Fluorophore
Excitation
Maximum
(nm)
Emission
Maximum
(nm)
Photons/Emission
Event
Photoactivation
Buffer(s)
Reference
Comments
2409(1)
UV-Violet(405nm)
Glycerol/OS(1)
1-3
BindsdsDNA.Lowcytotoxicityandcellpermeable.
Doesnotrequirephotoactivationbutcanbeapplied.
SyntheticDyes
488nmExcitation
VybrantDyeCycle
369(B),~488(G) 437(B),~510(G)
Violet
DAPI
364(B),~488(G) 454(B),~510(G)
--
UV-Violet(405nm)
Glycerol/OS(4)
4
Hoescht33342
350(B),~488(G) 461(B),~510(G)
--
UV-Violet(405nm)
Glycerol/OS(4)
4
Hoescht33258
355(B),~488(G) 465(B),~510(G)
--
UV-Violet(405nm)
Glycerol/OS(4)
4
SYTO-13
488
509
--
--
50mMMEA/OS(5)
5
YOYO-1
489
509
--
--
50mMMEA/OS(7)
6,7
YO-PRO-1
491
509
--
--
50mMMEA/OS(7)
7
AlexaFluor488
495
519
1193(8)
UV-Violet(405nm)
10mMMEA/OS(8)
100mMMEA/OS(9)
8,9,44
Atto488
501
523
1341(8)
UV-Violet(405nm)
10mMMEA/OS(8)
8,9
OregonGreen
501
526
900(10,Live)
UV-Violet(405nm)
Live-OS
10
Picogreen
502
524
(Live)
--
11
Atto520
516
538
868(8),1000(12)
UV-Violet(405nm)
Live-OS+AA(11)
143mMBME/OS(8)
100mMMEA/OS(12)
532nmExcitation
AlexaFluor532
Atto532
532
532
552
552
---
---
100mMMEA/OS
100mMMEA/OS
8,12
13
9,14
DNAminorgroovebinder.Cellimpermeant.
DNAminorgroovebinder.Cellpermeant.
DNAminorgroovebinder.Cellpermeant.
DNAminorgroovebinder.Cellpermeant.Alsobinds
RNA.
DNAminorgroovebinder.Cellimpermeant.Also
bindsRNA.Dimeric.
DNAminorgroovebinder.Cellimpermeant.Also
bindsRNA.Monomeric.
Highperformance488nm-excitabledyeforSTORM
Highestperformance488nm-excitabledyeforSTORM
Recommendforlivecellonly(cellpermeable),better
greendyesforfixedwork.BODIPYderivative.
BindsdsDNA.Cellimpermeant
RecommendAtto488orAlexaFluor488instead,if
possible
Excitation
Maximum
(nm)
Emission
Maximum
(nm)
Photons/Emission
Event
Photoactivation
Buffer(s)
Reference
541
560
--
UV-Violet(405nm)
10mMMEA/OS(15)
15
546
575
551
569
720
UV-Violet(405nm)
Live-OS
17
554
576
--
UV-Violet(405nm)
Live-OS
17
AlexaFluor555
555
580
2500(2)
UV-Violet(405nm)
Glycerol/OS(2)
1,2,44
CF555
DY-547
555
557
565
574
UV-Violet(405nm)
UV-Violet(405nm)
559
570
10mMMEA/OS(15)
10mMMEA/OS(15)
10mMMEA/OS(8)
143mMBME/OS(8)
15
15
Cy3B
--1365(8,MEA)2057(8,
BME)
CF568
562
583
--
UV-Violet(405nm)
FLIP-565
565
580
--
UV-Violet(405nm)
LysoTrackerRed
577
590
820
UV-Violet(405nm)
AlexaFluor568
578
603
2826(8),1700(10,Live)
UV-Violet(405nm)
MitoTrackerRed
578
599
790
UV-Violet(405nm)
PhysiologicalBufferor
CultureMedium
Live-OS
10mMMEA/OS(8)
Live-OS(10)
Live-OS
ER-TrackerRed
587
615
820
UV-Violet(405nm)
Live-OS
17
635
658
--
--
100-200mMMEA/OS
(20)
20
644
665
--
UV-Violet(405nm)
Live-OS
17
644
665
--
UV-Violet(405nm)
SiR
645
661
630
--
HMSiR
645
661
2,600
--
Cy5
649
670
4254(8,MEA),5873(8,
BME)
UV-Violet(405nm)
AlexaFluor647
650
665
3823(8,MEA),5202(8,
BME),2400(25),18050
(26)
UV-Violet(405nm)
CF647
650
665
--
--
Fluorophore
561nmExcitation
CF543
TAMRA/TMR
(Tetramethyl
Rhodamine)
DiI
MitoTracker
Orange
4884(8)
1100(10,Live)
--
UV-Violet(405nm)
10mMMEA/OS(8)
Live-OS(10)
10mMMEA/OS(15)
8,10,16
8,15
15
Comments
Needsfurthertesting
TMRconjugates(TMRStar)oftenusedforSNAP-tag
labeling
Livecellstainforplasmamembranes
AgoodreddyeforSTORM,alsoworkswellin
thiol+oxygenscavengerbufferwell(unpublished).
Needsfurthertesting
Needsfurthertesting
HighestperformingreddyeforSTORM.
Verypromisingredalternative,butneedsfurther
testing.
18,19
17
8,10,15
17
Livecellstainforlysosomes.BODIPYderivative.
GoodreddyeforSTORM
Specificformitochondria,forlive-cellimaging
Specificforendoplasmicreticulum,forlive-cell
imaging
647nmExcitation
DY-634
MitoTrackerDeep
Red
DiD
Needsfurthertesting.
Specificformitochondria,forlive-cellimaging
Live-OS
17
Specificforplasmamembranes,forlive-cellimaging
PhysiologicalBufferor
21
CultureMedium
Siliconrhodamine,doesn'trequirereducingbuffer.
PhysiologicalBufferor
Higherperformancesiliconrhodaminederivative,
22
CultureMedium
doesn'trequirereducingbuffer.
10mMMEA/OS(8)
8,23,24
143mMBME/OS(8)
OneofthehighestperformingdyesforSTORM
10mMMEA/OS(8)
143mMBME/OS(8) 8,15,24,25,
TCEP(25)
26,44
Vectashield(26)
CurrentlyregardedasbestdyeforSTORM
10mMMEA/OS(15)
15
Fluorophore
Excitation
Maximum
(nm)
Emission
Maximum
(nm)
Photons/Emission
Event
Photoactivation
DyLight650
652
672
--
--
Atto655
663
684
1105(8),1200(10,Live)
UV-Violet(405nm)
CF660C
667
685
--
--
DY-678
674
694
--
--
Atto680
680
700
1656(8)
UV-Violet(405nm)
CF680
681
698
--
--
AlexaFluor700
696
719
--
--
Atto700
700
716
--
--
750nmExcitation
Cy7
DiR
743
748
767
780
997
--
UV-Violet(405nm)
UV-Violet(405nm)
AlexaFluor750
749
775
703(8),2800(25)
UV-Violet(405nm)
DyLight750
752
778
749
UV-Violet(405nm)
PA-GFP
504
517
313(32,Live)
UV-Violet(405nm)
PA-TagRFP
562
595
906(32,Live)
PA-mCherry1
570
596
706(32,Live)
PAmKate
586
628
--
Dronpa
503
518
262(32,Live)
UV-Violet(405nm)
mGeosM
503
514
248(32,Live)
UV-Violet(405nm)
Buffer(s)
Reference
Comments
100-200mMMEA/OS
20
(20)
10mMMEA/OS(8)
8,10,27,28,
Live-OS(10)
Oxazinedye,firstdemonstratedinlivecellsin
29
CultureMedium(26)
conjunctionwithTMPtag
100-200mMMEA/OS
20
(20)
10mMMEA/OS(15)
15
10mMMEA/OS(8)
8,15,27,28,
CultureMedium(26)
29
Oxazinedye,similartoAtto655
10mMMEA/OS(15)
100-200mMMEA/OS
15,20
(20)
10mMMEA/OS(15)
15,30
100mMMEA/OS(29)
10mMMEA/OS(15)
15,29
143mMBME/OS(8)
Live-OS
143mMBME/OS(8)
TCEP(25)
143mMBME/OS(8)
8
17
8,25
DoesnotperformwellwithoutTCEPbuffersystem
8
Fluorescent
Proteins
PA-FPs
PhysiologicalBufferor
CultureMedium
PhysiologicalBufferor
CultureMedium
PhysiologicalBufferor
UV-Violet(405nm)
CultureMedium
UV-Violet(405nm)or PhysiologicalBufferor
Blue(445nm)
CultureMedium
UV-Violet(405nm)
31,32
32,33
32,34
35
Recommendedonlyforcertainlivecellandmulticolor
experimentsasperformanceforSTORMisrelatively
low.
Recommendhigherperforminggreen-redPC-FPs
exceptforcertainmulticolorexperiments.
Recommendhigherperforminggreen-redPC-FPs
exceptforcertainmulticolorexperiments.
Onlyviablefar-redFPforSTORM.
PS-FPs
PhysiologicalBufferor
CultureMedium
PhysiologicalBufferor
CultureMedium
31,32
32,36
Reversiblyswitchabledark-green.WerecommendPSCFP2insteadformostSMLMapplications.
BetterthanDronpabuthigherdutycyclethanPSCFP2
Fluorophore
Dreiklang
Excitation
Maximum
(nm)
515
Emission
Maximum
(nm)
529
Photons/Emission
Event
700(Live)
mIrisFP
486(G),516(R) 546(G),578(R)
--
NijiFP
469(G),526(R) 507(G),569(R)
--
Photoactivation
Buffer(s)
UV(365nm)to
activateandViolet PhysiologicalBufferor
(405nm)to
CultureMedium
deactivate
UV-Violet(405nm)
fordark-greenand PhysiologicalBufferor
green-to-red.Blue
CultureMedium
(488nm)fordark-red
UV-Violet(405nm)
fordark-greenand PhysiologicalBufferor
green-to-red.Blue
CultureMedium
(488nm)fordark-red
Reference
37
38
39
Comments
UniqueFPwhere515nmlightelicitsfluorescence,
butcanbeactivatedordeactivatedwithtwo
differentwavebands.
Photoconvertablefromgreentoredstate,bothgreen
andredstatesreversiblyphotoswitchabletodark
state.DemonstratedforcombinedSMLM+pulse
chaseexperiments.
Photoconvertablefromgreentoredstate,bothgreen
andredstatesreversiblyphotoswitchabletodark
state.
PC-FPs
PS-CFP2
400(C),490(G)
468(C),511(G)
tdEos
506(G),569(R) 516(G),581(R)
mEos2
506(G),573(R) 519(G),584(R)
mEos3.2
507(G),572(R) 516(G),580(R)
mEos4b
505(G),569(R) 516(G),581(R)
mMaple3
489(G),566(R) 505(G),583(R)
Dendra2
490(G),553(R) 507(G),573(R)
PhysiologicalBufferor
CultureMedium
Live-OS(10)
1200(10,Live)
UV-Violet(405nm) PhysiologicalBufferor
774(32)
CultureMedium
1200(10,Live)
PhysiologicalBufferor
UV-Violet(405nm)
745(32)
CultureMedium
PhysiologicalBufferor
809(32,Live)
UV-Violet(405nm)
CultureMedium
PhysiologicalBufferor
850
UV-Violet(405nm)
CultureMedium
PhysiologicalBufferor
675(32,Live)
UV-Violet(405nm)
CultureMedium
UV-Violet(405nm)or PhysiologicalBufferor
686(32,Live)
Blue(488nm)
CultureMedium
223(32,Live)
UV-Violet(405nm)
32,40
10,31,32
10,32,41
32,42
43
32
32
PS-CFP2iseasilyimagedwithoutphoto-activation
andisagreatcandidateformulticolorimaging.
Tandemdimersopossibleartifacts.Highestphoton
outputinclass.
Tendstodimerize,recommendmEos3.2ormMaple3
instead.
AlongwithmMaple3,consideredbestofthegreenredPC-FPs.
OsmiumTetroxideresistant,engineeredfor
correlativeEMimaging
MostrecentlyintroducedPC-FPforSMLM,veryhigh
detectableFP:totalFPratio.
Bluelightactivationandgoodperformancemakes
Dendra2goodcandidateforlivecellSMLM.
Imaging
Buffers
Component 1
Component 2
Component 3
Component 4
Component 5
Total
Volume
Fixed Cell Imaging
790uLBufferB
200uLMEAStock(200mM)
MEA/OS
890uLBufferB
100uLMEAStock(100mM)
10uLGLOXStockorPOCStock
(OS=GLOXorPOC) 940uLBufferB
50uLMEAStock(50mM)
980uLBufferB
10uLMEAStock(10mM)
790uLBufferC
200uLMEAStock(200mM)
MEA/OS
890uLBufferC
100uLMEAStock(100mM)
25uLPCDStock
(OS=PCD)
940uLBufferC
50uLMEAStock(50mM)
980uLBufferC
10uLMEAStock(10mM)
970uLBufferB
20uLBMEStock(286mM)
BME/OS
980uLBufferB
10uLBMEStock(143mM)
10uLGLOXStockorPOCStock
(OS=GLOXorPOC) 985uLBufferB
5uLBMEStock(71.5mM)
989uLBufferB
1uLBMEStock(14.3mM)
920uLBufferC
20uLBMEStock(286mM)
BME/OS
930uLBufferC10uLBMEStock(143mM)
25uLPCDStock
(OS=PCD)
935uLBufferC
5uLBMEStock(71.5mM)
939uLBufferC
1uLBMEStock(14.3mM)
TCEP
920uLBufferD
50uLTCEPStock(25mM)
10uLGLOXStock
10uLMVStock(1mM) 10uLAAStock(1mM)
Glycerol/OS
800uLBufferE
190uLPBS
10uLGLOXStock
(OS=GLOXorPOC)
720uLPBS(pH=8OxEA
50uLMEAStock
30uLOxyFluor™
200uLSodiumDL-lactate
8.5)
1mL
1mL
1mL
1mL
1mL
1mL
1mL
Live Cell Imaging
Live-OS
(OS=GLOXorPOC)
990uLBufferF
OptionalMEA
Live-OS
(OS=GLOXorPOC)
990uLBufferF
OptionalBME
Live-OS+AA
(OS=GLOXorPOC)
980uLBufferF
OptionalBME
10uLGLOXStockorPOCStockOPTIONAL:6uLMEAstock(6mM)
-
-
1mL
10uLGLOXStockorPOCStock
OPTIONAL:5uLBMEstock(71.5mM)
-
-
1mL
10uLGLOXStockorPOCStock
OPTIONAL:5uLBMEstock(71.5mM)
10uLAAStock(1mM)
-
1mL
Stock Solutions
Composition
Storage
Notes
BufferA
10mMTris(pH8)+50mMNaCl
RoomTemperature,longterm
BufferB
50mMTris(pH8)+10mMNaCl+10%glucose
RoomTemperatureor4degC,longterm
BufferC
50mMTris(pH8)+10mMNaCl
RoomTemperatureor4degC,longterm
BufferD
200mMTris(pH9)+5%glucose
RoomTemperatureor4degC,longterm
BufferE
120mg/mLGlucoseinGlycerol
4degC
BufferF
L-15Medium+2-10%glucose
4degC,protectedfromlight,~1month
Forliveimaging,cansubstituteforothergrowth
medium+75mMHEPES.Usephenolred-freemedia.
BufferG
100mMTris(pH8)+50mMKCl+1mMEDTA+50%glycerol
RoomTemperatureor4degC,longterm
ForstorageofPCD.
77mgMEA+1.0mL0.25NHCl
4degCfor~2weeks
100xStockSolution
Dilution/Storage Buffers
Recommendtesting10-200mMTris,higherpHmay
helpduetoOS-inducedpHdrop.
Reducing and Oxidizing Reagent
Stock Solutions
MEAStock(1MMercaptoethylamine)
BMEStock(14.3MBetamercaptoethanol)
TCEPStock(0.5Mtris(2carboxyethyl)phosphine)
Providedbysupplieras~14.3Mstock.
4degC,longterm
100xStockSolution
Providedbysuppliersas0.5Mampoules.
4degC,usesamedayampouleopened.
20xStockSolution
MVStock(0.1Mmethylviologen)
25.7mgMV+1.0mLddH2O
4degCfor~2weeks
100xStockSolution
AAStock(0.1Mascorbicacid)
17.6mgAA+1.0mLddH2O
4degCfor~1month
COTStock(cyclooctatetraene)
20.8mgCOT+1.0mLDMSO
-20degC
100xStockSolution
BufferadditiveforimprovedAlexaFluor647photon
statistics.100xstocksolution.
Oxygen-Scavenging System Stock
Solutions
GlucoseStock
45%(w/v)solution
CatalaseStock
17mg/mLCatalaseindH2O
4degCfor~1month
GLOXStock
56mg/mLGlucoseOxidase+3.4mg/mLCatalaseStockinDilutionBufferA
4degCfor~2weeks
POCStock
112mg/mLPyranoseOxidase+3.4mg/mLcatalaseinDilutionBufferA
4degCfor~2weeks
PCDStock
1.4mg/mLProtocatechuate3,4-DioxygenaseinDilutionBufferG
-20degC
PCAStock
15.4mg/mLProtocatechuicAcidinddH2O(adjustpHto9.0with10NNaOH)
4degC
SodiumDL-lactate
60%(w/w)solution
4degC
OxyFluor™
Unknown
-20degC
ForuseinGLOXandPOCStocksolutions.
Thoroughlyvortexcontentsandcentrifugeatmax
speed,harvestsupernatantforuse.
Thoroughlyvortexcontentsandcentrifugeatmax
speed,harvestsupernatantforuse.
Donotagitatevigorouslyorexceedwarming
temperatureof37degC
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