral
diffusion, 15, 16frecipes, 17troubleshooting, 14
Neuronal population activity, imaging inawake and anesthetizedrodents, 839–849
application example, 847, 848fdata analysis, 843–847, 844f
action potential detection, 845–847correction for movement artifacts, 845estimation of noise levels and neuropil
contamination, 845normalization of fluorescence values,
843–844experimental procedures, 840–843
cell-attached electrophysiology,842–843, 844f
dye preparation for injection, 842–843habituation and training for awake
experiments, 840head plate design, 840–841, 841fhead plate implantation, 841–842surgical considerations, 842targeting sensory areas, 842
future prospects, 848–849Neuronal structural plasticity, two-photon
imaging of, 949–957NeuronIQ, 378Neurons
ballistic delivery of dyes for structure-function studies, 447–456
calcium imaging in neuronal endoplasmicreticulum, 339–344
imaging action potentials with calciumindicators, 369–374
imaging calcium waves and sparks incentral, 281–285
imaging green fluorescent protein–labeledusing light and electronmicroscopy (protocol), 228–236
discussion, 235experimental protocol, 230–234finding imaged neuron in sections,
231–232, 232ffixation and immunochemistry, 230fixation of imaged slices, 230–231imaging dendrites in EM, 234, 235fimaging setup, 228
materials, 228–229recipes, 236resin embedding for EM, 231serial sectioning and imaging in EM,
233–234, 233ftroubleshooting, 234–235
imaging neocortical neurons through achronic cranial window,319–328
imaging neuronal activity with geneticallyencoded calcium indicators(GECIs), 63–73
microbial opsins for optical control ofneural activity, 41–50
multiphoton stimulation of, 411–415optogenetic modulation of dopamine,
877–886, 879frecombinant fluorescent rabies virus
vectors for tracing, 83–98structure-function analysis of genetically
defined neuronal populations,377–385
two-photon imaging neuronal excitationin cerebellar cortex of awakemobile mice, 745–760
type-specific staining with voltage-sensitive dye, 486
uncaging calcium in neurons, 393–401NeuronStudio, 378Neuropeptide Y (NPY), 659Neuropeptide Y–enhanced green fluorescent
protein (NPY-eGFP), 183, 185,189, 191, 192f
NeuroPlex, 288, 479Neurosurgery, use of optical imaging in, 912Neurotransmitter uncaging. See UncagingNeutral-density filter, 1034NGM agar medium (recipe), 775
low-peptone, 775Nicotinic acetylcholine receptor (nAChR),
photoisomerizable, 36Ni-DAQ Tools, 267NIRF (near-infrared fluorescence)
tomography, of Alzheimer’sdisease, 989, 990
Nitrophenyl-EGTA (NP-EGTA), 393, 394f–396f,395–399, 399f, 644, 644f, 651
NK3630, 479–480NK2367 dye, 162, 162fNoise
signal-to-noise ratio (SNR)of calcium indicators, 64, 145–146confocal spot detection, 147–149, 148fdefined, 64GCaMPs, 558, 584improving
by increasing incident illumination,336
by increasing pixel dwell time,336–337
by line averaging, 336–337random-access pattern scanning,
554–555
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Noise (Continued)of voltage-sensitive dyes, 801, 802, 810
voltage-sensitive dye, imaging withdark noise, 472, 474noise in incident light, 472shot noise, 474, 474fvibrational noise, 472
Normal rat Ringer (NRR) solution, 540, 555NP-EGTA (nitrophenyl-EGTA), 393, 394f–396f,
395–399, 399f, 644, 644f, 651NpHR, 866, 868NRR (recipe), 540, 555Nystatin, 593
OObjective-coupled planar illumination
(OCPI) microscopy, 573–580,576f, 577t, 578f
Objective lens, 856, 1039–1040Odor coding pattern transformation with
olfactory bulb, 501–502, 504,507f, 508f
OGB-1. See Oregon Green BAPTA-1Olfactory receptor neurons, synapto-pHluorin
expression in, 21–22, 22fOlfactory system
calcium imaging in Drosophila with agenetic indicator, 557–563
overview, 557–558, 558f, 559fprotocol, 560–563
antennae–brain preparation, 561discussion, 563imaging setup, 560materials, 560–561nerve stimulation preparation,
561–562olfactory stimulation and imaging,
561recipes, 563
calcium imaging in intact olfactory systemof zebrafish and mouse,565–571
imaging methods, 567multiphoton imaging, 567wide-field fluorescence imaging,
567loading calcium indicators, 566–567
bolus loading of neurons down-stream from glomeruli, 567
dextran-coupled (protocol),568–571
genetically encoded indicators, 567selective loading of neurons
projecting to glomeruli, 566overview, 565–566, 566f
calcium imaging in populations ofolfactory neurons by planarillumination microscopy,573–580
overview, 573–575, 574f, 576fprotocol, 577–580, 577t, 578f
fluorescent calcium indicator protein(FCIP) expression, 586f, 597, 603
Operant conditioning, for assessing reward-related behavior, 883–884
Opsins. See also Optogeneticsbiophysical properties, 48–49chimeric, 867, 878gene expression and targeting systems,
867–868light-gated ion channels, 46–47light-gated ion pumps, 44–46in neuroscience, 864–867overview, 41–42, 42fphotoreaction mechanisms, 43fprinciples of operation, 41–42, 43–44step-function, 866table of, 864t–865ttype I (microbial type), 44type II (animal type), 44
Optical equipment, cleaning, 1039–1040Optical fiber
fiber-optic calcium monitoring ofdendritic activity in vivo,897–905
overview, 897–898protocol, 899–905
uncaging system, optical fiber-based,405–408
advantages of, 408assembly and optimization, 406, 407fexperiments utilizing the system,
406–408, 407flimitations of, 408materials, 406
in vivo calcium recordings and channel-rhodopsin-2 activation throughan optical fiber, 889–895
application examples, 895overview, 889protocol, 890–894, 893f
Optical imaging based on intrinsic signals,907–919
combining with other techniques, 917,918f
limitations of, 919outlook for, 917, 919overview, 908–913
in awake behaving animals, 912chronic imaging, 910developmental studies, 912example studies, 908, 910, 911fexperimental setup, 908, 909fhuman clinical studies, 912–913
sources of intrinsic signals, 913–915Optical imaging spectroscopy, 913Optically induced occlusion, of single blood
vessels in neocortex, 939–947Optical neural interface, fiber-optic-based,
868–873, 870fOptical parametric oscillator, 982Optical window. See also Cranial window;
Craniotomychoice of type, 975–976preparation protocol, 966–968
materials, 966–967
sealed craniotomy method, 968thinned skull method, 967–968troubleshooting, 968
Optodes, 912Optogenetics
Cre mice for targeting reward circuitry,878, 879t
described, 41, 42establishing a fiber-optic-based optical
neural interface (protocol),869–873, 870f
experimental method, 871–873material, 869–871, 870ftroubleshooting, 873
in freely moving mammals, 877–885future outlook, 874, 886modulation of dopamine neurons,
877–886, 879ffunctional integration during optical
control, 884–885, 885foptical measurement of dopamine
nerve terminals in brain slices,884–885
optical measurement of dopaminerelease in vivo, 885
integrating behavioral readouts withreward circuit modulation,880–884
conditioned place preference,881–883, 882f
experimental design, 880–881operant conditioning, 883–884
overview, 863–868tools, table of, 864t–865t
OptoXRs, 867, 878Oregon Green BAPTA-1, 521–523, 531, 534,
535f, 566, 579in Alzheimer’s disease imaging, 1000in vivo recordings and channelrhodopsin-
2 activation through an opticalfiber, 890, 891f
Oregon Green BAPTA-1 AM, 531, 534, 535fin Alzheimer’s disease imaging, 993–994,
994f, 1002–1005, 1003f, 1004fimaging calcium excitation in cerebellar
cortex, 758f, 759imaging neuronal activity and motor
behavior in zebrafish, 784, 787multicell bolus loading of, 495, 495f, 497in vivo recordings and channelrhodopsin-
2 activation through an opticalfiber, 893f
Oregon Green BAPTA-6F, 278Oregon Green BAPTA-5N, 147Organic dyes, labeling and imaging receptors
using, 2, 4–7, 4tORION, 378OSN dye solution (recipe), 571Oxidative stress, in Alzheimer’s disease, 994,
995fOxonol dyes
NK3630, 479–480RH155, 476
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Oxygen saturation of hemoglobin, 913–914,915–916
PParaformaldehyde (recipe), 385Paraformaldehyde solution 8% (recipe), 652Parallel fibers, 699, 702–704Parallel Worm Tracker, 773Patch-clamp pipette filling solution (recipe),
391Patch-clamp recording
calcium imaging in neuronal endoplasmicreticulum, 339–344
dye loading technique, 347–353infrared-guided neurotransmitter
uncaging on dendrites, 387–391two-photon targeted patching and
electroporation in vivo,459–467
targeting single cells with patchpipettes in vivo (protocol),461–467
advantages and limitations,466–467
application examples, 466experimental method, 462–464,
463f, 464fmaterials, 461practical considerations, 464–465recipes, 467
Patch pipettesdye loading with, 348–353targeting single cells with patch pipettes in
vivo (protocol), 461–467Pavolvian conditioning. See Conditioned place
preferencePBS. See Phosphate-buffered salinePentobarbital, 603, 724Perfusion system, for live cell and tissue slice
imaging, 334, 334f, 335fPET. See Positron-emission tomographyPFA (paraformaldehyde) (recipe), 385PHI (photothermal heterodyne imaging), 2, 6pHluorins, 19–24
advantages and limitations, 22–23application examples, 21–22, 22fecliptic, 19–20gene expression, 21optical imaging, 21overview, 19–20phogrin tagged with, 660ratiometric, 20synapto-pHluorins, 19–24targeting module, 20VGLUT1 tagged with, 658, 663–666, 665f,
667–669, 669fPhogrin, 660Phosphate-buffered saline (PBS) (recipe), 7,
110, 181, 193, 605PBS-MK 1x (recipe), 60510x, 223, 605
Photoactivation of Rose Bengal, 949, 954–955,955f, 957
Photobleaching, G-CaMP and, 558, 558fPhotoconversion, FM dye, 176–178, 176fPhotodamage
G-CaMP and, 558, 558freducing with antioxidants, 337SNR compromise, 336–337
Photoisomerizationlight-regulated ion channels, engineering,
33–38of retinal, 43–44, 43f
Photolysisacousto-optical deflector (AOD)-based
patterned UV neurotransmitteruncaging, 255–270
detection calcium signals from photolysisof caged compound in singleastrocyte, 644, 644f
infrared-guided laser stimulation ofneurons in brain slices, 388–390
light sources for, 399–400measuring effectiveness of, 401spatially resolved flash photolysis via
chemical two-photon uncaging,25–31
overview, 25–26, 26fprotocol, 27–30
two-photon, 246, 246f, 256uncaging calcium in neurons, 393–401
Photolyzable Ca2+ chelator, 151–152, 156f, 157Photon energy, 1031Photorhodopsin, 45Photoswitchable molecules and engineering
light-regulated ion channels,33–40
modifications to existing light-gatedchannels, 37–38
photoswitchable tethered ligands (PTLs),35
practical considerations, 38strategies, 33, 34ftargeted proteins, 36
ionotropic glutamate receptor, 36nicotinic acetylcholine receptor, 36voltage-gated potassium channel, 36
Photoswitchable tethered ligands (PTLs), forlight-regulated ion channelengineering, 35
ligand, 35modifications to existing light-gated
channels, 37–38engineering ion channel targets, 37photochromic ligands, 37–38photoswitch tuning, 37
photoswitch, 35practical considerations, 38
cysteine-maleimide reactivity, 38light, 38
reactive moiety, 35Photothermal heterodyne imaging (PHI), 2, 6Photothermal imaging microscope, 3Photothrombosis, 939, 941, 942f, 943–944,
945f, 947, 949, 954–955, 955fPhototoxicity
in FM dye imaging, 174in stimulated emission depletion (STED),
242voltage-sensitive dye s, 480
Piezoelectric bending element, 855Pipette solution for biocytin staining (recipe),
653Pipette solution for Fluo-4 staining (recipe),
653Pipette staining solution (recipe), 631Pittsburg compound B (PiB), 990, 992, 1003f,
1007Planar illumination microscopy, calcium
imaging in populations ofolfactory neurons by, 573–580
overview, 573–575, 574f, 576fprotocol, 577–580, 577t, 578f
Plaques. See Alzheimer’s diseasePlasma-mediated ablation, 940, 941, 942f,
944–945, 947Pluronic F-127 in DMSO, 492p75 neurotrophin receptor (p75NTR), 84Pockels cell, 412, 534Point spread function (PSF), 144Positron-emission tomography (PET)
of Alzheimer’s disease, 989, 990low spatial resolution of, 908
Potassium channelfluorescent Shaker (FlaSh), 54synthetic photoisomerizable azobenzene
regulated, 36ppHcrt promoter, 867Presenilins, 999Pressure injection, of AM calcium indicators,
371–372Promoters
aldehyde dehydrogenase (Aldh1L1), 687αCaMKII, 585, 586ffor Brainbow construct expression, 103CamKIIα, 867cytomegalovirus (CMV) immediate-early
(IE), 707, 708f, 718for expression in astrocytes, 687glial fibrillary acidic protein (GFAP), 687,
708f, 867glutamate transporter 1 (GLT1), 687ppHcrt, 867tetracycline, 585, 588, 588f–589f, 590, 593UAS, 557
Pronuclear injection, generation of Thy1constructs for, 214–216
cloning of fluorescent reporter proteinsequence, 215–216
experimental method, 215–216materials, 214–215preparation of DNA for pronuclear
injection, 216troubleshooting, 216
ProSense, 1025Proteorhodopsins, 45Proton pumps, light-driven, 45Pseudotyping, of rabies virus, 84f, 85, 95–96PSF (point spread function), 144
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Psychtoolbox, 784PTLs. See Photoswitchable tethered ligandsPulsed nitrogen laser, 400
QQD streptavidin conjugate solution (recipe),
17QDT. See Quantum dot trackingQuantum dots
imaging single receptors with, 11–17labeling neuronal membrane receptors
(protocol), 13–17experimental method, 14imaging setup, 13interpretation of imaging data, 15,
16fmaterials, 13measuring GlyR (glycine receptor)
lateral diffusion, 15, 16frecipes, 17troubleshooting, 14
overview, 11–12limitations to quantum dot staining, 12properties of, 11–12
blinking, 12color, 11fluorescence strength, 11mercury lamp excitation, 11
Quantum dot tracking (QDT)advantages and limitations, 7application example, 7, 8fdescription, 2image acquisition, particle tracking, and
analysis, 6labeled cell preparation, 5signal characterization, 5
RrAAV. See Recombinant adeno-associated
virusRabies virus
genome organization, 84–85, 84fneurotropism of, 83protocols, 87–98
application examples, 97, 97fdiscussion, 97pseudotyping, 95–96recipes, 97–98recovery of G-gene deleted virus,
93–94recovery of replication-competent
rabies virus from cDNA, 87–92pseudotyping, 84f, 85, 95–96recombinant fluorescent vectors for
tracing neurons and synapticconnections, 83–98
biosafety issues, 86complementation of gene-deleted
vectors, 85, 85fenvelope switching, 85genetic engineering of, 86protocols, 87–96pseudotyping, 84f, 85
recovery of G-gene deleted virus(protocol), 93–94
experimental method, 93–94materials, 93
recovery of replication-competent rabiesvirus from cDNA (protocol),87–92
cell culture, 89fixation of cells and direct
immunofluorescence, 90materials, 88–89transfection and virus recovery, 89–90virus stock preparation, 92virus titration, 91, 91f
titration of, 91, 91ftransynaptic transmission, 83–84vaccination, 86
Random-access multiphoton (RAMP)microscopy, 695
Random-access pattern scanning (RAPS)technique, 543, 545–555
Ratanesthesia, 534, 871, 931CNS-1 cell line, 1011–1017, 1016fdurotomy, 517
recA gene, 115Recipes
AAV plasmid preparation, 604–605anesthetic for rodents, 526artificial cerebrospinal fluid (ACSF), 129,
244, 315, 415, 424, 486–487,527, 652, 681–682, 696, 705,733, 760, 976, 988
HEPES-buffered, 719high-divalent, 425holding, 486–487modified, 487modified, free of carbonate and
phosphate, 936BES-buffered saline 2x (BBS), 605blocking solution, 445caged glutamate stock solution, 425calibrating solutions, 344Coomassie Brilliant Blue staining solution,
605cortex buffer, 328, 620culture medium, 192cutting solution, 425destaining solution, 605dissection medium, 742DMEM3+, 98DM-Nitrophen, 159Drosophila adult hemolymph-like saline
(AHLS), 561dye-containing pipette solution, 352dye-making recipe, 527dye solution, 905embryo medium, 789Evans solution, 789external solution, 352–353Fast Green (1% stock solution), 129, 138filter culture medium (FCM), 742fixative, 652
fluorescent gel solution, 445GMEM4+, 98HEPES-buffered culture medium (HCM),
742HEPES-KRH buffer, 670high-divalent ACSF, 425high-K+ Ringer’s solution, 192internal pipette solution, 206internal recording solution, 278, 467internal solution, 192intracellular saline containing SBFI, 303intrapipette solution, 344, 824isolectin B4 (IB4) derived from Giffonia
simplicifolia seeds, 742Krebs-Ringer solution, 391KX mixture, 315lead citrate, 236low-Ca+ Ringer’s solution, 193MEM, 670MEM medium for quantum dot imaging,
17microinjection buffer, 223microinjection solution, 696modified balanced salt solution (MBSS), 742Mowiol 4-88 mounting medium, 385MQAE cuvette calibration solutions, 631Na+-free extracellular saline containing
ionophores, 304NGM agar medium, 775NGM agar medium, low-peptone, 775NRR, 540, 555OSN dye solution, 571paraformaldehyde (PFA), 385paraformaldehyde solution 8%, 652patch-clamp pipette filling solution, 391PBS-MK 1x, 605PBS 1x, 605PBS 10x, 605phosphate-buffered saline (PBS), 7, 110,
181, 193, 223, 605pipette solution for biocytin staining, 653pipette solution for Fluo-4 staining, 653pipette staining solution, 631QD streptavidin conjugate solution, 17recombinant adenovirus containing FCIP
sequence, 719Ringer’s solution, 824
Ca2+-free, 509high-K+, 192Krebs-Ringer solution, 391low-Ca2+, 193mouse, 168normal rat Ringer (NRR), 540, 555standard, 1931x, 22310x, 223
RPMI 1640 cell culture medium, 988SDS-PAGE gel, 605–606SDS-PAGE Laemmli buffer (5x), 606SDS-PAGE 10x SDS running buffer, 606slice solution, 653standard external saline, 499, 631, 1009standard extracellular saline, 344
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standard extracellular solution forvertebrate neurons, 294
standard intracellular solution forvertebrate neurons, 294
standard pipette solution, 499, 1009Terrific Broth (TB-A), 604Terrific Broth (TB-B), 604titration to estimate purity of DM-
Nitrophen, 159in vitro calibration solutions, 631
Recombinant adeno-associated virus (rAAV)fluorescent calcium indicator protein
(FCIP) expression and,588–590, 588f–589f
in optogenetics studies, 878, 879fReconstruct software, 105, 107–108Red fluorescent protein (RFP), for dense-core
granule staining, 659–660RESOLFT (reversible saturable optical
fluorescence transitions), 238Retinal, 43–44, 43fRetinal Schiff base, 43, 43f, 44Retrograde staining with voltage-sensitive
dyes, 484–485Reverse tetracycline transactivator (rtTA), 585,
587, 589f, 590, 595Reversible saturable optical fluorescence
transitions (RESOLFT), 238Reward circuits
genetic strategies for controlling, 878,879f, 879t
optogenetic modulation of dopamineneurons, 877–886, 879f
functional integration during opticalcontrol, 884–885, 885f
optical measurement of dopaminenerve terminals in brain slices,884–885
optical measurement of dopaminerelease in vivo, 885
integrating behavioral readouts withreward circuit modulation,880–884
conditioned place preference,881–883, 882f
operant conditioning, 883–884reagents and transgenic mouse lines for
targeting, 879tRFP (red fluorescent protein), for dense-core
granule staining, 659–660RH1691, 481–482RH155 dye, 476Rhod-2 AM, 690Rhod-2 dextran, 138Rhodopsin, 43. See also Microbial opsinsRinger’s solution recipes, 824
high-K+, 192low-Ca+, 193mouse, 168normal rat Ringer (NRR) solution, 540, 555standard, 1931x, 22310x, 223
Rose Bengal, 944, 949, 950, 953f, 954–955,955f, 956, 957
RPMI 1640 cell culture medium (recipe), 988rtTA. See Reverse tetracycline transactivatorRuthenium-bipyridine-trimethylphosphine-
glutamate (RuBi-glutamate),431–432
SSample chamber, for live cell and tissue slice
imaging, 332–334, 333fSample drift, in stimulated emission depletion
(STED), 242SBFI (sodium-binding benzofuran
isophthalate), 202–206, 205f,297–302, 297–303, 301f
Scanning confocal microscopes, limits ofcurrent, 142
SDS-PAGE gel (recipe), 605–606SDS-PAGE Laemmli buffer (5x) (recipe), 606SDS-PAGE 10x SDS running buffer (recipe),
606Secretogranin II, 659Semiconductor quantum dots, labeling and
imaging receptors using, 2, 4–7,4t
Semliki Forest virus, 613Sensory rhodopsin I, 42f, 45–46Sensory rhodopsin II, 42f, 45–46SFOs (step-function opsin genes), 47Shadowpatching, 459–467Shot noise, in voltage-sensitive dye imaging,
474, 474fSignal-to-noise ratio (SNR)
of calcium indicators, 64, 145–146confocal spot detection, 147–149, 148fdefined, 64GCaMPs, 558, 584improving
by increasing incident illumination, 336by increasing pixel dwell time, 336–337by line averaging, 336–337
random-access pattern scanning, 554–555of voltage-sensitive dyes, 801, 802, 810
Single compartment model of calciumdynamics, 355–366
application examples, 363dynamics of single [Ca2+]i transients, 360extensions to model, 364–366
buffered calcium diffusion, 364deviations from linear behavior,
364–365, 365fsaturation of buffers and pumps,
364slow buffers, 365
measurement of calcium-drivenreactions, 365–366
materials, 356model application, 361–363
estimates of amplitude and totalcalcium charge, 362–363
estimates of endogenous Ca2+-bindingratio and clearance rate,
361–362, 362festimates of unperturbed calcium
dynamics, 363model assumptions and parameters,
356–360, 358t–359tbuffering, 358clearance, 360influx, 357–358
overview, 355–356schematic of model, 357fsummation of [Ca2+]i transients during
repetitive calcium influx,360–361
Single-condition mapping, 916–917Single-molecule epifluorescence microscope, 3Single-molecule tracking (SMT), for labeling
and imaging receptors usingnanolabels
advantages and limitations, 7application example, 7, 8fdescription, 2image acquisition, particle tracking, and
analysis, 6labeled cell preparation, 5signal characterization, 5
Single-nanoparticle photothermal tracking(SNaPT), for labeling andimaging receptors usingnanolabels
advantages and limitations, 7application example, 7, 8fdescription, 2image acquisition, particle tracking, and
analysis, 6labeled cell preparation, 5signal characterization, 5
Single photon confocal spot detection, 142Single-photon emission tomography
(SPECT), of Alzheimer’sdisease, 990
Skull holder, 308f, 309, 312–313Skull thinning, 967–968, 975–976Slice cultures, imaging microglia in, 735–742Slice solution (recipe), 653SLM (spatial light modulator), 258, 695SLMVs. See Synaptic-like microvesiclesSMT. See Single-molecule trackingSNaPT. See Single-nanoparticle photothermal
trackingSNR. See Signal-to-noise ratioSodium-binding benzofuran isophthalate
(SBFI), 202–206, 205f, 297–303,301f
Sodium channel protein-based activityreporting construct (SPARC),55, 55t, 56f
Sodium-free extracellular saline containingionophores (recipe), 304
Sodium imagingone-photon imaging in axons and
dendrites, 201–206loading cells and detecting [Na+]i
changes (protocol), 202–206
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Sodium imaging (Continued)application example, 204, 205fcalibration of SBFI fluorescence
changes, 204discussion, 204–206experimental method, 203limitations of method, 204–206materials, 202–203recipes, 206
overview, 201–202two-photon imaging in dendritic spines,
297–304calibration and measurement of [Na+]i
within neurons using SBFI(protocol), 300–304
discussion, 303experimental method, 300–302, 301fimaging setup, 300materials, 300recipes, 303–304troubleshooting, 302
overview, 297–299Sodium methohexital, 724–725SPARC (sodium channel protein-based
activity reporting construct),55, 55t, 56f
SPARK (synthetic photoisomerizableazobenzene regulatedpotassium) channel, 36
Spatial light modulator (SLM), 258, 695Spatially optimized line scans, generation of,
934–935Spatially resolved flash photolysis via chemical
two-photon uncaging, 25–31overview, 25–26, 26fprotocol, 27–30
Spatial resolution, of voltage-sensitive dye,801–802
SPECT (single-photon emission tomography),of Alzheimer’s disease, 990
Spinal cord, two-photon imaging/microscopyof neurons and glia in, 721–733
overview, 721–722preparation of mouse spinal column for
repetitive imaging using 2pLSM(protocol), 727–728
preparation of mouse spinal column forsingle imaging using 2pLSM(protocol), 722–726
anesthetizing and intubating themouse, 724–725
experimental method, 724–726materials, 723–724preparing the spinal cord for imaging,
725–726, 725ftroubleshooting, 726
in vivo two-photon imaging of mousespinal cord (protocol), 729–732
experimental method, 730–731imaging setup, 729materials, 729microscopy and image acquisition,
730–731, 731f
mounting the vertebral column, 730preparation for imaging, 730
SPQ (6-methoxy-N-(3-sulphopropyl)quinolinium),624
SR101. See Sulforhodamine 101Standard external saline (recipe), 499, 631,
1009Standard extracellular saline (recipe), 344Standard extracellular solution for vertebrate
neurons (recipe), 294Standard intracellular solution for vertebrate
neurons (recipe), 294Standard pipette solution (recipe), 499, 1009STED. See Stimulated emission depletionStep-function opsins (SFOs), 47, 866Stimulated emission depletion (STED),
237–244application to astrocyte imaging, 695dyes, STED-compatible, 243future prospects for, 243image acquisition and analysis, 240–241imaging of dendritic spines, 237–244
application example, 243, 243fin living hippocampal tissue
(protocol), 239–244discussion, 243experimental protocol, 241imaging setup, 239–241materials, 241recipes, 244troubleshooting, 242
laser sources, 243microscopy setup, 239f, 240overview of, 237–238principles of microscopy, 239
Strokeimaging long-term changes caused by
induced, 957laser-targeted focal photothrombotic
model, 954–955, 955fStructure–function analysis of genetically
defined neuronal populations,377–385
overview, 377protocol, 378–385
application examples, 381–384, 382fcircuit analysis, long-range
projections, and inputmapping, 384
histological reconstruction ofbiocytin-filled cells, 381
reconstruction of 3D morphology,381, 383f, 384
single-cell electrophysiology andmorphological analysis ofgenetically defined neurons,381, 382f
discussion, 384–385experimental method, 379–380imaging setup, 378materials, 378–379recipes, 385
Structure–function studies, ballistic deliveryof dyes for, 447–456
Styryl dyes. See FM dyesSubcellular targeting, using genetically
encoded calcium indicators(GECIs), 70
Sulforhodamine, 179Sulforhodamine 101 (SR101), 497, 498
chemical structure, 674ffor imaging calcium excitation in
cerebellar cortex, 758f, 759specificity of, 677fstaining of astrocytes in a brain slice, 640,
641f, 642for three-dimensional imaging of
neuronal network activity, 534,535f
for two-photon calcium imaging in visualsystem, 521–523
for two-photon imaging of neural activityin awake mobile mice, 830–831
in vivo imaging of astrocytes (protocol),688–689
in vivo labeling of cortical astrocytes with,673–682, 674f
application example, 680, 680foverview, 673–674, 674fprotocol, 675–682
Synaptic connections, recombinantfluorescent rabies virus vectorsfor tracing, 83–98
Synaptic inhibition, imaging with a geneticallyencoded chloride indicator,75–80
Synaptic-like microvesicles (SLMVs)identification of, 657imaging exocytosis and recycling in
astrocytes, 662staining (protocol), 657–658
Synaptic vesicle recycling, imaging with FMdyes, 171–181
Synapto-pHluorins, 19–24advantages and limitations, 22–23application examples, 21–22, 22fgene expression, 21optical imaging, 21overview, 19–20targeting module, 20
Synthetic photoisomerizable azobenzeneregulated potassium (SPARK)channel, 36
TTamoxifen
induced recombination in mice, 106–107toxicity, 106–107
Tau protein, 989, 992–993, 999tdRFP, immune cell expression of, 987, 987fTellurium dioxide, 257, 261Temporal resolution, of voltage-sensitive dye,
801–802Terrific Broth (TB-A) (recipe), 604Terrific Broth (TB-B) (recipe), 604
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Tetracycline (Tet)-inducible system, forfluorescent calcium indicatorproteins (FCIPs), 583, 585–587,586f–587f
Tetracycline promoters, 585, 588, 588f–589f,590, 593
Tetracycline (Tet) transactivator (tTA), 585,587, 588f–589f, 594t, 595
Tetrodotoxin (TTX), 433, 497, 497fTexas Red, 991fTexas Red dextran, 132f, 138, 952T helper 17 (Th17) cells, 981, 984Thinned skull imaging, 305–315. See also
Transcranial two-photonimaging of living mouse brain
Thioflavin-S, 991–993, 991f, 1000, 1002, 1003,1003f, 1004f, 1005, 1007
Three-dimensional imaging of neuronalnetwork activity, 529–540
application example, 538, 538fmeasuring neuronal population activity
using 3D laser scanning(protocol), 531–537
animal preparation and 3D imagingprocedure, 534
assigning fluorescence signals to cellswithin the scan volume, 535f
creating 3D scan trajectories, 532–533,532f
discussion, 539experimental method, 532–536imaging setup, 531laser intensity adjustment, 533–534materials, 531–532, 532frecipe, 540signal assignment and 3D visualization
of network dynamics, 534, 536troubleshooting, 536–537
overview, 529–530principles of 3D laser scanning, 532f
Thy1 mice, 207–225expression vector, 208–209, 208flines, 211–213
generating, 212–213overview of, 211–212table of, 212t
overview, 207–211protocols
generation of Thy1 constructs forpronuclear injection, 214–216
cloning of fluorescent reporterprotein sequence, 215–216
experimental method, 215–216materials, 214–215preparation of DNA for pronuclear
injection, 216troubleshooting, 216
generation of tissue sections forscreening Thy1 lines, 217–220
experimental method, 218–220imaging setup, 217materials, 217–218troubleshooting, 220
imaging of Thy1 lines using an acuteneuromuscular explant,221–222
experimental method, 222imaging setup, 221materials, 221
recipes, 223stochastic subset labeling, 209–211, 210f,
211fTIRFM. See Total internal reflection
fluorescence microscopyTN-XL, 584TN-XXL, 64, 65f, 67, 67t, 584
calcium imaging of neurons in visualcortex, 611–620, 612f
in vivo expression of, 612–613Total internal reflection fluorescence
microscopy (TIRFM)advantages of, 191imaging exocytosis with, 183–193, 184f
application examples, 191, 191f, 192fimaging setup, 185–186loading and TIRF imaging of
dissociated bipolar cells(protocol), 187–188
experimental method, 187–188materials, 187troubleshooting, 188
overview, 183–185, 184frecipes, 192–193transfection and TIRF imaging of
cultured bovine chromaffincells (protocol), 189–190
experimental method, 189–190materials, 189troubleshooting, 190
limitations of, 191–192microscope objective selection, 185monitoring exocytosis in astrocytes,
655–670optical design considerations, 184optical design for dual-shutter prismless,
663principle of, 183–184safe operation of microscope, 1037
Transcranial two-photon imaging of livingmouse brain, 305–315
application example, 310f, 311f, 314challenges of preparing an animal for,
314–315cranial window imaging compared, 314overview, 305–306protocol, 307–315
discussion, 314–315experimental method, 308–312imaging setup, 307mapping imaging area for future
relocation, 310, 310fmaterials, 307–308recipes, 315recovery, 312re-imaging, 312thinned skull preparation, 308–310, 308f
TPLSM imaging of neuronal or glialstructures, 310f, 311, 311f
troubleshooting, 312–313Transfection
of astrocytes, 658, 659–660calcium phosphate, 597, 599–600genetically encoded calcium indicators,
595, 597, 599–600Transsynaptic tracing, with recombinant
fluorescent rabies virus vectors,83–98, 85f
Tricaine methanesulfonate, 569–570Troponin
TN-XL, 584TN-XXL, 584, 611–619
TTA. See Tetracycline (Tet) transactivatorTTX (tetrodotoxin), 433, 497, 497fTwo-photon fiberscopes, 851–860
application examples, 857–859, 858ffluorescent dye labeling in vivo, 857imaging setup, 852–857, 853t, 854f
components, 852, 853fdesign considerations, 852–857, 854ffluorescence detection, 856mechanical attachment to animal, 857miniaturized fiber-scanning devices,
855–856small microscope objectives, 856two-photon excitation through optical
fibers, 853, 855Two-photon imaging/microscopy
acousto-optical deflector (AOD)-basedmicroscopy for high-speedcalcium imaging of neuronalpopulation activity, 543–555
of astrocytic and neuronal excitation incerebellar cortex of awakemobile mice, 745–760
of blood flow in cortex, 927–936, 930f, 933fcalcium imaging in Drosophila olfactory
system with a genetic indicator,557–563
calcium imaging in vivo dendritic imagingin fly visual system, 777–780
calcium imaging of dendritic spines,273–278
calcium imaging of neurons in visualcortex using troponin C–basedindicator, 611–620, 612f
chloride imaging using MQAE, 623–632chloride imaging with Clomeleon, 77, 79electron microscopy reimaging of green
fluorescent protein–labeledaxons and dendrites, 227–236
fluorescent calcium indicator protein(FCIP) expression, 597, 603
of genetically encoded calcium indicators(GECIs), 70
for glioma imaging, 1013–1017, 1016fimaging action potentials with calcium
indicators, 373, 373fimaging astrocyte calcium and vacuole-
like vesicles, 639–653
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Two-photon imaging/microscopy (Continued)imaging neocortical neurons through a
chronic cranial window,319–328
of immune cells in neural tissue, 981–988mapping of neural circuits, 429–434measuring neuronal population activity
using 3D laser scanning(protocol), 531–537
of microglia in mouse cortex in vivo,961–976
miniaturization for imaging in freelymoving animals, 851–860
multiphoton stimulation of neurons andspines, 412
of neural activity in awake mobile mice,827–836
of neural networks in mouse model ofAlzheimer’s disease, 999–1009
of neuronal population activity in awakeand anesthetized rodents,839–849
of neuronal structural plasticity in miceduring and after ischemia,949–957
of neurons and glia in spinal cord in vivo,721–733
sodium imaging in dendritic spines,297–304
staining of astrocytes in the intact rodentcortex with SR101 (protocol),675–682
of structural and functional properties ofastrocytes, 685–696, 686f
of structure and function in Alzheimer’sdisease, 989–995
targeted patching and electroporation invivo, 459–467
transcranial of living mouse brain,305–315
in vivo calcium imaging in visual system,511–527
in vivo calcium imaging using multicellbolus loading of fluorescentindicators, 491–499
Two-photon laser-scanning microscopy(TPLSM, 2pLSM). See alsoTwo-photonimaging/microscopy
all-optical in situ histology of brain tissue,439–444, 440f, 444f
imaging neocortical neurons through achronic cranial window,319–328
mouse spinal cord preparation forrepetitive imaging, 727–728
NADH intrinsic fluorescence imaging,692–694, 693f
of neurons and glia in spinal cord,721–733
overview, 721–722preparation of mouse spinal column
for repetitive imaging using
2pLSM (protocol), 727–728preparation of mouse spinal column
for single imaging using 2pLSM(protocol), 722–726
in vivo two-photon imaging of mousespinal cord (protocol), 729–732
Two-photon photolysis, 246, 246f, 256Two-photon uncaging microscopy, 245–253
application examples, 251–253, 251f, 253fimaging of AMPA receptor densities in
hippocampal neurons, 251–252,251f
induction of structural plasticity atsingle dendritic spines, 252,253f
chemical, 25–31disadvantage of, 30overview, 25–26, 26fprotocol, 27–30
application example, 29, 29fdiscussion, 29–30experimental method, 28imaging setup, 27materials, 27–28troubleshooting, 28
development of, 245–246imaging and uncaging of dendritic spines
(protocol), 247–250experimental method, 250imaging setup, 247–249
electrophysiology setup, 249microscopy setup, 247–248, 247fmode-locked laser beam, 248software, 248–249
materials, 249mapping of neural circuits, 429–434overview, 245–246properties of caged glutamate for two-
photon photolysis, 246, 246fTyrosine hydroxylase, 878, 880
UUAS promoter, 557Ultraviolet (UV) light
acousto-optical deflector (AOD)-basedpatterned UV neurotransmitteruncaging, 255–270
circuit mapping by ultraviolet uncaging ofglutamate, 417–425
infrared-guided neurotransmitteruncaging on dendrites, 387–391
optical fiber–based uncaging system,405–408
Uncagingacousto-optical deflector (AOD)-based
patterned UV neurotransmitteruncaging, 255–270
calcium in neurons, 393–401Ca2+ uncaging in nerve terminals, 151–159characteristics of caged compounds, 256chemical two-photon uncaging, 25–31infrared-guided neurotransmitter
uncaging on dendrites, 387–391
optical fiber–based uncaging system,405–408
two-photon mapping of neural circuits,429–434
two-photon uncaging microscopy,245–253
Urethane, 504, 534, 550, 603, 931, 952
VVaccinia virus, 86VChR1
activation spectrum of, 864deactivation rate, 866
Velate protoplasmic astrocytes, in vivocalcium imaging with syntheticand genetic indicators,707–719, 708f
Venus, 584, 1011, 1012, 1013, 1016, 1016f,1018–1020, 1020f
Vercuronium, 526Vesicle recycling. See Synaptic vesicle recyclingVesicular glutamate transporter-1 (VGLUT1),
657–658VGLUT-mCherry, 663, 664–665, 665fVGLUT-pHluorin, 663–666, 665f,
667–669, 669fVessel occlusion
optically induced occlusion of single bloodvessels in neocortex, 939–947
two-photon imaging of neuronalstructural plasticity in miceduring and after ischemia,949–957
Vibrational noise, in voltage-sensitive dyeimaging, 472
Viral delivery using rAAV, 588–590, 588f–589fVisual cortex
calcium imaging of neurons in usingtroponin C–based indicator,611–620, 612f
future perspectives, 618–619overview, 611–612repeated two-photon imaging of TN-
XXL-labeled neurons(protocol), 614–620
application examples, 612f, 618,619f
cranial window implantation, 615data analysis, 618experimental method, 615–617imaging setup, 614imaging visually driven calcium
signals, 616mapping of visual cortex, 615–
616materials, 614–615recipes, 620repeated imaging, 617two-photon imaging, 616
intrinsic optical imaging of functionalmap development inmammalian, 633–637, 635f,636f
1082 / Index
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Index / 1083
Visual systemorientation preference in 3D neural
networks of visual cortex, 538,538f
in vivo dendritic calcium imaging in fly,777–780
in vivo two-photon calcium imaging,511–527
overview, 511–512protocol, 513–527
anesthesia induction in cats,514–515
anesthesia induction in rodents,514
contact lens insertion, 516craniotomy in cats, 516craniotomy in rodents, 516, 516fdiscussion, 526durotomy, 517dye loading, 517–519, 518fexperimental method, 514–522imaging setup, 513initial surgery in cats, 515initial surgery in rodents, 515materials, 513–514neuropil signal, 521–522, 521frecipes, 526–527signal from astrocytes, 521somatic signal from neurons, 520troubleshooting, 522–525visual stimulation, 519–520
Voltage-gated potassium channel, syntheticphotoisomerizable, 36
Voltage imaging, dendritic, 287–294imaging setup, 288–289overview, 287–288staining individual vertebrate neurons
(protocol), 290–294advantages and limitations, 294example, 292f, 293experimental method, 290–291, 291fmaterials, 290recipes, 294troubleshooting, 291
Voltage-sensing phosphatase, 57, 58Voltage-sensitive dye (VSD)
Aplysia abdominal ganglion actionpotentials, 472, 473f, 476–477
dendritic voltage imaging, 287–288,290–291, 291f, 292f, 293
imaging of cortical spatiotemporaldynamics in awake behavingmice, 817–824
application example, 822–823, 822foverview, 817–818, 818fprotocol, 819–824
discussion, 823experimental method, 820–821materials, 819recipes, 824
imaging of neocortical activity, 799–812distributed processing, 803–804, 804f
dynamics of shape processing atsubcolumnar resolution,805–806, 806f
long-range horizontal spread oforientation selectivity controlby intracortical cooperativity,804–805, 805f
long-term imaging of corticaldynamics in behaving monkeys,810–811
microstimulation in frontal and motorcortex, 811–812
outlook, 812overview, 799–800selective visualization of neuronal
assemblies, 806, 808–809, 808fspatial and temporal resolution,
801–803from in vitro single-cell recordings to
in vivo population imaging,800–801
without signal averaging, 809–810imaging with, 471–487
future directions of, 486imaging of population signals in brain
slices (protocol), 478–480bleaching, 480data acquisition and analysis, 479dye staining, 480experimental method, 479–480imaging setup, 478–479materials, 479phototoxicity, 480sensitivity, 480slice preparation, 479–480total recording time, 480
protocols, 476–487recipes, 486–487recording action potentials from
imaging neurons ininvertebrate ganglia (protocol),476–477
retrograde staining (protocol),484–485
in vivo imaging of mammalian cortexusing “blue” dyes (protocol),481–483
neuron type-specific staining, 486overview, 471–475
camera choice, 475dark noise, 472, 474noise in incident light, 472shot noise, 474, 474fvibrational noise, 472
Voltammetry measurement of dopaminerelease, 884, 885f
Volvox carteri, 47, 864VSDI (voltage-sensitive dye imaging). See
Voltage-sensitive dyeVSFPs (voltage-sensing fluorescent proteins),
54–55, 55t, 56f, 57
WWater-soluble dyes, ballistic delivery of, 452Wavelength, 1031Whisker movement, voltage-sensitive dye
(VSD) imaging in, 812–823,822f
Wide-field calcium imaging in olfactorysystem of zebrafish and mouse,566f, 567, 568
Wide-field charge coupled device (CCD)camera–based imaging, ofcalcium waves and sparks incentral neurons, 281–285
loading cells and detecting [Ca2+]i changes(protocol), 282–285
application examples, 284–285, 284f,285f
discussion, 284–285experimental method, 283materials, 282–283
overview, 281Wollaston prism, in interferometry, 196Woodchuck hepatitis virus post-
transcriptional control element(WPRE), 588f
Worm Tracker (software), 773, 774fWorm tracking, 763–775
experimental methods, 765, 765fprinciples of single-worm tracking,
764–765protocols
illumination for worm tracking andbehavior imaging, 768–769
preparation of samples for wormtracking, 766–767
recipes, 775tracking movement behavior of
multiple worms on food,770–772
WPRE (woodchuck hepatitis virus post-transcriptional controlelement), 588f
XXenon arc lamps, as light sources for
photolysis, 399–400Xylazine, 308, 315, 322, 324, 514, 526, 569,
595, 602, 688, 711, 714, 871,892, 952
Xylocaine, 322
YYC3.6, in Alzheimer’s disease imaging, 993,
994fYC3.60, 64, 65f, 67, 584Yellow Cameleons, development of, 583–584Yellow fluorescent protein (YFP). See also
Venuscircularly permuted variants, 584in Clomeleon, 75, 76f, 77, 79–80, 80fin stimulated emission depletion (STED),
238, 241–243
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Thy1-YFP, 209, 210f, 211f, 212tin VSFPs (voltage-sensing fluorescent
proteins), 55, 55t, 56f, 57Yellow Cameleons, 583–584
ZZebrafish
advantages of use, 783–784anesthesia, 569calcium imaging in intact olfactory system
of, 565–571imaging methods, 567
loading calcium indicators, 566–567overview, 565–566, 566f
confocal calcium imaging of neuronalactivity in larval, 791–797
imaging setup, 792protocol, 793–797
application example, 796, 796fdiscussion, 797experimental method, 793–794materials, 793troubleshooting, 794–795
imaging neuronal activity and motorbehavior, 783–789
imaging setup, 784–785calcium imaging of tectal neuronal
population activities, 784, 785fsimultaneous recording of tectal
Ca2+ dynamics and motorbehavior, 785
visual stimulation, 784–785protocol, 786–789
discussion, 789materials, 786–787methods, 787–788recipes, 789troubleshooting, 788
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