Compact FLIM and FCS Upgrade Kit for Laser Scanning ...Bülter, Felix Koberling, Rainer Erdmann, PicoQuant GmbH Introduction Already for more than 20 years, confocal laser scanning

Technical Note

Compact FLIM and FCS Upgrade Kit for Laser Scanning Microscopes (LSMs)

Benedikt Krämer, Volker Buschmann, Uwe Ortmann, Michael Wahl, AndreasBülter, Felix Koberling, Rainer Erdmann, PicoQuant GmbH

Introduction

Already for more than 20 years, confocal laserscanning microscopy is a widely used tool in bio-chemistry, cell biology and other related sciences.However, the usually applied continuous-wave(cw) laser scanning microscopy, which measuresfluorescence intensity, is strongly depending onthe knowledge of system parameters for a quan-titative analysis. A technique disregarding thesedrawbacks is therefore preferable. Fortunately,fluorophores can not only be investigated by theiremission wavelength or overall intensity, but alsoby their fluorescence lifetime.

The use of time-resolved techniques in confocallaser scanning microscopy will grant the followingadvantages:

• Identification of fluorophores by theirfluorescence lifetime

• Discrimination of fluorescence light frombackground scattering by temporal separation

• Reduction of needed detectors: One detectorcan determine different lifetimes simultaneously,however, only one spectral range.

• Fluorescence lifetime as a quantitative measureenhances the accuracy of analyticalmeasurements

• Quantitative Förster Resonance EnergyTransfer (FRET) efficiency through fluorescencelifetime analysis

Useful applications of lifetime as a parameter canbe found in the investigation of environmentalparameters. Typical examples are the mapping ofcellular conditions such as pH [1], ion concentra-tion or oxygen saturation by fluorescencequenching [2]. Förster Resonance Energy Transfer(FRET) [3,4] between different fluorophores allowsto study the binding of reaction partners in livingcells as well as measurements of distances in therange of only a few nanometres.

Alternatively, the temporal resolution can be usedto monitor fluorescence intensity fluctuations, e.g.for Fluorescence (Lifetime) Correlation Spectro-scopy (F(L)CS) [3,5]. The method can be appliedto investigate diffusion processes and to measurethe concentration of selected molecules.

With the ongoing technical development of time-resolved instrumentation, the combination of LSMswith time-resolved techniques was immanent. Thistechnical note describes how common LaserScanning Microscopes can be upgraded towardsFLIM, FCS and other time-resolved methods usingthe dedicated products from PicoQuant.

© PicoQuant GmbH, 2009 Technical Note: LSM upgrade kit v. 3.0 Page 1

Fig. 1: Selected subcomponents of the FLIM & FCS upgrade kit for LSMs

Technical aspects

The upgrade kit is based on the method of Time-Correlated Single Photon Counting (TCSPC) fordata acquisition and sensitive photon counting de-tectors. Along with a suited short pulsed laser athigh repetition rates, the upgrade kit offers anexcellent detection efficiency at high temporalresolution. It can be used with one photon and twophoton excitation schemes using pulsed diodelasers or Titanium:Sapphire lasers.

The complete set up consists of the followingcomponents:

• Commercial Laser Scanning Microscope suchas

- FluoView FV 1000 or FluoView FV 300 fromOlympus

- SP5 from Leica

- A1 or C1(si) from Nikon

- LSM 510 / 710 from Zeiss

• Excitation sources, which are either picoseconddiode lasers from PicoQuant (LDH Series) alongwith a dedicated driver (PDL Series) orTitanium:Sapphire lasers

• Single Photon Avalanche Diodes (SPAD) orphoton counting Photomultiplier Tubes (PMT)

• TCSPC data acquisition system (PicoHarp 300,HydraHarp 400)

• Data acquisition and analysis software(SymPhoTime)

• Set of necessary cables

• Computer

The upgrade kit is added as an external extensionto the LSM, as schematically shown in Fig. 2. Toensure an accurate system performance, severalpreconditions must be met:

1. It must be possible to guide the pulsedexcitation light into the LSM. For single photonexcitation schemes this is realised by one ormore fibre input ports. Such ports are standardfor each LSM allowing to connect the cw lasersfrom the LSM laser combiner to one or more ofthese ports. Titanium:Sapphire lasers for two-photon excitation are usually coupled via a freespace entrance port.

2. It must be possible to guide the fluorescencelight out of the microscope. This is crucialbecause the build-in detectors of the LSM aretypically not suited for photon countingapplications. The external detectors can beattached in two ways: either via a fibre exit portfor descanned detection, which is the usualconfiguration, or directly to a side port for non-descanned detection (NDD, mainly used incombination with two-photon excitation).

3. It must be possible to synchronize the dataacquisition with the movement of the laserscanner in order to actually generate the image.Typically synchronization signals at thebeginning and end of each line and as well asat the beginning of each frame are used (“linestart, line stop and frame start”). Thesesynchronization signals are available or can bemade available at the LSM controller.

For all above mentioned LSMs these technicalissues are addressed by the upgrade kit fromPicoQuant. It might, however, in some cases benecessary to modify the LSM itself, e.g. by addingan entrance or exit port or modifying the LSMcontroller firmware.


Fig. 2: Sketch of the general layout of the FLIM & FCS upgrade kit

Driver of the PDL-Series

Microscope

LSMController

Computer with PicoHarp 300 and analysis software

Synchronisation

Line and Frame clock

Excitation via fibre

Emission via fibre

Detector signal

LSM ControlComputer

La

ser

he

ad

Laser Scanner

Fibre coupling unit (FCU II)

LSM lasercombiner

La

ser

he

ad

Lase

r h

ead

Lase

r h

ead

Detector Unit

Router

Special considerations for each supportedLSM

Leica SP5

The external detectors are attached via multimodefibres to the X1 exit port of the SP5.

Pulsed UV excitation at 405 nm is coupled to theSP5 via a corresponding UV fibre port. All otherexcitation wavelengths (407, 640 nm) are coupledto the SP5 via a multi function port (MFP), whichacts alternatively for two photon excitation.

For FLIM, the SP5 must be equipped with a“Scanner Trigger Unit”, which is available fromLeica. This box generates the necessary markersignals for the synchronization between dataacquisition and movement of the scanner. Leicaalso provides a laser pulse unit which enables ROIscanning and allows to control the laser repetitionfrequency by the SP5 software.

PicoQuant and Leica have also introduced theTCS SMD series, which integrate hard- and soft-ware from PicoQuant with the confocal systemTCS SP5. In the TCS SMD, the complete dataacquisition is controlled by one single software.Straightforward application wizards guarantee acomfortable performance and allow for automatedrecording of FLIM volume stacks and FLIM lambdastacks for spectral and time resolved imaging. FCStime series performed at data points predefined inthree dimensions enable automated measure-ments of diffusional parameters in living cells.

Nikon C1si

The Nikon C1si contains two fibre exit ports. Theupgrade kit from PicoQuant is attached to thespectral exit port. In case the system is alreadyequipped with the spectral detection box, it isnecessary to swap fibres between spectral and

FLIM / FCS operation.

The Nikon laser combiner must be equipped with afibre switch with an open AFC connector to allowthe incoupling of the PicoQuant pulsed lasers intothe C1si. This switch is not standard and can besupplied by PicoQuant upon request. Due to thisfibre switch pulsed and internal cw lasers can notbe used simultaneously.

Nikon A1

The external detectors are attached via a multi-mode fibre to the AUX fibre exit port of the scanhead, which needs to be modified by PicoQuantwith a special APC fibre coupler. Nikon mustremove the focusing lens from the AUX port priorto installation.

Pulsed laser diodes ranging from 405 nm up to640 nm can be coupled into the Nikon scan headvia the VIS fibre input port. The Nikon lasercombiner must be equipped with a fibre switchfrom PicoQuant with an open AFC connector toallow the incoupling of the PicoQuant pulsed lasersinto the A1. Due to this fibre switch pulsed andinternal cw lasers can not be used simultaneously.For the implementation of pulsed laser diodes at485 nm and 532 nm special main dichroics arenecessary which must be built in by Nikon prior tothe installation.

Olympus FluoView FV 300

The upgrade of the FluoView FV 300 requires aspecial fibre outcoupler, which is not offered byOlympus. PicoQuant is able to provide such asolution for newer versions of the FluoView


FV 300. The fibre outcoupler is added to the topcover of the scan unit. In addition PicoQuantdelivers a modified dichroic detection slider whichguides the light to the exit port.

The pulsed lasers (405 nm - 640 nm) are coupledto the FluoView FV 300 using the UV/IR port,which must be available. A specially developedpolarisation combiner is inserted in the FluoViewFV 300 and allows the simultaneous usage ofpulsed and cw lasers.

Olympus FluoView FV 1000

The upgrade of the FluoView FV 1000 requires theintegration of a fibre exit port, which is provided byOlympus.

The pulsed lasers are coupled to the FluoViewFV 1000 via the existing UV and IR ports. The UVLaser (375 nm) is directly connected to the UV portand the VIS wavelengths ranging from 405 nm to640 nm are coupled into the FluoView FV 1000 viathe IR port. The corresponding ports musttherefore be present and unused. A speciallydeveloped polarisation combiner is inserted in theIR port and allows the simultaneous usage of (VIS)pulsed and cw lasers.

The serial number on the PSU (LSM controller)backplane must be 5H... , 6H... and higher and theconstruction year of the system should be laterthan 2005.

Zeiss LSM 510

The upgrade requires a LSM 510 based on aninverted microscope body with the scan unitattached to the left side port. The system must fur-ther be equipped with a free space exit port alongwith a suited tubing. These parts are available fromZeiss upon request. If these preconditions are met,the fibre outcoupler is added externally byPicoQuant. Therefore the system must stand on anoptical table with either metric or inch threads.Furthermore, the breadboard must extend 20 cmto the left side over the LSM 510 unit.

If the system is equipped with a Confocor 3, anoptical switch from Zeiss with a free space exit portmust be implemented between the LSM 510 andthe Confocor 3. The free space exit port mustcontain a tubing.

For UV lasers (375 - 440 nm) an additional UV portmust be added by Zeiss. Two separate FCUs fromPicoQuant are needed each for the UVwavelengths (375 nm - 440 nm) and the VISwavelengths (470 nm - 640 nm). The lasers fromthe Zeiss laser combiner must be guided throughthe PicoQuant Fiber Coupling Unit (FCU) using thebypass option.

On the LSM 510 controller port B must be free.The data base has to be changed by Zeiss to allowthe generation of marker signals for the line andframe synchronization at port B. Due to thisalterations the 477 nm line of the Ar-Ion laser willnot be available for the LSM any more. Thecontroller must further have at least a hardwareversion of 3.5, available since 2004.

Zeiss LSM 710

The system must be equipped with a free spaceexit port along with a suited tubing. These partsare available from Zeiss upon request. If thesepreconditions are fulfilled, the fibre outcoupler isadded externally by PicoQuant. Therefore thesystem must stand on an optical table with eithermetric or inch threads. The breadboard shouldextend 50 cm around the LSM 710 unit for easyaccess.

If the system is equipped with a Confocor 3, anoptical switch from Zeiss with a free space exit portmust be built in between the LSM 710 and the


Confocor 3. The free space exit port must containa tubing.

Only pulsed lasers provided by Zeiss can beintegrated into the set up.

Detailed description of the involvedcomponents for the upgrade kit

In the following pages, the individual components,that are part of the upgrade kit are described inmore detail. These subsystems include theexcitation unit, the detector configurations as wellas the measurement device for Time-CorrelatedSingle Photon Counting (TCSPC) along with thecorresponding hardware and system software.

Excitation

As excitation source a short pulsed laser with ahigh repetition rate is the system of choice for time-resolved measurements using Time-CorrelatedSingle Photon Counting (TCSPC). A short laserpulse excites the fluorophore and the subsequentfluorescence light decay can be measured.Typically, picosecond diode lasers are used, whichare available as a combination of a laser driver unit(PDL Series) and a variety of laser diode heads(LDH Series) ranging from 375 nm to 800 nm (seeFig. 3). These laser heads work in pulsed mode atvariable repetition rates from single shot up to80 MHz or even in cw mode. They are controlledby a driver of the PDL Series, which are availableas single channel or multi-channel versions withmanual or computer control. All drivers allow tochange the the laser output power – an essentialfeature to avoid photo-bleaching. Furthermore thedrivers enable to adjust the repetition rate of thelaser diodes to match the excitation pulse period tothe fluorescence decay of the fluorophore. Themulti-channel driver versions further permit to useseveral laser heads simultaneously for advancedexcitation schemes like Pulsed InterleavedExcitation (PIE). All drivers directly generate asynchronization signal for each pulse, suitable forTCSPC measurements.

The laser heads are typically included in a specialfibre coupling unit (FCU II) that can couple up tofive different laser heads into one polarisationmaintaining single mode fibre (see Fig. 4). Inaddition the FCU II allows to control the laserintensity via a ND-filter wheel as well as a scaffold.

If only a single laser diode should be implemented,it can be directly coupled via a fibre into the LSM(see Fig. 5). This compact coupler also includes ascaffolding for fine tuning the laser intensity. Thisconfiguration requires a free fibre coupling port atthe LSM for the corresponding wavelength.

The laser safety of the additional laser sources isguaranteed since under normal operationconditions all laser light is shielded and guidedthrough optical fibres.

As an alternative to pulsed diode lasers, especiallyfor two photon excitation schemes, short pulse la-ser systems such as Titanium:Sapphire lasers canbe integrated as well. In some cases an additionaltrigger diode is necessary to generate a stabletrigger signal with optimal timing performance forTCSPC measurements.

For all excitation wavelengths it is mandatory thatthe LSM contains all needed dichroics or beamsplitters (also for Leica LSMs with AOBSbeamsplitter). Any necessary change of the LSM configurationmust, however, be performed by the manufacturerof the LSM itself and can not be realised byPicoQuant.


Fig. 5: Compact and direct fibre coupling of a single diode laser head

Fig. 4: Fibre Coupling Unit (FCU II) – in normal operation theFCU will be closed by a suited lid to ensure laser safety

Fig. 3: Laser head of the LDH Series and single channel laser driver of the PDL Series

Detection

The standard photomultiplier detectors presentwithin the LSM are operated in analogue mode,where the current signal from the photon detectoris used as a measure of fluorescence intensity.This allows an easy change of the detectorsensitivity by variation of the supply voltage (HV) ofthe detector, but on the other hand prevents theusage of these detectors in time-resolvedmeasurements. For these purposes speciallydesigned photon counting detectors such asSingle Photon Avalanche Diodes (SPADs) orphoton counting Photomultiplier Tubes (PMTs) areused. These detectors are in most cases notintegrated in the LSM, but must be addedexternally via an exit port and a multimode opticalfibre. A suitable exit port is available as an optionfor all mentioned LSMs.

The upgrade kit can be configured for a single ordual channel detector version (Fig. 6 and 7).Typically SPAD detectors are used for the upgradekit as these detectors feature a very high detectionefficiency up to 50% (wavelength dependent),which is essential for e.g. single molecule studieslike Fluorescence Correlation Spectroscopy (FCS).The best currently available types are the PDMmodules from MPD, which also feature anextremely high temporal resolution of 50 ps FWHM(at 640 nm). SPADs in general have a rather smallactive area in the micrometer range, whichtherefore requires an accurate opto-mechanicalset up. Thus the detectors are mounted into a self-contained housing, which also includes filterholders for optical filters (bandpass, longpass, etc.,Fig. 6). The applicable filters have a standarddiameter of 25 mm (one inch) and can easily be

changed to account for different excitation ordetection schemes. The dual channel set upadditionally contains suited mounts for beamsplitting optics, which are included in a three-position tower, that can be operated from outside.Different beam configurations can thus be easilyrealized without greater efforts.

As an alternative to SPADs, the PhotomultiplierTubes of the PMA-M series can be used.Compared to SPADs they have a lower detectionefficiency and are thus not suitable for singlemolecule applications like FCS. However, for FLIMthey are well-suited. All PMTs feature an excellent RF shielding as wellas a built-in pre-amplifier. Since PMTs have arelatively large active area of several millimetres,they don't need sophisticated optics and opto-mechanics like SPADs and are thus not included ina self-contained housing (Fig. 7).

Eventually, PMT detectors can further be attachedto the microscope in a so called “non-descannedmode (NDD)”. Here the detector is coupled via adichroic mirror and appropriate filter set to a non-confocal exit port of the microscope. In thisdetection mode only two photon excitation can beused, where the confocal volume is no longermainly defined by the detection pinhole, but ratherby the extremely small volume where thenecessary high excitation radiation is present. Thisoption allows to upgrade a multi-photon excitation(MPE) LSM towards photon counting andfluorescence lifetime capability without the need ofa fibre exit port.


Fig. 7: PMT units: single channel detection (left) and dual channel detection (right)

Fig. 6: SPAD units: single channel detection (left) and dual channel detection (right)

Data acquisition and synchronization

The data acquisition of the upgrade kit is based onthe principle of Time-Correlated Single PhotonCounting (TCSPC). TCSPC is the most powerfuland sensitive method to measure fluorescencelifetimes. Briefly, the method is based on theprecise measurement determination of the timedifference between the moment of excitation andthe arrival of the first fluorescence photon at thedetector (“TCSPC time”). The measurement of thistime difference is repeated several million times toaccount for the statistical nature of fluorescenceemission and all TCSPC times are sorted into ahistogram. Such a histogram of photon arrivaltimes is generated for each image pixel, whichdoes of course require to synchronize the dataacquisition with the movement of the laserscanner. For FLIM, the histogram is analysed toextract the fluorescence lifetime, which isdisplayed in a false colour code.

From a technical point of view the synchronizationof the data acquisition with the movement of thelaser scanner can be realized in several ways. Themost flexible solution is a special measurementmode called Time-Tagged Time-Resolved mode(TTTR), whose basic ideas are (see Fig. 8):

• add an additional timing information (the “timetag”) to each TCSPC time, which re-presentsthe arrival time of each photon relative to thebeginning of the experiment. This time tag istaken from a second, continuously running clockwith nanosecond resolution.

• do not immediately form a histogram for eachpixel during the measurement, but write themeasured data (“TCSPC time” andcorresponding “time tag”) into one data file.

• include external synchronization signals(“marker”) from the laser scanner at thebeginning and end of each line as well as thebeginning of each frame into the data file. A timetag needs to be added to each marker signal inorder to sort the marker signal into the temporalorder of the events.

• in case of a multi-channel set up add a channelidentifier (“which detector?”) to each measuredTCSPC time

One photon event in the TTTR data stream there-fore consists of four individual pieces of infor-mation: the “TCSPC time”, the “time tag”, channelinformation and synchronization information. Sor-ting photons into their corresponding “image pixel”is now a simple mathematical procedure, whichcan be summarized for each detection channel as:

• split the measurement data into framesaccording to the marker signals for “frame start”

• split each frame into lines according to themarker signals “line start” and “line stop”

• calculate the difference of the time tags from“line start” and “line stop” to get the “scanningtime per line”

• divide the “scanning time per line” by the pixelnumber per line to get the “scanning time perpixel”


Fig. 8: Scheme of the TTTR mode

• sort each photon into the corresponding pixelusing the time tag and the “scanning time perpixel”

This procedure does of course require the know-ledge about the image size and that the scannermoves with a constant speed. The data analysis isalready possible during the measurement by a par-allel data processing and allows an online view ofthe lifetime image, FCS curves as well as the fluor-escence intensity time trace. As great advantageof the TTTR approach the complete photondynamics are preserved. The concept of TTTRallows the data storage without redundancy andwithout any loss of information, in contrast to e.g.on-board histogramming. Therefore, virtually allalgorithms and methods for the analysis of fluores-cence dynamics can be implemented and a verysophisticated offline data analysis is possible (seeFig. 9).

The data acquisition is done using the Pico-Harp 300 USB 2.0 stand-alone module [6]. Thisunique device features a temporal resolution of4 ps and allows to resolve fluorescence lifetimeswell below 100 ps – even down to 60 ps in combin-ation with a very fast detector of the PDM Series. Aspecial routing device, PHR 800, allows to connectmore than one detector to the TCSPC unit andthus enables multi-channel detection (Fig. 10 aand b).

Software

The SymPhoTime software is used for dataacquisition and analysis. During FLIM measure-ments the software already calculates and displaysa FLIM image (“online-FLIM”), which is calculatedby the mean TCSPC arrival time of the fluores-cence photons. It permits a quick assessment ofthe image quality and lifetime contrast. Theadvanced analysis functions of the SymPhoTimesoftware for FLIM measurement include fitting thedata in each image pixel to an exponential decayfunction to extract the fluorescence lifetime. TheSymPhoTime software supports fitting of the mea-surement data to an exponential decay function upto the 4th order including numerical reconvolutionalgorithms. A special fitting routine can beemployed to account for areas with low signalintensity. The fitting results (amplitudes, lifetimesor lifetime distributions) can be visualizedindividually or combined with freely adjustablecolouring schemes. Besides the whole image, it isalso possible to define several arbitrary areas(ROI) inside the image for a detailed analysis.

Also during FCS measurements an online corre-lation is performed and displayed for fast assess-ment of the measurement data. For detailedanalysis, the SymPhoTime software provides anefficient correlator for FCS and supportsautocorrelation and cross correlation between twochannels. A user defined time gate can also beapplied, which can be very useful to suppressscattered light contributions. As an advancedmethod, the software includes analysis routines forFluorescence Lifetime Correlation Spectroscopy(FLCS) [5]. FLCS allows background and after-pulsing suppression without the need for a crosscorrelation. In addition, for dyes with differentlifetimes but overlapping spectral properties, indi-vidual FCS curves can be calculated. Also crosscorrelation between these dyes is possible. Fittingroutines for different established FCS models arealready included and all results can be exported forfurther offline analysis.

The SymPhoTime software contains further ana-lysis routines for many fluorescence relatedprocedures. A special scripting language alsopermits to generate special user-defined analysisprocedures.


Fig. 9: Analysis possibilities based on the TTTR mode

Fig. 10: b) PHR 800 router

Fig. 10: a) PicoHarp 300 TCSPC unit

Experimental results

1) FLIM-FRET measurements with cw acceptorbleaching

In a FLIM-FRET measurement only the fluores-cence lifetime of the donor molecule is monitored.If donor and acceptor fluorophores are closeenough to each other (in the range of severalnanometers), the excitation energy can betransferred from the donor to the acceptormolecule. As a result of the energy transfer, theFRET process can be identified by a decrease ofthe fluorescence lifetime (quenching) of the donorin comparison to the lifetime of the individualmolecule. Since the lifetime is an absolutemeasure, the FRET efficiency can be quantifiedfrom the ratio between the lifetime of moleculesshowing FRET and molecules that are notundergoing FRET in each image pixel. A directapproach to obtain the lifetime image of the FRETmolecules not undergoing FRET is obtained byphoto-bleaching the acceptor dye with a secondlaser. FRET analysis with the lifetime approachallows for quantitative results, no correction factorshave to be applied.

The second method to obtain FRET efficiencies isusing the ratio of the fluorescence intensity of do-nor and acceptor applying two detector channels.

However, in order to quantify FRET efficiency byintensity based FRET measurements correctionfactors as for the quantum efficiencies of dyes anddetectors have to be used which are difficult toobtain.

The example (Fig. 12) demonstrates FLIM-FRETmeasurements of fluorescent proteins inside livingcells (12V HC Red cells), labelled with EGFP andRFP. The data are taken with an upgradedFluoView 1000 from Olympus using an Apo 60xobjective (1.4 N.A. Oil immersion) with fluores-cence detection between 500 nm and 540 nm.

The left image in Fig. 12 shows the fluorescencelifetime image of EGFP in the presence of RFPmolecules. The mean lifetime value is found to be2.2 ns indicative of FRET. If the acceptor moleculeis bleached by strong laser irradiation (i.e. cwexcitation at 568 nm) no energy can be transferredfrom the donor to the acceptor any more. Thus thefluorescence lifetime of EGFP increases (see rightimage in Fig. 12). The mean lifetime value is nowfound to be 2.9 ns.


Fig. 11: Screenshots from the software system SymPhoTime

2) Protein interactions of human CentromereProteins via FLIM-FRET

As a second example of FLIM-FRET measure-ments, protein interactions between humanCentromere Proteins (CENP) have been analysed.These proteins are part of the chromosome andensure correct segregation of every chromosomeduring cell division. For this example, U2OS cellshave been transfected with two centromere pro-teins, that were fused to Cerulean(CENP-B-Cerulean) as the donor and EYFP(EYFP-CENP-A) as the acceptor. The data weretaken with an upgraded FluoView FV 1000 fromOlympus using a 1.35 N.A. oil immersion objectiveand a dual channel detection set up to measurethe fluorescence from the donor and acceptormolecules simultaneously.

The results in Fig. 13 clearly allow to distinguishdifferent cells. In cell 1, the fluorescence lifetime inthe donor as well as in the acceptor channel was

found to be approx. 3 ns, which corresponds to thelifetime of “pure” CENP-B-Cerulean as shown byan independent measurement. It can therefore beconcluded that this cell is not transfected byEYFP-CENP-A. The situation is different for thesecond cell. Here, the fluorescence lifetime wasfound to be 1.2 ns in the donor channel and 2.8 nsin the acceptor channel. A more detailed analysisrevealed a rise of the fluorescence intensity atearly times in the acceptor channel with a timeconstant of approx. 0.5 s. This rise is due to thetime needed for the energy transfer from the donorto the acceptor fluorophore and is thus (along withthe different lifetimes) a clear indicator that in thiscell both CENP-B-Cerulean and EYFP-CENP-Awere present and undergo FRET. The mean FRETefficiency E of cell 2 can be calculated to be E= 1 –(1.2 ns / 3 ns) = 0.6. The data thus reveal acentromere specific clustering between CENP-Aand CENP-B centromere proteins.


Fig. 13:

FLIM-FRETmeasurements of

Cerulean/EYFPusing a dual

channel setup.

Data courtesy ofSandra Orthaus,former member

of Fritz LipmannInstitute, Jena,

Germany

Fig. 12: FLIM-FRET measurements of EGFP/RFP with cw bleaching of the acceptor molecule. Data courtesy of Philippe Bastiaens, MaxPlanck Institute of Molecular Physiology, Dortmund, Germany

3) Afterpulsing removal with FluorescenceLifetime Correlation Spectroscopy (FLCS)

Fluorescence Lifetime Correlation Spectroscopy(FLCS) is basically a fusion of Time-CorrelatedSingle Photon Counting and Fluorescence Corre-lation Spectroscopy. This method uses picosecondpulsed excitation in conjunction with time-resolvedfluorescence detection for separating different FCScontributions. The core of the method is a statist-ical separation of different photon contributionsdue to their picosecond timing information, per-formed on a single photon level.

One possible application of FLCS is the removal ofdetector afterpulsing artefacts common to SPADdetectors. These contributions are visible at shortlag times and thus often prevent the correctdetermination of e.g. triplet lifetimes or dyeconcentration. One possible solution in order tocircumvent these influences is to split the fluo-

rescence signal equally onto two detectors and toperform a cross-correlation. However, with FLCSthe different temporal properties of the afterpulsingevents in comparison to fluorescence photonsallows to statistically filter the acquired data beforethe correlation calculation. The result of such acalculation is free from afterpulsing effects withoutthe need for a second detection channel.

An example of a corresponding FLCS mea-surements of a 10 nM solution of ATTO 488 in wa-ter is shown in Fig. 14. The data is taken with anupgraded Nikon C1si and the measurement timewas 60 s. The left image in Fig. 14 shows a clas-sical autocorrelation function for FCS. Clearly theinfluence of detector afterpulsing is visible at earlylag times and an evaluation of e.g. the concen-tration of the dye via the fitting of the data is pro-blematic. If the same data is evaluated usingFLCS, the influence of detector afterpulsing is re-moved as can be seen in the right graph of Fig. 14.

Further reading

[1] Ryder, A.G., Power, S., Glynn, T.J., Morrison, J.J., Proc. SPIE, Vol. 4259, p. 102-109, (2001)

[2] Gerritsen, H.C., Sanders, R., Draaijer, A., Levin, Y.K., J. Fluorescence, Vol. 7, p. 11-16 (1997)

[3] Lakowicz, J. R., Principles of Fluorescence Spectroscopy, Third Edition, Springer Science+Business Media, New York, ISBN 0-387-31278-1 (1999)

[4] Herman, B., Fluorescence Microscopy, Second edition, Springer Verlag, New York, (1998)

[5] Kapusta P., Wahl M., Benda A., Hof M., Enderlein J., J. Fluo., Vol.17, p.043-048 (2007)

[6] Wahl M., Rahn H.-J., Gregor I., Erdmann R., Enderlein J., Rev.Sci.Inst., Vol.78, 033106 (2007)

Fig. 14: Example of FLCS to remove the effect of detector afterpulsing. Classical autocorrelation-FCS (left) clearly shows the influenceof afterpulsing, whereas FLCS (right) removes this artefact.

Copyright of this document belongs to PicoQuant GmbH. No parts of it may be reproduced, translated or transferred to third partieswithout written permission of PicoQuant GmbH. All Information given here is reliable to our best knowledge. However, no responsibilityis assumed for possible inaccuracies or ommisions. Specifications and external appearences are subject to change without notice.

PicoQuant GmbHRudower Chaussee 29 (IGZ)12489 BerlinGermany

Phone +49-(0)30-6392-6560Fax +49-(0)30-6392-6561Email [email protected] http://www.picoquant.com

Afterpulsing

Documents

Compact FLIM and FCS Upgrade Kit for Laser Scanning ...Bülter, Felix Koberling, Rainer Erdmann, PicoQuant GmbH Introduction Already for more than 20 years, confocal laser scanning