Download SPC3 User Manual - Micro Photon Devices

MICRO PHOTON DEVICES
SPC3
Single Photon Counting Camera
© Micro Photon Devices S.r.l.
Via Stradivari, 4
39100 Bolzano (BZ), Italy
email [email protected]
Phone +39 0471 051212 • Fax +39 0471 501524
SPC3 User Manual Version 1.0.1 - October 2015
Micro Photon Devices S.r.l. – Italy
All Rights Reserved
SPC3:SinglePhotonCountingCamera64x32
TableofContents INTRODUCTION
3
SINGLEPHOTONAVALANCHEDIODE
4
Furtherreadings
6
SPC3HARDWARECHARACTERISTICS
7
Electricalconnections
7
Cameramechanicaldimensions
10
SPC3OPERATION
11
Standardoperation
11
Gatedoperation
12
FLIMOperation
FurtherReadings
14
18
Acquisitionparameters
Integrationtime
Dead-timetimeanddead-timecorrection
Backgroundsubtraction
18
18
20
23
Additionalinformation
CalculationoftheSnapsizeandtheContinuousAcquisitionDatarate
Cameraauto-protection
23
23
24
SPC3SOFTWAREINTERFACE–VISUALSPC3(WINDOWSONLY)
25
Installation
25
SoftwareInterface
Mainwindow:ControlPanel
MainWindow:PlotsettingsandPlaybackControl
MainWindow:ControlButtons
MainWindow:AcquisitionParametersSummary
CounterWindow
FLIMWindow
25
26
28
30
31
31
32
SPC3-SDK,FILEFORMATSANDIMAGEJPLUGIN
33
SYSTEMREQUIREMENTS
36
COPYRIGHTANDDISCLAIMER
36
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Introduction
SPC3isasinglephotoncountingcamerabasedona2-Dimagingarrayof64x32smartpixels.Eachpixel
comprisesasingle-photonavalanchediode(SPAD)detector,ananaloguefront-endandadigitalprocessing
electronics. This on-chip integrated device provides single-photon sensitivity, high electronic noise
immunity, and fast readout speed. The imager can be operated at full resolution with a maximum frame
rate of about 100.000 frames per second with negligible blind time (dead-time). Inside each pixel three
independent counters are integrated. Each one of them can be independently gated throughan external
signal; counter 1 can also be gated by an internally generated signal (e.g. for FLIM mode acquisition).
Counters 2 and 3, instead, can also count downward, with their direction controlled through an external
signal.
SPC3pixels featurehigh photon-detection efficiency (PDE) in the visibleandnear-UV spectral region, and
low dark-counting rates, even at room temperature. The imager is easily integrated into common optical
setups thanks to a Thorlabs® SM1 thread, a C-mount mechanical adapter and a high-speed USB 3.0
computerinterface.ThecameraisshowninFigure1.
ThecameradiffersfromconventionalCharge-CoupledDevices(CCD)orCMOSsensorsbecauseitperforms
a“fullydigital”acquisitionofthelightsignal.Eachpixeleffectivelycountsthenumberofphotonswhichare
detectedbythesensorduringtheacquisitiontime.
Figure1.
SPC3:SinglephotonCountingCamera.
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SinglePhotonAvalancheDiode
TheSPADisap–njunctionwhichisreverse-biasedwellaboveitsbreakdownvoltage.Underthisoperating
condition, the absorption of a single photon generates an electron-hole pair, which is accelerated by the
highelectricfieldacrossthejunction.Theenergyofthechargecarriersiseventuallysufficienttotriggera
macroscopic avalanche current of few milliamperes through the device. The SPAD biasing electronics,
namely the Active Quenching Circuit (AQC), is integrated on the same silicon chip. The AQC senses the
avalanche current, quenches it and resets the diode to its initial state. The avalanche is quenched by
decreasing the voltage across the SPAD junction below breakdown for an adjustable time (hold-off), that
rangesfromfewtenstofewhundredsofnanoseconds.TheSPADbiasvoltageisthenrestoredtotheinitial
state,andthusthediodeisreadytodetectthenextphoton.Thetotaltime,whichisrequiredtorestorethe
initial state of the SPAD after the detection of a photon, is named dead-time. During the dead-time, no
photonscanbedetected.TheAQCprovides,additionally,avoltagepulsetoanassociatedcounter,whichis
integratedineachpixel.
ThemeasurementoflightsignalsbyaSPADhasseveraladvantagesconcerningthesignaltonoiseratio.No
analoguemeasurementofvoltageorcurrentisneeded,sincethedetectoractslikeadigital“Geiger-like”
counter. It follows that no electronic noise is added by analogue to digital converters or amplifiers while
measuringthesignal.Additionally,thedetectorislesssensitivetotheelectromagneticinterferenceorthe
electricalnoisegeneratedbyexternalequipment,differentlyfromchargecoupleddevices.
TheprimarynoisesourcesforSPADsare:
1) Dark counts. This noise source comprises all processes which can start an avalanche across the
SPADjunctionbutwhicharenotcausedbythedetectionofaphoton.Thetypicalsourceofdark
countsisthethermalcarriergenerationprocess.Darkcountsarethedominantnoisesourceforthe
SPADsoftheSPC3.
2) Afterpulsing. The detection of a photon can trigger, with a certain probability, an additional
detection event within few microseconds. This spurious event is caused by charge carriers, that
flew through the junction during the avalanche, remained trapped and are then subsequently
released. These carriers are accelerated by the high electric field across the junction and might
trigger another avalanche like the photo-generated electron-hole pairs, when released after the
dead-time. The afterpulsing probability depends on the detector dead-time and it is usually
reduced to about 1-2% at the normal operating conditions. Longer dead-times decrease the
afterpulsingprobability.
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3) Cross-talk.Duringtheavalancheprocess,photonsareemittedbyhotcarriers.Thesecanpropagate
through the silicon and trigger a detection event in SPADs which are placed in close proximity.
Practically,theCross-talkprobabilityisverylowamongthepixelsoftheSPC3(about10-5),dueto
thelargedistancebetweentheSPADs.Itscontributiontothetotalnoiseismostlynegligible.
Since the read-out noise of CCD or CMOS cameras is normally slightly higher than one electron (per
integrationtime)andsincethedarkcountingratesoftheSPADpixelsoftheSPC3isaboutonehundredper
second,itfollowsthattheoptimaloperatingconditionforthecameraisatfastormoderateframe-rates.
Particularly, the dark-counts contribution is low with exposure times of about 0.1s. By reducing even
furthertheintegrationtime,thedarkcountnoisebecomesnegligible.
Due to the dead time, the SPAD has a linear working range, i.e the number of detection events within a
defined time period (T) depends linearly on the illumination intensity, only at low or moderate photon
fluxes.Atlargephotonfluxesthenumberofdetectedphotonsdeviatesfromlinearityandsaturatestoa
constantvalue,whichisthereciprocalofthedead-time(Tdead).Asamatteroffact,sincethediodeisofffor
aperiodequaltoTdeadeverytimeitdetectsaphoton,themaximumcountratehappenswhenthedetector
oscillateswithaperiodTdead.SupposingNimpthenumberofimpingingphotonsonaSPADactiveareaduring
anintegrationtimeT,Ncountedthephotonscountedbythedetector,andtakingintoaccountthePDE,Ncounted
isequaltoNimpreducedbythePDEandmultipliedbytheratiooftheactualintegrationtimedividedbythe
setintegrationtimeT:
𝑁"#$%&'( = 𝑁*+, ∗ 𝑃𝐷𝐸 ∗
𝑇 − 𝑁"#$%&'( ∗ 𝑇('3(
𝑇('3(
= 𝑁*+, ∗ 𝑃𝐷𝐸 ∗ (1 − 𝑁"#$%&'(
)
𝑇
𝑇
𝑁"#$%&'( =
𝑃𝐷𝐸 ∗ 𝑁*+,
𝑇
1 + 𝑃𝐷𝐸 ∗ 𝑁*+, ∗ ('3(
𝑇
𝑒𝑞. 1
ByexpressingNimpasafunctionofNcountedoneobtainsalso:
𝑁*+, =
1
𝑁"#$%&'(
∗
𝑒𝑞. 2
𝑇('3(
𝑃𝐷𝐸 1 − 𝑁
∗
"#$%&'(
𝑇
Thetotalnumberofdetectedeventsisthussmallerthanthetotalnumberofphotonswhichcrossthep-n
junction,becauseofthesaturationeffectandthePDE.When𝑁*+, ∗ PDE =
@
@ABCA
,thenumberofcounted
eventsisalreadyreducedof50%.SinceaSPADisblindforTdeadaftereachtriggering,itisalsoclearthatina
timeperiodT,themaximumnumberofphotonsthatcanbecountedis:
𝑁"#$%&'(,+3E =
𝑇
𝑇('3(
𝑒𝑞. 3
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Figure2.
Numberofmeasuredphotonsinanimageof1msexposureanddead-timeof100nsasafunctionof
thenumberofincomingphotons(PDE=100%forsimplicity).
Let’ssupposeforexampleanexposuretimeT=1ms,aPDE=50%forsimplicityandadead-timeTdead=100
ns.Inthiscase,themaximumnumberofphotonsthatcanbecounted(ormoreingeneralthemaximum
numberofSPADtriggerings)is10’000.Inourexamplealso𝑁*+, ∗ PDE =
@
@ABCA
correspondsto10’000;this
means that when a photon flux of 20’000’000 photons/s hit the detector, during the integration time T,
only20’000(Nimp)photonscrosstheactiveareabutonly10’000canbedetectedduethePDEof50%,and
finallyonly5’000eventsare,onaverage,counted(Ncounted)becauseofsaturation.See,forexample,Figure
2thatshowsthenumberofcountedphotonsasafunctionofthenumberoftheimpingingphotonsforan
exposuretimeT=1ms,adead-timeof100nsandaPDEof100%forsimplicity.
Furtherreadings
M.Ghioni,A.Gulinatti,I.Rech,F.Zappa,S.Cova,“ProgressinSiliconSingle-PhotonAvalancheDiodes”,IEEE
JournalofSelectedTopicsinQuantumElectronics,2007,13,852-862.
D.Bronzi,F.Villa,S.Tisa,A.Tosi,F.Zappa,D.Durini,S.Weyers,W.Brockherde,"100000Frames/s64×32
Single-PhotonDetectorArrayfor2-DImagingand3-DRanging”,IEEEJournalofSelectedTopicsinQuantum
Electronics,2014,vol.20,no.6,pp.354-363.
M.Vitali,D.Bronzi,A.J.Krmpot,S.N.Nikolic,F.J.Schmitt,C.Junghans,S.Tisa,T.Friedrich,V.Vukojevic,L.
Terenius, F. Zappa, R. Rigler, "A Single-Photon Avalanche Camera for Fluorescence Lifetime Imaging
Microscopy and Correlation Spectroscopy", IEEE Journal of Selected Topics in Quantum Electronics, 2014,
vol.20,no.6,pp.344-353.
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SPC3hardwarecharacteristics
Electricalconnections
TheSPC3haspowerandUSBconnectionsononeside,andamulti-coaxialconnectorontheothersidefor
variouscontrolsignals,asshowninFigure1andFigure3.SPC3isprovidedwithacableadapterfromthe
multi-coaxialconnectortostandardSMAconnectorsasshowninFigure4.Adescriptionoftheinputand
theoutputscanbefoundinTable1.TheElectricalspecificationsoftheinputandoutputsarereportedin
Table2.
Table1.
SPC3InputsandOutputsdescription.
USB
High-speedUSB3.0connector.ItisrecommendedtouseshortUSBcables(<2m).
+12VDC
Jackforconnectingtheprovided+12Vpowersupply.
SYNCOUT
Coaxialoutputwhichcandrive50Ohmterminatedtransmissionlines.A50OhmDCload
termination is required. Electric pulses of 3.3V LVCMOS are generated at this output for
synchronizingthecamerawithanyexternaldevice.TheSPC3canbeconfiguredbytheuser
to output the internal 50MHz reference clock, synchronised with the internal 100MHz
master clock, for synchronizing any external instrument (such a laser) with the internal
generated gate. The same output can be configured also to generate a signal (pulse)
synchronous with the start of each hardware integration frame. The delay between the
actualstartoftheintegrationframeandtherisingedgeofthispulseisshowninFigure5.
For more details on gated operation, see the corresponding paragraph in the SPC3
Operationsection.
TRIGIN
Thiscoaxialinputrequiresa3.3Vpulse(5Vtolerated)ofminimumdurationof40nstostart
thecameraacquisitionusingasignalfromanexternaldevice(e.g.ashutter,alaser,etc.).
Thefirstframestartsbetween20nsand40nsaftertheTRIGINrisingedge.SincetheTRIG
IN signal is asynchronous respect to the internal clock, such delay is of course notdeterministic (unless it is synched with the SYNC OUT signal). The input impedance is 50
Ohm.SeeFigure5forreference.
Active-lowinputswith50OhmDCinputimpedance.Eachinputcontrolsoneofthethree
GATEIN1
in-pixel counters. When GATE IN is ‘1’, the incoming photons are not counted by the
GATEIN2
measuring electronics. 3.3V LVCMOS signals are accepted but the inputs are 5V tolerant.
GATEIN3
The minimum duration of a GATE IN pulse is 5 ns. The actual internal gate is delayed by
about15ns.
Inputtoselectthecountingdirectionofcounters2and3.Theinputhasa50OhmDCinput
DIRECTION impedance; 3.3V LVCMOS signals are accepted but the input is 5V tolerant. A low value
meansUPcounting.DIRECTIONtimingsTBD.
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Figure3.
Cameraside-views:hardwareconnections.
Figure4.
CamerawiththecableadapterandtheSMAoutputdescription.
Figure5.
TimingofTRIGINandSYNCOUTsignalswithrespecttotheactualstartoftheacquisition.
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Table2.
ElectricalCharacteristicsoftheSPC3measuredatroomtemperature(25°C)
Symbol
Parameter
Signal
TRIG IN,
DIRECTION
TRIG IN,
DIRECTION
GATE,
Min
Typ
Max
Unit
2.4
3.3
V
0
0.8
V
VIH
Inputlogic-highvoltage(5Vtolerant)
VIL
Inputlogic-lowvoltage
VOH
Outputlogic-highvoltage(50Ωload)
SYNCOUT
2.7
V
VOL
Outputlogic-lowvoltage(50Ωload)
SYNCOUT
0
V
tPI
PropagationdelayfromTRIGINtoacquisitionstart
TRIGIN
20
40
ns
tIN
TRIGINpulseduration
TRIGIN
40
ns
tPO
PropagationdelayfromacquisitionstarttoSYNCOUT
SYNCOUT
5
10
ns
tOUT
SYNCOUTpulseduration(framesyncmode)
SYNCOUT
40
ns
tir
Inputrisetime(10%-90%)
TRIGIN,GATE
5
ns
tif
Inputfalltime(10%-90%)
TRIGIN,GATE
5
ns
tor
Outputrisetime
SYNCOUT
1
ns
tof
Outputfalltime
SYNCOUT
1
ns
tgate
GATEpulseduration
GATE
5
ns
tgate_delay
GATEdelay
GATE
10
15
20
ns
DIRECTIONdelayXXX
DIRECTION
XX
XX
ns
Internalreferenceclock-frequency
SYNCOUT,internal
50
MHz
Internalreferenceclock-duty
SYNCOUT,internal
50
%
tdir_delay
-
-
GATE,
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Cameramechanicaldimensions
Figure6.
SPC3mechanicaldimensions.Alldimensionsinmm.
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SPC3Operation
The SPC3 camera features three different modes of operation that can be optimised through the use of
severalworkingparameters,allcontrollablefromacomputerusingeitherthedeliveredSDKlibraryorthe
providedWindows®PCsoftware.Thefollowingparagraphsdescribeindetailallthesefeatures.
Standardoperation
The camera is operated in its standard mode when each pixel is simply used to count photons in a user
defined integration time and the counters are not gated (see the next paragraph for details). When the
SPC3isoperatedinthismode,threedifferentacquisitionmodesarepossible:
•
Live acquisition: single images from the camera are acquired and downloaded to the computer.
SinceinthismodeitisthePCthatcontinuouslyasksfornewimages,thetimebetweentwoimages
cannotbepreciseandstronglydependsonhowthePCsoftwarebehavesandonthePC’scurrent
workload.Thismodeshouldnotbeusedforactualscientificacquisition,butitisinsteadusefulfor
acquiringalowframerate“live”movie(i.e.real-time),forinstancewhenaligningthecamerainthe
optical setup or adjusting the acquisition parameters with the current illumination conditions.
Considering the practical limitations of the USB connection when transferring small data blocks,
typical achievable frame-rates are in the range of 50-100fps (actual values depend on the
performance of both the PC hardware and the software employed). As a consequence, the PC
requestsofliveimagesshouldbelimitedtoamaximumof1every10msorlongertime.
•
Snap: a sequence of images is acquired and stored into the internal memory of the camera. The
dead period between subsequent frames (inter-frame dead-time) is less than 10 ns, and the
frames’timingisaccuratelydeterminedbytheinternalclock.Oncetherequirednumberofframes
ismeasured,theelectronicinterfacetransfersthedatatothecomputer.Theimagescanthenbe
displayedandsavedonthehard-drive.Thismodecannotbeusedforliveacquisition,sincealldata
will be downloaded to the PC only at the end of the entire snap, which can last few seconds.
Instead,thisapproachallowsdataacquisitionsatthemaximumframe-ratepossible,sinceitdoes
notdependontheactualthroughputoftheUSBlinkorPCsavespeed.Snapsizeislimitedbythe
capacityoftheinternalmemory(128MiB).Formoredetailsonhowtocalculatesnapsize,seethe
paragraphonCameraConfiguration.IfyouneedtoacquiremoredatayoushoulduseContinuous
Acquisitionmode,asdescribedbelow.
•
Continuous Acquisition: a continuous acquisition of data between a START command and a STOP
commandfromthePCisperformed.Oncetheacquisitionisstarted,theinternalmemoryisusedas
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a buffer, and data should be continuously read by a PC. In case the internal memory gets full
becausethePCfailstodownloadthedataswiftlyenough,datalossoccursandanerrorisissued.
The maximum achievable frame rate strongly depends on PC performance (specially on the HDD
writespeed).ThankstoUSB3.0itispossibletoacquirethefullarrayatthemaximumspeed(about
100kfps).However,inordertosaveallgenerateddataatthishighthroughputitisalsocrucialthat
theemployedmass-storageunitisveryfast(seetheparagraphonCameraConfigurationformore
detailsonhowtocalculateactualdatarates).SSDstorageunitsarethusrecommended.IfyourPC
cannotsustainsuchahighthroughputyouwillhavetoreducetheframerateortosaveonlyhalf
the array. If this is not possible you must resort to the Snap mode, at the expense of the
experimentduration.
In both Snap mode and Continuous Acquisition mode it is possible to trigger the actual start of the
acquisitionwithanexternalsignal.
Inallthreemodesitispossibletooutputapulsesynchronouswiththestartofeachintegrationtime.Itis
alsopossibletointernallysubtractabackgroundimagepreviouslystoredintothecamera.
Gatedoperation
Normally,thecountersofeachpixeloftheSPC3willjustcontinuouslycountallthepulsesgeneratedbythe
SPADs.Thisoperatingmodeiscalledfree-runninganditisthedefaultoperationmode.Anyway,eachofthe
three integrated binary counters which register the detected number of photons in each pixel can be
enabledordisabledbyagatesignal.Thisoperatingmodeiscalledtime-gating.
Thegatesignalforcounters2and3isprovidedonlybythetwocoaxialinputsGATEIN2andGATEIN3.
Thegatesignalforcounter1insteadisthelogicalORbetweentwoindependentdigitalsignals:GATEIN1,
i.e.oneofthecameracoaxialinputs,andSoftwaregate,generatedinternallybytheSPC3electronics,which
canbesetbytheuserthroughtheprovidedsoftware. EachofthethreeexternalGATEINsignalscanbe
periodicoraperiodicanditwillbeappliedasynchronouslyanddirectly(throughtheORgate)totheSPADs.
TheSoftwareGateisaperiodicdigitalsignal,whichissynchronizedwiththeinternalReferenceclock(20ns
period).TheusercansettheDelay,i.e.thetimeshiftbetweentherisingedgeoftheReferenceclockand
the high-to-low transition of Software gate, and the parameter Width, which defines the duration of the
“counter-enabled”Softwaregatepulse.Essentially,theSoftwaregategeneratesaperiodicgatesignalofa
givenWidthandDelayfromthereferenceclock.NotethattheWidthisthedurationoftheSoftwareGate’s
lowstateasshowninFigure7.
TheWidthoftheSoftwareGate,whichisgeneratedbythecontrolelectronics,canbevariedintherange
0÷100%ofthereferenceclock,instepsof1%.Actually,duringafactorycalibration,theGatewidthhas
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been internally discretized with 1000 points over the full range. In this way, whenever one of the 100
nominalselectablevaluesisrequestedbytheuser,theSPC3cameraissettotheclosedpossibleone,i.e.
the one with the smallest possible error. Note that, however, values below 2ns could be unreliable.
ParticularlytheminimumachievableGatesignalisabout1.5ns.Theactualsetvaluecanbereportedtothe
user by the software. The Delay can be varied in the range -40%÷+40% of the Software Gate period, in
nominalstepsof0.1%,correspondingto20ps.Theactualgate-delaystepcanactuallyvarydependingon
theusedcamera,howeveraninternalcalibrationensuresthattheintegralnonlinearityindelaysettingis
always less than +/- 20ps. An initial offset may also be present. This offset is on purpose not calibrated,
since it will sum up with other setup-depended offsets (e.g. from different cable lengths). The overall
systemoffsetcanbeevaluatedthroughopticalmeasurementsemployingashort-pulselaser(i.e.lessthan
100pspulsewidth).
WhentheSPC3isoperatedingatedmode,thesamethreeacquisitionmodesofthestandardoperationare
possible,thistimeinconjunctionwiththeuseofthethreegates.
Figure7.
Hardwaregatingexample:Delay=5nsandWidth=5ns(bothof25%oftheReferenceClockperiod).
ThereferenceclockhasbeensenttotheSYNCOUToutputofthecamera.Additionally,aGATEINsignal
was provided by the user. Only the photons, which are detected when the “Gate signal” is on, are
counted.Examplevalidforcounter1;forcounter2and3theexampleremainsthesamewithoutthe
softwaregate.Insuchcaseonlythe4thphotonwouldnotbetdetected.
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FLIMOperation
Fluorescence Lifetime IMaging (FLIM) is an imaging technique based on the measurement of the
exponentialdecayrateofthefluorescencefromafluorescentsample,ratherthanofitsemissionintensity.
Thefluorescencelifetimeofamoleculeisameasurementoftherateofdecayoftheemission,whichisa
propertyoftheindividualsinglemolecule,andthusitisunaffectedbychangesinprobeconcentrationor
excitationintensity.FLIMcanbeusedforinstanceasanimagingtechniqueinconfocalmicroscopyandtwophotonexcitationmicroscopy.
AtypicalapplicationofFLIMisthestudyofFörsterResonanceEnergyTransfer(FRET),themechanismof
energy transfer between two chromophore molecules with the emission band of one overlapping the
absorptionbandoftheother.Whenthistwocomponentsareincloseproximity,thefirstone,calleddonor,
initiallyinitselectronicexcitedstatemaynon-radiatively(withoutemissionofthelight)transfertheenergy
to the second one, called the acceptor. The result is the quenching of the donor fluorescence, thus the
decreaseofthedonoremissionlifetime.Theefficiencyoftheenergytransferisinverselyproportionalto
the sixth power of distance between donor and acceptor, making the effect noticeable only at distances
shorterthan10nm.FRETisusedtodeterminetheproximityinthenano-meterrangebetweenproteinsor
other elements of interest associated with suitable fluorophores labelled as donors and acceptors. Such
measurements are used as a research tool in fields such as biology and chemistry. Different ways of
performing FRET measurement exist. The obvious problem with the intensity-based steady-state FRET
measurementsisthattheemissionbandofthedonorextendsintotheemissionbandoftheacceptorand
the absorption band of the acceptor extends into the absorption band of the donor. Furthermore, the
concentrations of the donor and acceptor and the fraction of donor molecules linked to an acceptor
molecule are variable and unknown. All this makes the intensity-based FRET measurements requiring
calibrationwiththesamplescontainingonlydonorsandacceptors,ormeasurementsinwhichthesecond
step is the destruction of acceptor by photo-bleaching, obtaining the FRET measurement as the relative
increase of donor fluorescence intensity. The FLIM-based FRET measurement has the advantage that the
resultsareobtainedfromasinglelifetimemeasurementofthedonorandarenotaffectedbythechanges
of the sample concentration, excitation intensity and other factors that limit the intensity-based FRET
measurements.
FLIM images are most often obtained by employing the Time Correlated Single Photon Counting (TCSPC)
techniqueandapointdetector(suchasaPMT),whichisthenscannedinordertoreconstructthedecay
histogramforeachimagepixel.Theexponentialdecaymodelisfittedtoeachpixelhistograminorderto
determine the lifetime. The colour of each image pixel represents the determined lifetime, giving the
possibility to obtain images with contrast between materials with different decay rates even if they
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fluoresceatthesamewavelength,or,ontheotherhand,identifytworegionofthesamematerialevenif
theyhavedifferentintensity.TheuseofanarrayofSPADshasmajoradvantagesforimagingapplications
since, as opposed to single pixel systems, it does not require any scan of the sample. A second major
advantageofthepresentedpixelstructureconcernstheshortdead-time,whichhasalowerlimitofabout
50ns,thusallowingthecountingofveryhighphotonfluxes.
TheembeddedshortgatecapabilityenablestheuseofSPC3asaFLIMcameraeventhoughtheemployed
SPAD imager chip is only counting the detected photons and it is not time-tagging them. In fact, it is
possibletoemploythegateinordertoimplementatime-gatedFLIMdetectionsystem.Intime-gatedFLIM
thedecayofthefluorescenceisreconstructedbyrepetitivelycountingthenumberofdetectedphotonsin
short time windows that are progressively shifted with respect to the excitation laser pulse, as shown in
Figure8.Inordertoperformthistypeofmeasurement,theSYNC_OUToutputformtheSPC3camerahasto
beconnectedtothetriggerinputofapulsedlasersourceabletogenerateopticalpulseswithwidthofat
mostfewhundredsofpicoseconds.Suchlaseristhenusedtoexcitethefluorescenceinthesampleunder
test.TheinternallygeneratedGatesignalactivatesthecountersafterthegenerationofthelaserpulsefor
thetimedefinedbygatewidth.Accordingly,thefluorescencedecaykineticsismeasuredbychanginggate
shift(position)overtime.Bothgateshiftandgatewidthhaveoptimalvaluesdependingonthelifetimeof
theexcitedstateofthefluorescentmoleculesandontheimagingframe-rate.
In order to speed-up FLIM acquisitions and increase the frame rate, the SPC3includes an automatic FLIM
modeinwhichtheinternalgatesignalisautomaticallygeneratedwithuser-definedstep-widthandshifted
oftheuser-desirednumberofsteps.
Figure8.
OpticalwaveformreconstructionusingtheGated-FLIMapproachwiththeSPC3camera.
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Each "FLIM acquisition" is thus composed by a sequence of FLIM elementary (step) frames, one for each
desiredgateposition(step),eachoneconsistingofanacquisitionwiththesameexposureparametersofa
standardacquisition.FLIMmodeacquisitionsarepossibleinSnapmodeandContinuousAcquisitionmode
whereasarealLivemodeinnotavailable,sinceeachFLIMacquisitioniscomposedbyseveralelementary
frames. However, the VisualSPC3 software (see below) offers a seamlessly equivalent Live mode which is
internallyimplementedperformingrepetitiveSnapacquisitions.
Duetothetimerequiredforinternalgateshifting(whichisalsodependentontheshiftparameters),the
timerequiredforafullFLIMacquisitionisnotsimplythesumoftheintegrationtimeofeachstepanditis,
instead,convenientlycalculatedbythecamerasoftwareandreportedtotheuser.Areferenceclockinside
thecameraguaranteesthateveryFLIMacquisitionstartsonlyafterthistimeiselapsed,thusensuringthe
isochronicityofFLIMacquisitions,andallowingtherecordingof“FLIMmovies”.
Exactly as for the gated operation, the gate signal is generated synchronized with the internal 50MHz
referenceclock.Itswidthcanbevariedintherange0÷100%ofthereferenceclockperiod(20ns),insteps
of1%,whilethegateshiftcanbevariedintherange-40%÷+40%ofthesameperiod,innominalstepsof
0.1%,correspondingto20ps.Ofcourse,inordertomaketheGated-FLIMwork,theexternallaserhastobe
synchronisedwiththegateandthus,incaseofFLIM,theSYNCOUToutputstheinternalreferenceclock.
Finally,asshowninFigure8,sincethereferenceclockperiodis20ns,thenominalmaximum“observation”
timewindowisalso20ns.Actually,sincethegateshiftrangeis-40%÷+40%,thegatecanbemovedfrom2
nsto18nsandthustheactualmaximumobservationwindowis16ns.Thereasonforthereducedrangeis
duetothefactthatforshiftslongerthan+/-40%ofperiod,theshiftaccuracyisnotsatisfactoryandthus
theSPC3FPGAdoesnotallowtheirselection.
Theactualwidthandphaseofthegatesignalobtainedforagivensetofparametersmaychangeamong
differentSPC3units,duetofabricationtolerancesoftheFPGA.That’swhythegatewidthiscalibratedby
MPDbeforedeliveringthecamera.
TheinitialpositionofthegateinaFLIMsequenceisalsointernallycalibratedandhasanaccuracybetter
than +/- 20ps plus a constant offset (see also paragraph on Gated Operation). This shift-offset is not
calibratedsinceincaseofaFLIMexperimentalset-upwhatisreallyimportanttomeasureisthefulloffset
ofthegatesignalwithrespecttothelaserpulse.Thisoffsetisnotonlydeterminedbytheinternalshiftoffset due to the camera, but also (and probably largely) by the delays introduced by the used external
interconnection cables and the laser itself. A calibration of the actual gate shift with respect to the laser
excitation is therefore necessary after the camera is mounted into the measurement setup. This can be
easilydonebydirectlyshiningthelaserontothearray,withoutinterposinganyfluorescentsampleorfilter,
andperformingafullmeasurementovertheentire20nsshiftrange.Byinspectingthemeasurementand
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finding the laser peak, it is possible to evaluate the shift between the actual laser pulse and the internal
reference clock. Actually it is not even important to measure it. What needs to be done is simply the
adjustmentofthecables’lengthfromtheSYNC_OUTofthecameratotheTRIG_INofthelaserinorderto
placethelaserpulseatthebeginningoftheFLIMobservationtimewindow.Inthiswaythefluorescence
decaywillbecentredinsidethe16nsFLIMwindow.Thiswouldalsopreventanyfoldingofthefluorescence
signalandtheassociatedmeasurementdistortion.Of course since the first and the last 2 ns of the FLIM
windowareignored,thelaserpulseisnotseenwhenitfallsthere.Ifthishappens,making1mlongerof
shorter the cable that connects the camera to the laser will make the laser pulse reappear in the FLIM
observation window. Such calibration could be useful also to zero any skew among pixels but, in many
applications, like the FLIM, it is usually not needed; should it be, the SPC3 user can contact MPD at the
followingemailaddressformoredetails:[email protected]
Finally,afactorycalibrationisperformedalsoontheactualaveragegate-shiftbin(i.e.minimumgateshift):
thisvalueisreportedtotheuserthroughtheprovidedsoftware,anditshouldbeusedasascalefactoron
thetimeaxisofthereconstructedFLIMwaveform.
FLIMdataacquiredcanbeeithersavedonamultipageTIFFwithembeddedacquisitionmetadataaccording
totheOME-TIFFformatorintheproprietarySPCFformat.Forbothformats,imagedataiscomposedbya
setofimagesfollowinga"FLIMfirst,timesecondscheme",i.e.withthefollowingframesequence:1stgate
shift of 1st FLIM measurement, 2nd gate shift of 1st FLIM measurement, ..., nth gate shift of 1st FLIM
measurement,1stgateshiftof2ndFLIMmeasurement,2ndgateshiftof2ndFLIMmeasurement,...,nthgate
shiftof2ndFLIMmeasurement,etc.
OME-TIFFfilecouldbeopenedwithanyimagereadercompatiblewithTIFFfile,sincemetadataaresaved
intotheImageDescriptiontaginXMLformat.InordertodecodeOME-TIFFmetadata,itispossibletouse
free OME-TIFF readers, such as OMERO or the Bio-Formats plugin for ImageJ. For more details see the
OME-TIFF web site: http://www.openmicroscopy.org/site/support/ome-model/ome-tiff/. OME-TIFF
metadata include the ModuloAlongT tag, which allows the processing of FLIM data with dedicated FLIM
softwares such as FLIMfit (see http://www.openmicroscopy.org/site/products/partner/flimfit). A tutorial
on basic usage of FLIMfit is available at http://help.openmicroscopy.org/flimfit.html. In order to import
OME-TIFF file generated by SPC3 camera, simply select “Load FLIM data…” from File menu. For further
supportonFLIMfitpleasecontactdirectlyitsdeveloper.
SPCF file are binary files composed by a header with acquisition metadata followed by raw image data,
containingthe8/16bitpixelvaluesinrow-majororder.SPCFfilecanbereadusingtheprovidedImageJ/Fiji
plugin. In addition, since the format is fully documented in SPC3-SDK documentation, users can directly
readdatafromtheirowncode.
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FurtherReadings
1. JosephR.Lakowicz.PrinciplesofFluorescenceSpectroscopy3rdedition.Springer(2006).
2. J.Pawley,HandbookofBiologicalConfocalMicroscopy,3rdEdition.NewYork:PlenumPress,2006.
3. T.W.J.Gadella,Ed.,FRETandFLIMTechniques,Volume33.Amsterdam:ElsevierScience,2008.
4. S.P.Chan,Z.J.Fuller,J.N.Demas,andB.A.DeGraff,“Optimizedgatingschemeforrapidlifetime
determinationsofsingle-exponentialluminescencelifetimes.”AnalChem,vol.73,no.18,pp.4486–
4490,2001.
5. D. D.-U. Li, S. Ameer-Beg, J. Arlt, D. Tyndall, R. Walker, D. R. Matthews, V. Visitkul, J. Richardson,
and R. K. Henderson, “Time- domain fluorescence lifetime imaging techniques suitable for solidstateimagingsensorarrays.”Sensors,vol.12,no.5,pp.5650–5669,2012.
Acquisitionparameters
Integrationtime
Each pixel of the SPC3 camera integrates three 9-bit binary counters (two of them with up-down
capability),whichmeasurethenumberofphotonsdetectedduringaspecifiedHardwareIntegrationTime
(HIT). The HIT is thus the time during which the impinging photons are counted without resetting the
integratedcounters,i.e.thetimefromwhenthesecountersareclearedandstartedtothetimewhenthey
arelatchedandread.ItcanrangefromtheHardwareReadoutTime(HRT)ofthearraytoabout0.65ms.
TheHardwareReadoutTimeofthearrayisproportionaltothenumberofcounters(Ncounters)inuseandit
alwayscorrespondstotheinverseoftheframe-rate.InotherwordstheHRTistheminimumtimeneeded
toreadthecountersinuseforallthepixels.HRTvalueisgivenby:
𝐻𝑎𝑟𝑑𝑤𝑎𝑟𝑒𝑅𝑒𝑎𝑑𝑜𝑢𝑡𝑇𝑖𝑚𝑒 = 𝑁"#$%&'RS ∗ (5𝑛𝑠 ∗ 𝑁,*E'W + 160𝑛𝑠)
Incaseofuseofasinglecounterperpixel,thistimeisequaltothetimeneededtoreadthe2048pixelsand
is10.40µs;for2countersitisequalto20.80µsandfor3countersitisequalto31.20µs.Itmustbealso
noted that even when saving only half the array, in order to reduce the mass storage bandwidth
requirement,thereadoutwillbecarriedoutfortheentireimagerandsotheHardwareReadoutTimewill
notbereduced.
It is also possible to increase the total integration time by internally (inside the camera’s internal FPGA)
accumulating several Hardware Integration Time, in order to obtain the so called Frame Exposure Time.
This is a viable solution since the SPC3 has no readout noise, thus there is no penalty in repeating the
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readout, apart for a negligible dead-time (or inter-frame dead-time) of less than 10ns between adjacent
HardwareIntegrationTime.ThenumberofHardwareIntegrationTimesthatareaccumulatedtoobtaina
FrameExposureTimecanrangefrom1to65534.AbeneficialsideeffectofaccumulatingseveralHardware
IntegrationTimesinsteadofusingalongerHardwareIntegrationTimeisthattheequivalentdynamicsof
the internal counter is increased. In fact, since each Hardware Integration Time can accommodate up to
511 photon detections, 2 Hardware Integration Time can hold 1022 detections, 10 Hardware Integration
Timecanhold5110andsoon.
Finally,itispossibletoemploytheinternaldetectorgatingcapabilityofCounter1inordertoachievean
EquivalentHardwareIntegrationTimesmallerthantheHardwareReadoutTime.ThisispossiblebygatingoffthedetectorduringaportionofHardwareIntegrationTime.EquivalentHardwareIntegrationTimecan
beasshortas10ns.Pleasenotehowever,thatusinganEquivalentHardwareIntegrationTimeshorterthan
theHardwareReadoutTimeresultsinacorrespondingincreaseintheinter-framedead-time,byanamount
equal to the difference between the Hardware Readout Time and the chosen Equivalent Hardware
IntegrationTime.Sinceinthisparticularworkingmodethegateiscontrolleddirectlybythecamera,itis
notrecommendedtoapplyanyexternalgatesignalortochangetheinternalgatesettings,eitherwiththe
PC software or with the standard gate-control procedures provided in the SDK. Particularly the new gate
settingswilloverwritethealreadyappliedonesthusinterferingwiththeEquivalentHardwareIntegration
Timegenerationprocess.
Inordertoconfigurethesethreeparameters,theSPC3canbesetintotwodifferentmodes.Innormalmode
the camera settings are constrained so that the Hardware Integration Time is equal to the Hardware
ReadoutTime,inordertoavoidor atleastreducetoaminimumtheprobabilityof theoccurrenceofan
overflow of the integrated counters. In fact, the internal 9-bit binary counters can overflow at long
HardwareIntegrationTimesandthusinduceartefactsinthedetectedimage.
As already shown, the maximum number of photons Nmax that can be counted per second, given a dead
timeofTdead,is:
𝑁+3E =
1
𝑇('3(
(𝑐𝑜𝑢𝑛𝑡𝑠/𝑠𝑒𝑐𝑜𝑛𝑑)
thusthemaximumnumberofphotonsthatcanbecountedduringaHardwareIntegrationTimeequaltoT
is:
𝑁+3E\]^@ =
1
𝑇('3(
𝑇
Forinstance,whenusingasinglecounter,Tisequalto10.40µsandwiththeminimumTdeadof50ns,the
maximumcountedphotonsis:
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𝑁+3E\]^@ =
𝑇
𝑇('3(
=
10.40µ𝑠
≅ 208 < 511
50𝑛𝑠
thus, any overflow is avoided since 511 is the maximum number that can be counted by a 9-bit counter
before overflowing. Instead, when using all the three counters, T is equal to 31.20µs and with the
minimumTdeadof50ns,themaximumnumberofphotonsthatcanbecountedis:
𝑁+3E\]^@ =
𝑇
𝑇('3(
=
31.20µ𝑠
≅ 624 > 511
50𝑛𝑠
andtheoverflowmayoccurwithverystronglight.
As a consequence, in order to minimise the probability of incurring in overflows, or even to completely
avoidtheminsomecases,acleverstrategyistoobtainlongFrameExposureTimesbyaccumulatingmany
Hardware Integration Times, each equal to the Hardware Readout Time, directly in the SPC3. Due to the
short inter-frame dead-time smaller than 10ns, this operating mode induces only negligible signal losses
anddoesnotincreasethenoise(sinceread-outnoiseisnotpresentintheSPC3Camera).
In advanced mode, both Hardware Integration Time and Frame Exposure Time are user adjustable, thus
particularcaremustbetakenbytheuserinordertoavoidartefactsduetotheoverflowoftheintegrated
9-bit counters, which is no more avoided or minimized. In addition, also the ability to achieve shorter
Equivalent Hardware Integration Time on Counter 1, by using the internal gate generation, is enabled.
InternalgateisautomaticallyenabledwhentheuserselectsaHardwareIntegrationTimeshorterthanthe
HardwareReadoutTime.Itisthenuptotheuserthecalculusoftheactualinter-framedead-time;forthis
purpose,sincetheHardwareReadoutTimeisequaltotheinverseoftheframe-rateinSnaporContinuous
Acquisitionmodes,itcouldbeusefultohavealookattheactualframe-ratebyenablingandcheckingthe
SyncOutoutput.Pleasealsonotethatwhenthisfeatureisenabled,onlyCounter1willbeautomatically
gated, whereas Counters 2 and 3 will have an actual Hardware Integration Time equal to the Hardware
Readout Time. Figure 9 summarizes the relationship between Hardware Integration Time, Readout Time,
FrameExposureTimeinthetwoworkingmodes.
Dead-timetimeanddead-timecorrection
SPADdead-timecanbeselectedbetween50nsand150nsbothinnormalandinadvancedmode.There
areabout60discretevaluesthatcanbeassumedoverthefullrange,thuswheneveravalueisinputbythe
user,theSPC3cameraissettotheclosestpossiblevalue,accordingtofactorycalibration.Theactualvalue
canbereportedtotheuserbythesoftware.
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Figure9.
Integrationtimesandworkingmodes.
The dead-time strongly affects the image quality. It limits the after-pulses generated in each single pixel
when set at high values, e.g. 150 ns, but it introduces a limitation on the total number of photons per
second that each pixel can detect. This induces a non-linearity, when bright spots are observed (see the
introductorysectionforfurtherdetails).
Itispossibletoimprovethequalityoftheimagebycorrectingforthenon-linearityintroducedbythedeadtime(seesection“Single-PhotonAvalancheDiode”).Infact,asimpleoperationasbackgroundsubtraction
might perform incorrectly when large numbers of photons per second are detected by the pixels. This
occurs because, for example, a cumulated flux of 10’000 photons (5’000 signal photons + 5’000 optical
backgroundphotons)mightsufferalargernon-linearityinthedetectionthantheacquisitionofonly5’000
opticalbackgroundphotons,aspredictedbyEq.1andshowninFigure2.Forsakeofclaritylet’sconsider,
asbefore,5’000photonsperintegrationwindowTbothforthebackgroundandforthesignal.Let’salso
assumeT=1ms,adead-timeof100nsandaPDE=100%forsimplicity.Inthissituationiffollowsthatonly
3’333photonsarecountedwhenacquiringthe5’000background(BG)photonsandonly5’000arecounted
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whenacquiringthecumulated10’000photonsofsignalandbackground(SG+BG),seeEq.1forreference.It
is thus clear that subtracting the measured 3’333 BG photons from the 5’000 SG+BG photons is totally
incorrect and results in the over-subtraction of the background which should have been 2’500 and not
3’333.Inanycase,thecorrectwayofproceedingistocorrect,bymeansofEq.2,forthedead-timeeffect
the actual measured number of photons and obtain the real number of impinging photons (10’000 and
5’000)forbothimagesandonlyAFTERperformthesubtraction.
By enabling the embedded dead-time correction, the effect of the non-linearity is mostly removed and a
betterandfasterprocessingofthedatabecomespossible.Figure10showstheimprovementoftheimage
qualityoftwo“white”frameswithautomaticbackgroundsubtractionenabled.Thereferenceimageshows
a noisier pattern compared to the dead-time corrected one. This is due to an “over-subtraction” of the
background,causedbydead-timeinducednon-linearity.
The effect is even more visible in the intensity histogram on the right of Figure 10. The histogram of the
reference image deviates from an expected Gauss-like profile and shows a remarked shoulder for lower
numberofphotons(greencurve).Conversely,thecorrectedimageisclosertotheidealGaussdistribution
(bluecurve).
Itmustbeobservedthatthedead-timecorrectionisonlyeffectiveunderamoderateilluminationofthe
sensor.ForthisreasonwehavedecidedthattheSWimplementeddead-timecorrection,doescompensate
the non-linearity shown in Figure 2 but it does not in any case expand the counting range beyond the
natural limit imposed by the hold-off time, as shown in Figure 11. As a consequence, when a strong
illuminationisapplied,thecorrectedvalueswillinanycasesaturatetotheexpectedsaturationlevelgiven
bythecurrenthold-offtime.
Figure10. Improvement of the image quality by using the dead-time correction algorithm. The intensity
histogramsofthetwoimagesareplotontheright.
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Figure11. Effectofthedead-timecorrectorinanimageof1msexposureanddead-timeof500nsasafunctionof
thenumberofincomingphotons.
Backgroundsubtraction
SPC3 can perform in-camera background subtraction. For this purpose the user must have previously
acquiredadarkimage(i.e.withtheshutterclose)withthesameparametersoftheactualacquisition(i.e.
thesameintegrationtime,hold-offandgate),andthenhastoenablethebackgroundsubtraction.
Additionalinformation
CalculationoftheSnapsizeandtheContinuousAcquisitionDatarate
Integration Time and Half-Array Saving Mode, directly influences the data size of a single Snap mode
acquisitionandthedatarateinContinuousAcquisitionmode.
Snapmodedatasize,inbytes,isgivenby:
𝑆𝑛𝑎𝑝_𝑆𝑖𝑧𝑒 = 𝑁,*E'W_i3j'( ∗ 𝑁kR3+'S ∗
𝐵𝑃𝑃
8
where Nframes is the number of frames in the Snap and bit-per-pixel (BPP) is 8 if there is no internal
accumulation of several Hardware Integration Times and both dead-time correction and background
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subtraction are off, 16 otherwise. Npixel_Saved is the number of saved pixel and currently can be 2048 (full
array)or1024(halfarray).
ContinuousAcquisitionmodedatarate,inbytes-per-second,isgivenby:
𝐶𝐴_𝐷𝑎𝑡𝑎𝑅𝑎𝑡𝑒 = 𝑁,*E'W_i3j'( ∗
𝐵𝑃𝑃
∗ 𝐹𝑟𝑎𝑚𝑒𝑅𝑎𝑡𝑒
8
where
𝐹𝑟𝑎𝑚𝑒𝑅𝑎𝑡𝑒 =
1
𝐻𝑎𝑟𝑑𝑤𝑎𝑟𝑒_𝑅𝑒𝑎𝑑𝑜𝑢𝑡_𝑇𝑖𝑚𝑒 ∗ 𝑁S$+S
beingNsumsthenumberofinternalaccumulations.
Cameraauto-protection
The SPC3 has a protection mechanism that shuts down the SPAD high voltage when too much current is
flowing through the array, for example when really high photon fluxes are impinging on the camera.
Solution:Reducetheamountoflightthatfallsonthesensor,disconnectandreconnectthecameratothe
hostPCandrestartthesoftware.
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SPC3Softwareinterface–VisualSPC3(Windowsonly)
Thecameraisprovidedwithanacquisitionandcontrolsoftware,whichisrunningonMicrosoftWindows
operatingsystems:VisualSPC3.Thissoftwareprovidesadirectaccesstoallthecamerafunctionsanditisa
basic tool to display images. It is recommended to use the Software Development Kit for a more flexible
camera control. Due to the start-up time needed by the internal firmware to be fully working, the
VisualSPC3 should be started at least 15 seconds after having powered the SPC3. Failing to do so will
generateanerrorbythesoftware.Simplycloseandrestartthesoftware.
Installation
Double-clickthedesiredinstaller(32-bitor64-bit)andfollowtheguidedinstallationprocedure.Pleasenote
thatdevicedriverfromOpalKelly,providedontheSPC3USB-Key,mustbeinstalledbeforeconnectingthe
cameratothePC.
SoftwareInterface
The VisualSPC3 interface is composed a main window divided in several sections as showed in Figure 12,
and a separate window for each active counter (see Figure 13). An additional window will also pop-up
whenFLIMmodeisenabled(see).
Figure12. Screenshotofthemainwindowofthegraphicaluserinterface.
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Mainwindow:ControlPanel
Thispanelallowssettingallthecamerahardwareparameters.
In the Exposure Parameters subsection it is possible to set the image integration parameters (exposure
time, number of frames for Snap mode and hardware binning). Either normal or advanced modes are
available. There are four numeric fields (see Table 3 for an example of settings), which can be either
editableorautomaticallyadjustedbythesoftwaredependingoftheselectedmode,namely:
ü Frameexposuretime(Normalonly):integrationtimeforeachframerequiredbytheuser.Thepossible
values range between 10.40 µs and 2.40 ms. This value does not correspond to the Hardware
IntegrationTimeofthesensor.Longerexposuretimesareobtainedbybinningavariablenumberof
frames.
ü Hardware Integration time: physical integration time of the SPC3 sensor. This value is fixed to the
HardwareReadoutTimeinnormalmode.Inadvancedmode,itcanrangefrom10nsto655.25µs.
ü Hardware binning: number of frames of duration equal to Hardware Integration Time that are
summed-up before being stored in the computer memory. In Advanced mode this field can be
changedbytheuser,inNormalmodeitisadjustedbythesoftwaredependingonthechosenFrame
ExposureTime
ü Numberofframes:numberofframesthatareacquiredduringaSnapevent.Thisnumberrangesfrom
1to65534for16bitimagesandto131070for8bitimages.
Table3.
PossibleconfigurationsoftheExposureParameterssubsection(NormalandAdvancedmode)
NormalAcquisitionControlBox
The duration of a frame is obtained by binning a sequence of frames of
shorter exposure times. In this example, a 52.00 µs Frame exposure is
obtained by summing 5 frames of 10.40 µs. This acquisition is then
repeated 1000 times and the data are transferred to the host computer.
Practically,5000framesof10.40µsareacquiredandbinnedbyafactorof
5. This acquisition mode does not degrade the signal-to-noise ratio of the
imagesbecausenoread-outnoiseispresent.
AdvancedAcquisitionControlBox
Theuserhasafullcontrolontheframeacquisition.
In the example, each frame has an effective duration of 103.7 µs and no
binningoftheframesisperformed(Hardwarebinning=1).Theacquisition
is then repeated 1000 times and the data are transferred to the host
computer.Frameexposuresdownto10nscanbeobtained.
WARNINGtheintegratedcounterscanoverflowatlongexposuretimes.
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Inthissubsectionthereisalsoasetofcheckboxestofurthercustomizingtheacquisition.Thefulllistand
theirexplanationcanbefoundinTable4.
Table4.
ListofcheckboxesintheExposureParametersubsectionandtheirexplanation
Acquirehalfarray
Only the central 32x32 portion of the array is acquired. Useful to
reducerequiredbandwidth.
EnableCounter2
Enable the extra in-pixel counters. When a counter is enabled a new
Counter window pop-ups, when a counter is disabled the
correspondingwindowisclosed.
EnableCounter3
Signeddata
WhenatleastCounter2isenabled,thischeckboxindicatesifthedata
fromCounter2and3hastobeconsideredsignedornot.Usefulwhen
thesecountersareemployedinup/downmode.
Waitforexternaltrigger
ThecamerawaitsforanexternaltriggerontheTRIGINinputbefore
startingtheacquisitionofanimagesequence.
Enablebackgroundsubtraction
The acquired dark frame is subtracted by the output image. A dark
frame acquisition must have been previously performed (see the
ControlButtonsparagraph).
Force8bitmode
AvailableonlyinAdvancedmode.Dataaretruncatedto8bit.Usefulto
reducerequiredbandwidth.
IntheSoftwareGatingsubsectionitispossibletoconfiguretheinternalgategeneration,whichappliesonly
toCounter1.Twogatedoperationmodearepossible,PeriodicGatemodeandFLIMmode.InPeriodicGate
modeaperiodicsignaliscontinuouslyappliedtoCounter1,asexplainedinTable5.
Table5.
PeriodicGatingsectionconfigurationexplained.
EnableGate
Enable/Disablethehardwaregating
Delay
Change the delay of the gate signal respect to the internal reference clock. The delay value is
expressedasapercentageofthereferenceclockperiod(20ns).E.g.Delay=+10%è+2nsdelay
Width
Durationofthegate-onsignal.Thisisexpressedasapercentageoftheinternalreferenceclock
period(20ns).E.g.Width=20%è4nswidth.
InFLIMmodetheinternalgateisautomaticallycontrolledinordertoperformagatedFLIMacquisitionwith
parametersexplainedinTable6.SeeparagraphonFLIMoperationforfurtherdetails.
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Table6.
FLIMmodesectionconfigurationexplained.
EnableFLIMmode
Enable/DisableFLIMmode.WhenFLIMmodeisenabledanadditionalFLIMwindowpopups(seeparagraphbelow).
GateWidth
Durationofthegate-onsignal.Thisisexpressedasapercentageoftheinternalreference
clockperiod(20ns).E.g.Width=20%è4nswidth.
FLIMsteps
NumberofstepstobeperformedforeachFLIMacquisition.
FLIMshiftperstep
GateshiftforeachFLIMstepsinthousandthsofreferenceperiod.E.g.Step=50‰è1
nsstep
FLIMStartposition
StartpositionoftheofthefirstFLIMstepwithrespecttotheinternalreferenceclock.
Thedelayvalueisexpressedasapercentageofthereferenceclockperiod(20ns).E.g.
Delay=-10%è-2nsdelay
IntheDeadtimesubsectionitispossibletoadjustthehold-offtime(intherange50÷150ns)andenable
theembeddeddead-timecorrection.
In the Synchronization Output subsection it is possible to enable the generation of voltage pulses on the
SYNCOUToutput.Twopossiblesynchronizationsignalsareavailable:
ü Framesync:a50nspulseatthebeginningofeachframe
ü Ref.Clock:theinternal50MHzclockisreplicatedtothisoutput.
MainWindow:PlotsettingsandPlaybackControl
Theplotsettingsapplyonlytotheimagesdisplayedonthescreen.Nochangesareintroducedtothe
datainthememorybuffer.Therefore,theseparametersdonotaffectthesavedimages.Allthissettings
canbechangedinreal-timeduringdisplay.TheplotsettingscanbeadjustedasexplainedinTable7.
After a Snap has been acquired, it is possible to review the images by using the controls shown in
Table8.Itisalsopossibletomovealongthedatausingthescrollbar.Thecurrentframenumberand
FLIMstep(ifinFLIMmode)arealsoshown.
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Table7. Plotsettingsandcontrolbuttons.
Automatically sets the black and white levels of the image according to the present
frame: the black level is assigned to the pixel with the minimum number of detected
photonsintheframewhilethewhitelevelisassignedtotheoneswiththemaximum
count.Thissettingwillremainappliedalsoforthefollowingframes.Sinceitispossible
to have in the following frames pixels with values lower than black or higher than
white,theywillberespectivelyindicatedinblueorred.
Resetsblackandwhitelevelstodefaultvalues:0÷255for8-bitimages,and0÷65535
for16-bitimages.
FlipX
FliptheimagealongtheX-axis.
FlipY
FliptheimagealongtheY-axis.
Rotate90°CW Rotatetheimageby90°clockwise.
KeepWindows
Ifselected,movingthemainwindowwillalsomovetogetheralltheotherwindows.
Docked
Table8.
Liveviewercontrolbuttons.
Gotothefirstacquiredframe
Gotothepreviousframe
Gotothenextframe
Gotothelastacquiredframe
Playtheimagesequencebackward
Continuous rewind. When the last (first) acquired frame is displayed, it restarts from
thefirst(last)frame
Playtheimagesequenceforward
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MainWindow:ControlButtons
Table9.
Cameracontrolbuttonsforstarting,recordingandsavingframeacquisitions.
Startthelive-modeacquisition.Oncetheacquisitionhasbeenstarted,thebuttonbecomes
aredbox.Pressitagaintostoptheacquisition.InFLIMmodetheaveragecountsoverall
FLIMstepsisvisualized.
Starts the acquisition of a sequence of frames (Snap mode). If no external trigger is
requested, the acquisition starts immediately and the button becomes a red box. If an
externaltriggerisrequired,thebuttonturnsfirstinanorangebox,theninaredoneonly
when the trigger is detected and the acquisition really starts. When the box is orange, a
clickonthebuttonstopsthewaitingforatrigger.Whentheboxisred,theusermustwait
fortheendoftheacquisition.
Starts the continuous acquisition of frames. If no external trigger is requested, the
acquisitionstartsimmediatelyandthebuttonbecomesaredbox.Ifanexternaltriggeris
required,thebuttonturnsfirstinanorangebox,theninaredoneonlywhenthetriggeris
detected and the acquisition really starts. For interrupting the waiting for the external
triggersignalortheacquisition,onceithasbeenstarted,pressthebutton.
WARNING:Continuousmodeacquisitionmaygeneratehugeamountofdatainshorttime,
dependingonacquisitionsettings.Check“Totalsaveddata”intheAcquisitionParameters
SummaryboxfortheactualsizeofdatasavedontheHDD.
Performstheacquisitionofabackgroundframe.Oncethelightispreventedtoreachthe
detector, only dark counts are measured. A dark frame is generated by averaging the
number of frames acquired. This frame is then sent to the acquisition electronics to
performhardwarebackgroundsubtraction.Theacquisitionofthebackgroundframemust
be performed using the same parameters that will be used for the actual measurement.
Anychangeintheacquisitionparameters,includingthegateparametersandthedead-time
correctionsettings,REQUIRESanewacquisitionofthebackgroundframe.Thebackground
frameisnotstoredinsidethecameraandislostwhenthecameraisswitchedoff.
SavestheacquireddatabyaSnapintoaSPC3formatfileorspecialSPC3FLIMformat.
SavesthedataacquiredbyaSnapintoamultipageTIFFfile.Thefileiscompliantwiththe
OME-TIFF specification, which allows embedding in the file metadata all the acquisition
parametersforthespecificmeasurement,aswellasFLIMmodeparameters.OME-TIFFfile
canbeopenedwithanyimagereadercompatiblewithTIFFfile,sincemetadataaresaved
intotheImageDescriptiontaginXMLformat.InordertodecodeOME-TIFFmetadata,itis
possibletousefreeOME-TIFFreader,suchasOMEROortheBio-FormatspluginforImageJ.
FormoredetailsseetheOME-TIFFwebsite:
http://www.openmicroscopy.org/site/support/ome-model/ome-tiff/.
OME-TIFFmetadataforFLIMmodeacquisitionincludetheModuloAlongTtag,whichallows
the processing of FLIM data with dedicated FLIM software such as FLIMfit (see
http://www.openmicroscopy.org/site/products/partner/flimfit).
3
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MainWindow:AcquisitionParametersSummary
In this section, the information on the acquisition, based on the actual parameters, is reported. The
explanationofalltheshownparametersisreportedinTable10.
Table10.
ExplanationoftheparametersshownintheAcquisitionParametersSummarypanel.
Framerate
ThisistheactualframerateofaSnaporContinuousAcquisition
TotalFrameTime
This is the actual frame exposure time (i.e. the real Hardware Integration
Time,multipliedbythenumberofinternalsums)
Totalacquisitiontime
ThisisthetotaltimerequiredtoconcludetheSnapacquisitionofNframes
Interframedead-time
Thisistheactualinter-framedead-time.Normallyitis<10nslong,butmay
increase in Advanced mode if very short Hardware Integration Times are
selected.
Bitperpixel
Numberofbitperpixel(8or16)
Snapfilesize
FiledimensionforaSnapacquisitionwiththecurrentparameters
Totalsaveddata
(onlyvisibleduringContinuous
Acquisition)
Thisistheactualsizeofdatasofarsavedtothedisk.Thenumberisupdated
inreal-timeaftereverywriteoperation.
CounterWindow
VisualSPC3willshowuptothreewindows(seeFigure 13)showingthemapoftheaccumulatedcountsin
eachcounter.WindowforCounter1isalwaysvisible,windowsforCounter2and3willpop-upifselected
intheExposureParameterssubsectionoftheControlPanel.InadditiontosettingsshowninTable7,itis
possibletomanuallyadjusttheblackandwhitelevelsoftheimageanditscontrastbyusingthecontrolson
the color-bar at the left of the image. After a Live or Snap mode acquisition is stopped, it is possible to
directlyreadthenumberofphotonsdetectedbyeachpixelbysimplymovingthemousepointeroverthe
image(availableonlyifwindowsizeissettothedefaultvalue.Check“Keepwindowsdocked”torevertto
defaultsize).
InFLIMmodeonlyCounter1windowisavailable.InLivemodetheaveragecountsoverallFLIMstepsare
visualized,whereasinSnapmodecountsforeachindividual FLIMstepareshown.Forthesakeofclarity
let’sconsideraSnapof10FLIMacquisitions(camerainFLIMmode)andlet’ssupposethateachacquisition
is composed by 50 steps (Gate Shifts, i.e. ‘Gate positions’): a total of 500 frames will be thus shown in
Counter1window,followinga"FLIMfirst,timesecondscheme".Withsuchscheme,theframesequence
wouldfollowthisordering:1stgateshiftof1stFLIMmeasurement,2ndgateshiftof1stFLIMmeasurement,
...,nthgateshiftof1stFLIMmeasurement,1stgateshiftof2ndFLIMmeasurement,2ndgateshiftof2ndFLIM
measurement,...,nthgateshiftof2ndFLIMmeasurement,etc.
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Figure13. ScreenshotoftheCounterwindow.
FLIMWindow
WhenFLIMmodeisenabled,anadditionalFLIMwindowpop-ups,asshownin.Thisgraphrepresentsthe
countedphotonsineachstep(GateShift)ofthecurrentFLIMacquisitionforaselectedpixel,anditisthus
the TCSPC histogram that reconstructs the waveform under investigation convoluted with the IRF of the
system.TheIRFisroughlyarectangularshapewithawidthequaltotheGatewidth.Tochangewhichpixel
isvisualized,simplyclickonthedesiredpixelinCounter1window.ThegraphisupdatedinrealtimeinLive
mode. In Snap mode it shows the entire FLIM waveform corresponding to the currently visualized FLIM
acquisition(i.e.itwillnotchangeif,inCounter1,adifferentFLIMstepfromthesameFLIMacquisitionis
visualized ). It is possible to choose between linear and logarithmic scale for the Y axis. The Accumulate
checkbox,ifselected,willcausethehistogramtoaccumulatecountsamongframes.
Figure14. ScreenshotoftheFLIMwindow.Inthiscase,theacquisitionistheIRFofthesystemwhenGatewidthis
setto2ns,and800gatestepsspanning16nsareacquired.
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SPC3-SDK,FileFormatsandImageJPlugin
SPC3 is provided with a Software Development Kit (SDK), composed by a shared library and few example
projects.TheSDKallowstheuseracquiredatafromtheSPC3andtochangeoperatingparameterdirectly
fromtheirownapplication.ItisavailableforWindows,MacOSXandLinuxoperatingsystems,bothin32-
and 64-bit versions. More information on available functions can be found in the related documentation
includedintheUSBkey.
Acquired data from Snap acquisitions can be saved both in OME-TIFF format and in a MPD proprietary
binaryfileformat.DatafromContinuousacquisitioncanonlybesavedinMPDproprietaryformat.
OME-TIFFfilecouldbeopenedwithanyimagereadercompatiblewithTIFFfile,sincemetadataaresaved
intotheImageDescriptiontaginXMLformat.InordertodecodeOME-TIFFmetadata,itispossibletouse
free OME-TIFF readers, such as OMERO or the Bio-Formats plugin for ImageJ. For more details see the
OME-TIFFwebsite:http://www.openmicroscopy.org/site/support/ome-model/ome-tiff/.
ProprietaryformatfilesproducedbySPC3cameraarebasedonthesamebinarystructure,butdifferentfile
extensions are used for differentiate among data. Photon counting acquisitions are stored in .spc3 files
whereasFLIMmodeacquisitionsaresavedin.spcffiles.Inaddition,eachcameraisprovidedwithan.spce
file containing measured photon detection efficiency. All these files contain binary data, composed by a
header with acquisition metadata followed by raw image data which contain the 8/16 bit pixel values in
row-majororder.Thebyteorderislittle-endianforthe16bitdata.Rawdatahavethefollowingmeaning:
.spc3files: data represents photon counts in each pixels during each Exposure Time, with acquisition
parameters reported in the header. In case more counters are used, data are interlaced, i.e.
thesequenceofframesisthefollowing:1stframeof1stcounter,1stframeof2ndcounter,1st
frameof3rdcounter,2ndframeof1stcounter,etc.
.spcffiles: data represents photon counts in each pixels during each Exposure Time, with acquisition
parametersreportedintheheader.Imagesfollowa"FLIMfirst,timesecondscheme",1stgate
shiftof1stFLIMmeasurement,2ndgateshiftof1stFLIMmeasurement,...,nthgateshiftof1st
FLIM measurement, 1st gate shift of 2nd FLIM measurement, 2nd gate shift of 2nd FLIM
measurement,...,nthgateshiftof2ndFLIMmeasurement,etc.
.spcefiles: data represent measured Photon Detection Efficiency for each pixel. Values range from 0 to
10000,i.e.a32.5%PDEwillbeexpressedas3250.Eachframerepresentsawavelength,whose
rangeandsteparereportedintheheader.
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Theheaderiscomposedbyasignatureof8bytes,andametadatasectionof32bytes,asreportedinTable
11.Notethatmultibytefieldsarelittle-endian.
Table11.
HeaderstructureofSPC3proprietaryfileformats.spc3,.spcf,.spce.
Byteoffset
Numberofbytes
Description
0
8
Filesignature:0x4d5044ff03000000
8
1
Numberofrows
9
1
Numberofcolums
10
1
Bitperpixel
11
1
Countersinuse
12
2
Hardwareintegrationtime(multiplesof10ns)
14
2
Summedframes
16
1
Deadtimecorrectionenabled 17
1
Internalgateduty-cycle(0-100%)
18
2
Holdofftime(ns)
20
1
Backgroundsubtractionenabled
21
1
Dataforcounters1and2aresigned
22
1
FLIMenabled
23
1
FLIMshift%
24
1
FLIMsteps
25
4
FLIMframelength(multiplesof10ns) 29
2
FLIMbinwidth(fs)
31
1
PDEmeasurement
32
2
Startwavelength(nm) 34
2
Stopwavelength(nm) 36
2
Step(nm)
38
2
Unused
Inordertoeasilyvisualizeandelaboratedatastoredintheproprietary.spc3,.spcfand.spcefileformats,a
pluginforImageJsoftware(http://rsb.info.nih.gov/ij/)isalsoprovided.Thepluginiscompatiblebothwith
ImageJ (tested on v. 1.49q) and with Fiji (http://fiji.sc/Fiji), which is essentially a distribution of ImageJ
togetherwithseveralusefulplugins.
Inordertoinstallthepluginfollowthisprocedure:
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•
forImageJ(testedonv.1.49q),launchImageJandselectthemenuitemPlugins→Install…,thenfollow
the instructions and select the file SPC3_Reader.java provided in SPC3 USB key. The "Save Plugin,
MacroorScript..."dialogappearswithalistofyourcurrentlyinstalledImageJplugins.Selectafolder,
such as the Input-Output folder, and save the SPC3_Reader.java file there. Do not change the name
SPC3_Reader.java,asitfollowsImageJnamingconventionsanditisnotseenbyImageJifitdoesnot
containanunderscore.
•
forFiji,launchFijiandselectthemenuitemPlugins→InstallPlugin…,thenfollowtheinstructionsand
selectthefileSPC3_Reader.javaprovidedinSPC3USBkey.RestartFiji.
In order to use the plugin select SPC3_Reader under the Plugins menu. It will be in the location you’ve
previouslychosenforImageJ,orattheendofthemenuforFiji.An"OpenSPC3file..."dialogappears.Select
the.spc3,.spcfor.spcefileyouwishtoload.ImageJwillnowaskyouhowtodealwithsigneddata.
Ifdatais8bit,therearethreeoptions:
•
Convertdatato32bitfloat
•
Importunsigneddataasisandconvertsigneddatato32bitfloat
•
Import all data as is (this means ImageJ will interpret signed data as unsigned, e.g. the number -1,
whichin8bitbinaryis11111111,willbeshownas255)
Ifdatais16bit,therearefouroptions:
•
Convertdatato32bitfloat
•
Importunsigneddataasisandconvertsigneddatato32bitfloat
•
Import all data as is (this means ImageJ will interpret signed data as unsigned, e.g. the number -1,
whichinbinaryis11111111,willbeshownas255)
•
ImportalldataasisanduseanImageJcalibrationfunctiontomapsigneddata(thismeansImageJwill
remapdatatoshowsignedvalues,e.g.thenumber-1,whichin16bitbinaryis1111111111111111and
willreadas65535withoutremapping,willbeproperlyshownas-1).
If the selected file is properly formatted and no error occurred, an image window for each SPC3 counter
enabledopens.Framescanbeskimmedusingthecontrolleratthebottomofeachwindow.Selectingfrom
themenuthecommandImage→ShowInfo…willshowallcameraparametersfortheacquisition.
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Systemrequirements
§
High-speedUSB3.0interfaceforfullspeedacquisition,compatiblewithUSB2.0
§
Hostcomputer(minimumrequirements)
§
o
2GHzprocessorand1GBofRAM
o
SSDrecommendedforfullspeedcontinuousacquisition.
Supportedoperatingsystems
o
VisualSPC3
§
o
MicrosoftWindows7,8,32or64bitversions
SDKandImageJPlugin
§
MicrosoftWindows,7,8,32or64bitversions
§
LinuxUbuntu 12.04 LTS, CentOS6.5,6.6,6.7 orcompatibledistributions,32or64
bitversions.Differentdistributionsshouldwork,butwerenottested.
§
§
MacOSX10.8andabove
TestedImageJversions(differentversionsshouldwork,butwerenottested)
o
StandaloneImageJ:version1.49q
o
Fijidistribution:version2.0.0-rc-28/1.50b
Copyrightanddisclaimer
No part of this manual, including the products and software described in it, may be reproduced,
transmitted,transcribed,storedinaretrievalsystem,ortranslatedintoanylanguageinanyformorbyany
means, except for the documentation kept by the purchaser for backup purposes, without the express
written permission of Micro Photon Devices S.r.l. All trademarks mentioned herein are property of their
respectivecompanies.MicroPhotonDevicesS.r.l.reservestherighttomodifyorchangethedesignandthe
specificationstheproductsdescribedinthisdocumentwithoutnotice.
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