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Modular FLIM Systems for Olympus FV1000 Laser Scanning

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1. _ Repeat ginny i E StopT Time CEN CFD SYNC P E Collection 3 Range Limit Low Freq Div Ovfl Control Stop 1 000 gt 50 00E 9 T 2000 34 1 Step CFD a E Fig 38 Access to system and control parameters from main panel Detector Control Parameters The detectors the shutters and the picosecond diode lasers of confocal one photon FLIM sys tems are controlled via the DCC 100 card 7 see Fig 22 The software panel of the DCC 100 is shown in Fig 39 Power Supply Connector 11 Connector2 Connector 3 Cosin Cooling of or a a tay on PMC 100 and Detectors H 5v E 5V E v Sly H7422P 40 E 5v ov E 5v 2 Detectors 100 E b7 100 Amps Laser Power E b6 or OVLD sips Gain Detector 2 EJ b4 Ej b3 0 a 079 5 Volts Er 0 b0 a al 90 68 a 500 DigOut Gain HV Cooler OVLD Gain Detector 1 Shutter 2 Enable Outputs Settings from auto set Outputs disabled Shutter 1 Enable out Fig 39 Detector control panel Depending on the system configuration the DCC 100 controls one or two detectors one de tector and one picosecond diode laser and one or two shutters see Fig 25 to Fig 29 and Fig 16 to Fig 20 Enable Outputs Button After the start of the DCC 100 software the outputs of the DCC 100 are disabled Please note that this is a safety function It avoids unintentionally switching on the output voltage of a high voltage power
2. Detector PMC 100 Detector PMC 100 Shutter Shutter Scan Clocks Photodiode SPC Board Power Suppl amp Control Power Supply Kocer amp Control 10 dB Fig 27 Dual PMC 100 NDD FLIM system Multiphoton NDD FLIM Systems al The gain control for the PMC 100 detectors is provided via connector 1 and connector 3 of the DCC 100 card The output pulses of the detectors are fed into two inputs of an HRT 41 router The routing signal generated by the router and the combined single photon pulses of the detectors are connected to into the routing input and the CFD input of the SPC 830 mod ule Important In order to maintain correct timing between the routing signals and the photon pulses the CFD cable must not be longer than the routing cable Dual R3809U system A beamsplitter is connected to the side port of the microscope via a shutter assembly R3809U detectors are attached to both outputs of the beamsplitter As in the setups described above the detectors and shutters are controlled via the DCC 100 detector controller card and the p box The high voltage for both R3809U detectors is provided by an FuG HCN 14 3500 A power supply The high voltage is controlled by a signal from connector 1 of the DCC 100 card The output pulses of the detectors are amplified in HFAH 26 01 preamplifiers and fed into two inputs of an HRT 41 router The routing signal generated by the router and the combined sing
3. High Performance Photon Counting User Handbook Modular FLIM Systems for Olympus FV1000 Laser Scanning Microscopes Becker amp Hickl GmbH l J II Becker amp Hickl GmbH Nahmitzer Damm 30 12277 Berlin Germany Tel 49 30 787 56 32 FAX 49 30 787 57 34 http www becker hickl com email info becker hickl com Ist Edition May 2006 This handbook is subject to copyright However reproduction of small portions of the mate rial in scientific papers or other non commercial publications is considered fair use under the copyright law It is requested that a complete citation be included in the publication If you require confirmation please feel free to contact Becker amp Hickl Contents Contents Introdu 11 OMS srsi beiwanesidsissauiek or a na a babandessnagauiehsanaadevdacuaushbabasias nasadcheusaadsviadeaushtabesunsss l Woy Use ELIM eenaa o a e a NR 1 Requirements tora FLIM Technigtt iici aare e E E anieicacuanlaies 4 Multi Dimensional Time Correlated Single Photon Counting cccccsssssseseseeeeeeeececeeeeeeeeeeeeeeeeaaas 6 One Photon FLIM versus Multi Photon PIM iscscss coceies ieasasat Seaeasenbaasdyatieaneish yeedaguieceoeeh inesdsiesceeb abies 8 The FV 1000 PLIM Systems cssiinvexsnnian el veninldddnniaaeniiasas ieee eet 1 Data AC CUTS INION Sy Ste ierics sf ni Maayan snnache tun tanhenaeh E A 1 One Photon Confocal FLIM Detection Systems cccccessssseececeeeeeeeeeeea
4. interacting time ps Fig 101 Fluorescence decay components in FRET systems Double exponential decay analysis delivers the lifetimes 7 and Tret and the intensity factors a and b of the two decay components From these parameters can be derived the true FRET efficiency Erret the ratio of the distance and the Forster radius r ro and the ratio of the num ber of interacting and non interacting donor molecules Nfret No E fret 1 T fet To OIS F per Co T jer or inf a 1 E fret N fet No alb Applications 91 Fig 102 shows the fluorescence decay curves in a selected spot array of 7 adjacent pixels selected by the blue crosshair of Fig 100 100000 120 alls E ps 77 2 5 1000 l am A 10 ae dps 0003 10 10000 Fig 102 Fluorescence decay curve in a selected spot of Fig 100 The decay profile is clearly double exponential The fluorescence decay is indeed double exponential with a fast lifetime component Tret of 712 ps and a slow lifetime component To of 1 84 ns Fig 103 shows the result of a double exponential analysis of the data The left image shows the ratio of the lifetimes of the non interacting and interacting donor fractions To Teret repre sented by t2 and t2 in the lifetime images The coulour of the image is thus directly related to the distance of the donor and acceptor molecules An image of the ratio of interacting and non interacting donor molecules Nfe No repre
5. Al Fig 99 Lifetime distribution window left and definitions in Preferences right M Analyse within color range Special Commands For more detailed analysis some additional commands were added to the menu which are available by clicking on the icons in the task bar Calculate Lock The Calculate gt Lock command should be used if individual data points are analysed F after the calculation of the decay matrix In the locked mode no recalculation is per formed if the user moves the blue crosshair to different pixels of the image This guarantees that the colour coding from the last calculation of the decay matrix is consistent with the fitting parameters given for the selected pixel Special Commands 87 Calculate Unlock After changing any parameter which may effect the result of the fitting process the matrix have to be recalculated If the calculation was locked by a previous action please use the unlock command in order to take the changes into effect Polygon definition The function allows you to define a polygon in which the parameter distribution is calculated Switch on Show Mask Polygon in Preferences when you use the func tion IRF definition Defines the curve currently displayed in the decay window as an IRF Conditions Store After the selection of the fit model the time range for the fitting procedure the region fi of interest in the image etc it is possi
6. Power Supply DCC 100 Board Power amp Control Single Mode Fibre into Laser Sync Output Microscope Power Supply amp Control PML SPEC Assembly Fibre SPC Board bundle Scan Clocks i from Microscope Fibre Fig 20 One photon multi wavelength FLIM system The PML 16C detector of the PML SPEC assembly obtains its gain control signal and power supply voltages from connector 3 of the DCC 100 detector controller The connecting cable also feeds the overload signal of the PML 16C into the DCC 100 In case of overload the DCC 100 shuts down the gain and the 12V power supply of the PML 16C One Photon Confocal FLIM Detection Systems 21 The routing signals and the photon pulses of the PML 16C are connected to the lower 15 pin connector and the CFD input of the SPC 830 module respectively Important In order to maintain correct timing between the routing signals and the photon pulses the CFD cable must not be longer than the routing cable 22 The FV 1000 FLIM Systems Multiphoton NDD FLIM Systems The bh FLIM systems work both for microscopes with one photon excitation and with for microscopes with multiphoton It should however be noted here that multiphoton excitation is covered by patents owned by Zeiss 43 and Leica 57 The patent situation and thus the availability of the multiphoton technique for the FV 1000 depends on the country you are liv ing in Synchronisation with t
7. files depending on the selected file format If you want to over write an existing file you can select it in the File Name field A history of previously saved files is available by clicking on the button File Info After selecting the file text can be written into the Author Company and Contents fields Both for SPC data and SPC setup the file information is saved in the file The file informa tion helps considerably to later identify a particular measurement among a large number of data files We therefore strongly recommend to spend a few seconds on typing in a reasonable file information If you have selected an existing file the file information contained in it is displayed in the File info window If you want to overwrite this file you can edit the existing file information Loading Setup and Measurement Data 45 Selecting the data to be saved Under What to Save the options All used data sets Only measured data sets or Selected data blocks are available The default setting is All used data sets which saves all valid data available in the memory of the SPC modules These can be measured data calculated data or data loaded from another file Except for special cases see 22 we recommend to use the All used data sets option All used data sets Y All used data sets Only measured data sets Selected data blocks Loading Setup and
8. lished FLIM results the count rates and the amount of photobleaching are rarely mentioned so that only a few examples can be given here CFP YFP FRET images of HEK cells presented in 16 21 were recorded at 50 10 s CFP YFP FRET in Caenorhabditis Elegans 35 was recorded at lt 10 s Two photon autofluorescence of skin delivered about 60 10 s 70 71 72 These count rates are by factor of 40 to 100 lower than the maximum recorded count rate of the bh TCSPC devices used It should be expected that much higher count rates are ob tained from stained tissue Nevertheless imaging of the pH in skin tissue by BCECF was per formed at an average rate of only 2 10 s 58 although the frequency domain technique used was capable of processing much higher rates Acquisition Time of FLIM From a single exponential fluorescence decay recorded under ideal conditions the fluores cence lifetime can theoretically be obtained with a relative standard deviation or coefficient of variation CV of CV lt t T l 1 y N Details of FLIM Data Acquisition 71 with N number of recorded photons 4 52 65 In other words the fluorescence lifetime can be obtained with the same accuracy as the inten sity Measurement under ideal condition means the decay function is recorded with an instrument response function of negligible width into a large number of time channels within a time interval considerably longer than the d
9. 1 e have reached the correct operating point of the detector Of course the optimum CFD threshold depends on the detector gain or the high voltage of the PMT In other words a decrease in the CFD threshold and an increase in the detector gain or high voltage are largely equivalent Higher detector gain usually yields shorter IFR and lower differential nonlinearity However afterpulsing possibly even instability and early detector overload may limit the gain that can practically be used Details are described in 22 Typical values of DCC gain and CFD threshold for FLIM systems are Detector DCC Gain Voltage CFD Threshold R3809U 50 52 80 to 86 2 9 to 3 0 kV 80 mV PMC 100 0 80 to 90 80 mV PMC 100 1 80 to 90 100 mV PMC 100 20 90 to 100 40 mV H7422P 40 80 to 90 100 mV PML 16C 0 85 to 95 100 mV PML 16C 1 90 to 100 50 mV Please note No CFD adjustments have to be done for single photon avalanche photodiode SPAD detectors These detectors have their own internal discriminators and deliver a pulse of defined amplitude and duration for each photon Once these pulses are detected by the CFD of the SPC module further adjustment has negligible influence on the efficiency and the IRF shape When you have found a reasonable set of CFD and TAC parameters do not forget to save the results CFD Zero Cross The CFD zero cross level defines the point at the at which the timing reference is taken from the input pulses We recomm
10. 2 3 24 25 26 Interactions between the PCK and NKKkB signalling pathways have been investigated in 80 FRET between GFP and RFP and FRET cascades from GFP via Cy3 into Cy5 are demonstrated in 1 and 88 The agglutination of red blood cells by monoclonal antibodies was studied using FRET between Alexa 488 and Dil 93 Interaction of the neuronal PDZ protein PSD 95 with the potassium channels and SHP 1 target interac tion were studied in 30 32 It has also been shown that FRET can be used to monitor con formational changes of proteins in cells by FLIM FRET 38 76 A detailed description of a TCSPC FLIM FRET system is given in 46 The system is used for FRET between ECPF EYFP and FM1 43 FM4 64 in cultured neurones FRET between ECFP and EYFP in plant cells was demonstrated in 34 FRET measurements in plant cells are difficult because of the strong autofluorescence of the plant tissue The au thors show that two photon excitation can be used to keep the autofluorescence signal at a tolerable level It has been attempted to obtain additional FRET information from the acceptor emission measured simultaneously with the donor emission in a dual detector TCSPC system 61 An alb image of the acceptor decay should display the ratio of the acceptor emission excited via FRET and excited directly The integral intensity of FRET excited acceptor emission could then be used as a second way to obtain the net energy transfer rate Unfortunate
11. Because the delays are not adjusted yet the curve may be shifted or cut off at the left or the right end Change the cable length in the SYNC path in order to centre the decay curve in the display window A number of cables of different length and adapters to connect the cables is delivered with the SPC module More cable length in the SYNC path shifts the curve left less cable length shifts it right see Fig 63 left and middle For single detector systems you may also change the length of the cable at the CFD input The effect is opposite to that of a change in the SYNC cable length The result of the delay adjustment should be a curve as shown in Fig 63 right The decay curve should fit well into the recorded time window Make sure that a few time channels are left before the rise of the fluorescence signal The data acquisition software needs these chan nels to correct for signal background Fig 63 Effect of the cable length in the SYNC path Left SYNC cable too short Middle SYNC cable too long Right Cable length correct When adjusting the cable length of a diode laser based system check that the position of the decay curve remains constant for different laser repetition rates If the curve shifts by more than a few 100 ps the cable length is wrong and you are stopping the time measurement with the wrong laser period Typically 8 m cable are required in the
12. Display Parameters If the image is stretched or compressed the line and pixel clock dividers are not set according to the ratio of the pixel numbers in the mi croscope scan and the SPC recording The settings under More Parameters allow for correc tion in all these cases Please see 22 Once you have recorded a reasonable image save it into a file You may use the File Format option Setup to save only the parameters or Data to save the recorded data together with the setup Creating Setup Files for Different FLIM Configurations Starting from the setup values obtained in the previous steps you may now create setup data for other images sizes for using both detectors of a dual detector system or for multi wavelength imaging Please see Fig 36 and Fig 37 page 37 or refer to 22 We recommend to put the settings into the list of Predefined Setups see page 46 FLIM Measurements 65 FLIM Measurements Steps of a FLIM Measurement 1 Turn on the computer that contains the FLIM system If the FLIM system is contained in a laptop based Simple Tau system turn on the extension box first and then the computer 2 Start the FV1000 software on the microscope computer and start the SPCM and DCC software on the FLIM computer Place the DCC panel in a convenient area of screen 3 One photon systems Turn on the picosecond diode laser Multiphoton systems Make sure that the Ti Sa laser is activated in the
13. SPIE 5139 88 96 2003 H Kaneko I Putzier S Frings U B Kaupp and Th Gensch Chloride Accumulation in Mammalian Olfactory Sensory Neurons J Neurosci 24 36 7931 7938 2004 L Kelbauskas W Dietel Internalization of aggregated photosensitizers by tumor cells Subcellular time resolved fluorescence spectroscopy on derivates of pyropheophorbide a ethers and chlorin e6 under femtosecond one and two photon excitation Photochem Photobiol 76 686 694 2002 J P Knemeyer N Marm M Sauer Probes for detection of specific DNA sequences at the single molecule level Anal Chem 72 3717 3724 2002 M K llner J Wolfrum How many photons are necessary for fluorescence lifetime measurements Phys Chem Lett 200 199 204 1992 K K nig P T C So W W Mantulin B J Tromberg E Gratton Two Photon excited lifetime imaging of autofluorescence in cells during UVA and NIR photostress J M1 crosc 183 197 204 1996 K K nig Multiphoton microscopy in life sciences J Microsc 200 83 104 2000 K K nig Laser tweezers and multiphoton microscopes on life science Histochem Cell Biol 114 79 92 2000 K Konig Cellular Response to Laser Radiation in Fluorescence Microscopes in A Periasamy Methods in Cellular Imaging Oxford University Press 236 254 2001 K Konig U Wollina I Riemann C Peuckert K J Halbhuber H Konrad P Fischer V Fuenfstueck T W Fischer P Elsner Optical tomography of human skin
14. Scan Sync Out is an imaging mode that actively controls a scanner It is implemented mainly for scanning with piezo driven scan stages However the Scan Sync Out mode can also be used to record and accumulate fast triggered sequences of decay curves With a large number of accumulation cycles sequences as fast as a few microseconds per curve can be recorded The mode can be used to record photochemical quenching transients in chlorophyll 21 pos sibly also effects of electro physiological stimulation in neurones Please see 22 for details The FIFO mode differs from all the other modes in that it does not build up any photon dis tribution Instead the FIFO mode stores information about each individual photon The in formation stored is the time in the laser period the time since the start of the experiment and if several detectors are used the number of the detector that detected the photon The FIFO mode is the key to the application of single molecule techniques It can be used to record FCS and FCCS curves in combination with fluorescence decay curves photon counting histo grams or BIFL burst integrated fluorescence lifetime data 19 21 23 49 89 These tech niques require parking the beam with extremely low beam jitter and a detection volume on the order of a femtoliter For details please see see 22 typical results are described in 21 23 Steps and Cycles The memory of the SPC modules provides memory space for
15. The operation mode selection panel of the bh TCSPC modules is shown in GgeationMed ii the figure right The mode used for FLIM recording is Scan Sync In zl SeanSynein Other modes may be used for special application of a FLIM system ae pe The Single mode records one decay curve for each of the detectors con ii nected to the SPC 830 module It can be used for fluorescence decay FLEXT measurement with the laser beam being parked in a pixel of interest If eee used in combination with scanning it delivers an average decay curve Over J Scan Sync ln Scan Syne Cut the complete scan area seen evr The Oscilloscope mode performs a repetitive measurement and displays the results like an oscilloscope The mode is an excellent tool for setup maintenance and alignment purpose The F t T mode runs a time controlled sequence of Single measurements It is useful for photobleaching experiments experiments of photodynamic therapy and for recording chloro phyll transients The F t EXT mode is implemented for recording sequences of curves in connection with ex ternal experiment control The Fi T and Fi EXT modes record time gated intensity curves The Scan Sync In mode is the mode for recording FLIM data in a scanning microscope The SPC 830 module records a photon distribution over the time in the laser period and over the coordinates of the scan area see Fig 6 page 7 Data Acquisition 35
16. may save computation time since only the area within the crosshairs is processed Analysing Fluorescence Lifetime Images 79 Instrument Response Function Measuring the IRF requires to record the laser pulses through the normal optical path of the detection system In a microscope this is difficult because the same beam path is used for the excitation and emission light The laser light 1s scattered at a number of optical surfaces inside the microscope so that a clean reflection from the sample plane is usually not obtained In multi photon microscopes second harmonic generation or Hyper Raman scattering can be used 54 but even then IRF recording is not easy The SPCImage data analysis software therefore estimates an IRF from the recorded data The instrument response function can either be calculated automatically of on command The deci sion is made under Options Preferences see Fig 88 Calculation on command is started by clicking into Calculation System Response Calculate Mask Conditions Decay Matrix F2 Calculate Instrumental Response automatically Improve Matix F3 Decay Window System Response F6 Lock Unlock W Display Instrumental Response Fig 88 Definition of the calculation and the display of the IRF left and calculation of the IRF on command right Unless you tend to forget to start the IRF calculation we recommend not to use the automatic calculation This gives you the chance to s
17. not solve the general problem of the FRET techniques that the total decrease of the donor fluorescence intensity or fluorescence lifetime depends both on the distance of donor and acceptor and the fraction of interacting donor molecules In the simplest case a fraction of the donor molecules may not be linked to their targets or not all of the acceptor targets may be labelled with an acceptor This can happen especially in specimens with conventional antibody labelling 73 But even if the labelling is complete by far not all of the labelled proteins in a cell are interacting and the fraction of interacting protein pairs varies throughout the cell TCSPC FLIM solves this problem by double exponential lifetime analysis The resulting do nor decay functions can be approximated by a double exponential model with a slow lifetime component from the non interacting unquenched and a fast component from the interacting quenched donor molecules If the labelling is complete as it can be expected if the cell is expressing fusion proteins of the GFPs the decay components directly represent the fractions of interacting and non interacting proteins The composition of the donor decay function is illustrated in Fig 101 non interacting Intensity free donor proteins interacting but no acceptor A interacting Donor proteins not interacting OM NO interacting f t ae Utfrety be t TO e t T0 Poin proteina non interacting labelled and
18. scan Fig 81 middle The pixels of the resulting lifetime data array contain the results of the fitting procedure Fig 81 right hm wwwe awe Fig 81 Analysis of FLIM data Left Raw data pixels contain decay curves Middle Fit procedure delivers lifetimes and amplitudes for individual pixels Right Lifetime data Pixels contain results of fit procedure T1 T2 a1 a2 Data photon numbers in time channels In the simplest case the decay curves of the individual pixels can be characterised by a single exponential model The result of the fitting procedure is then a single fluorescence lifetime Lifetime images created from such data use the number of photons per pixel the intensity as brightness and the fluorescence lifetime as colour see Fig 82 76 Data Analysis Intensity Lifetime Intensity Lifetime Fig 82 Combination of the intensity and the lifetime information Left Intensity image brightness represents total number of photons per pixel Middle Pure lifetime image colour represents fluorescence lifetime Right Combined image brightness represents number of photons colour represents fluorescence lifetime Mouse kid ney sample stained with Alexa Fluor 488 wheat germ agglutinin and Alexa Fluor 568 phalloidin Two photon excitation at 860 nm Fluorescence decay curves of biological samples often contain fluorescence components of several fluorescing species The decay
19. 33 34 35 36 II References W Becker Adcanced time correlated single photon counting techniques Springer Ber lin Heidelberg New York 2005 W Becker The bh TCSPC handbook Becker amp Hickl GmbH 2005 www becker hickl com W Becker A Bergmann E Haustein Z Petrasek P Schwille C Biskup L Kelbauskas K Benndorf N Kl cker T Anhut I Riemann K K nig Fluorescence lifetime images and correlation spectra obtained by multi dimensional TCSPC Micr Res Tech 69 186 195 2006 O Berezovska P Ramdya J Skoch M S Wolfe B J Bacskai B T Hyman Amyloid precursor protein associates with a nicastrin dependent docking site on the presenilin 1 y secretase complex in cells demonstrated by fluorescence lifetime imaging J Neurosci 23 4560 4566 2003 O Berezovska B J Bacskai B T Hyman Monitoring Proteins in Intact Cells Science of Aging Knowledge Environment SAGE KE 14 2003 O Berezovska A Lleo L D Herl M P Frosch E A Stern B J Bacskai B T Hyman Familial Alzheimer s disease presenilin 1 mutations cause alterations in the conforma tion of prenesilin and interactions with amyloid precursor protein J Neurosci 25 3009 3017 2005 K M Berland P T C So E Gratton Two photon fluorescence correlation spectros copy Method and application to the intracellular environment Biophys J 68 694 701 1995 T Bernas M Zarebski R R Cook J W Dobrucki Minimizing photobl
20. 78 The maximum continuous count rate of the R3809U is about 1 10 photons per second Although this is enough for the majority of applications it should be noted that the R3809U is not a solution to fast acquisition FLIM PMC 100 The PMC 100 detector 22 is the standard detector for all bh FLIM systems It delivers an IFR of 150 ps FWHM Lifetimes down to about 200 ps are resolved The PMC 100 features excellent timing stability at high count rates It can therefore be used up to the highest count rates applicable with the bh TCSPC boards without noticeable degradation in the IRF 22 Typical applications are pH imaging oxygen imaging and ion concentration measurements The R3809U can be used at count rates up to 3 MHz 21 22 However the output current at this count rate is beyond the permissible maximum specified by Hamamatsu In FLIM applications high count rates normally oc cur only in a few pixels of the image Immediate damage under these conditions appears unlikely nevertheless life cannot be guaranteed 24 The FV 1000 FLIM Systems via fluorescence quenching The PMC 100 works well also for single exponential FRET measurements i e experiments that do not require separation of the interacting and non interaction donor fraction In double exponential FRET measurements the longer IRF makes the data analysis more difficult and less accurate than for the R3809U Autofluorescence im aging is possible as well though with some
21. 98 Control of the lifetime image display Left Colour panel right Intensity panel Please note that the colour and intensity parameters influence only the display of the data ob tained in the previous lifetime analysis Thus you can change the parameters without re analysing the FLIM data Mode Continuous defines a continuous colour scale over the specified parameter range Discrete allows you to assign red green and blue colour to specified ranges of the pa rameter displayed Range Parameter range of continuous colour scale The colours are assigned to the pa rameter selected under Coding of Direction The direction of the colour scale can be red green blue or blue green red Coding of The colours of the image can be assigned to any of the decay parameters ob tained in a single double or triple exponential fit Moreover arithmetic expressions of two parameters can be defined 86 Data Analysis Brightness and Contrast The sliders change the brightness and contrast of the intensity image and the lifetime image Scaling Assigns the brightness scale to the photon number defined in the photons per pixel field Autoscaling sets the scale automatically Reverse X scale Reverse Y scale The parameters reverse the images in X and Y Interpolate pixels For images of small pixel numbers the colour and brightness is interpo lated between the individual pixels We reco
22. Biomed Opt 8 472 478 2003 M Minsky Memoir on inventing the confocal microscope Scanning 10 128 138 1988 80 8l 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 References 99 P E Morton T C Ng S A Roberts B Vojnovic S M Ameer Beg Time resolved mul tiphoton imaging of the interaction between the PKC and NFKB signalling pathways Proc SPIE 5139 216 222 2003 J D Miller Y Chen E Gratton Resolving Heterogeneity on the single molecular level with the photon counting histogram Biophys J 78 474 586 2000 D V O Connor D Phillips Time correlated single photon counting Academic Press London 1984 G H Patterson D W Piston Photobleaching in two photon excitation microscopy Bio phys J 78 2159 2162 2000 R J Paul H Schneckenburger Oxygen concentration and the oxidation reduction state of yeast Determination of free bound NADH and flavins by time resolved spectros copy Naturwissenschaften 83 32 35 1996 J Pawley ed Handbook of biological confocal microscopy 2nd edn Plenum Press New York 1995 A Periasamy Methods in Cellular Imaging Oxford University Press Oxford New York 2001 M Peter S M Ameer Beg Imaging molecular interactions by multiphoton FLIM Biol ogy of the Cell 96 231 236 2004 M Peter S M Ameer Beg M K Y Hughes M D Keppler S Prag M Marsh B Vo jnovic T Ng Multip
23. FCS x Pa FIFO frame lerigtri A Tinne Div Decay i x Correlation ro Time ms E 10 000 Algorithm OD ON ma Pp oe r iain ADC sE i fore of Ej None Fe Zoorr c ist F Each start of sequence cycle CE Edd only Interval Fiouting chari i FIDA Bi Tho fd 3 1 000 Interval Time per Macro Time Clock 3 SYNC freq nn mn on AKKA Dead Time Corperications Fig 78 System parameters for combined decay and FCS measurement Main Parameters Display Start Interrupt Stop Exit SPC 830 FIFO Mode fes setup3 sdt 1 1 i 1 i 1 10 100 1000 10000 i Time ps le fal E a i a Device state Measurement Fifo Overflow 2 Trace Statistics E Repeat ae E E aea A0 SYNC Fifo Usage I Stopt Can n j S 00E 7 Oviic j ic roa Displaying data from file e bhtspc manualidatattypical setups fcs setup3 sdt all Gunite 1e4 1 03E 4 noe 13 0 0 E Er Dig Zom Hi g E f ast SER Display aE E Range aa a ZC Level EE E Freq Div Disp Page E 1 SYNC CFD TAC ADC J 100 Sf 5004 9 J sa FL MeasPage Sf 4 Fig 79 Main panel configuration for combined decay and FCS measurement For cross FCS with two detectors suppression of Raman scattering by gated FCS FCS with continuous lasers and other details please see 22 For FCS measurements it is important that the beam be parked in the spot of interest and that the detection volume is confined to less than a few femtolite
24. FV1000 control panel Turn the laser power down to a few For older Ti Sapphire lasers make sure that the laser is correctly pulsing The SYNC rate displayed by the SPCM software should correspond to the nominal repetition rate of the laser 4 Load a setup file a predefined setup or a data file of a successful measurement into the SPCM software Please see Loading Setup and Measurement Data page 45 5 Open the Acquisition Setting panel of the FV1000 software see Fig 69 Define an image size of 512 x 512 pixels You may use other frame sizes in this case make sure that the scan control parameters of the SPCM software correspond to that frame size Record an image of the sample in the FV1000 software Adjust the focus and zoom into the region of interest see also Fig 77 page 72 AcquisitionSetting x Mode lt OANE 2 a lt lt Fast 10 0us Pixel Slow gt gt lt gt AutoHv Z P 10 0us L 6 240ms F 3 264s 3 264s Size Aspect Ratio 1 1 C arbitrary x a gt 512 by 5 12 Area al Rotation oom Fa al l 0 0 o PanxX a 22 um o al Pany E zl 25um 0 3 0 1 E p g Fig 69 Acquisition settings in the FV 1000 control software 6 Open the Light Path amp Dyes panel of the FV1000 software Configure the beam path as shown in Fig 70 This sends the fluorescence light to the FLIM detector M LightPath
25. Fi Emeen Se Se es Se ee a a ee Sle Mo l Fix 5 4 I Scatter ons E Offset 14 4 Fix Multiexponential Decay Components E alii 338 5 tilps 6887 I Fix a2 35 3 me t2 ps 25334 4 I Fix a J25 5 t3lps 25734 I Fix Shit 10 HT Fi Scatter jong Tl Fix Offset i4 Fix Fig 96 To bottom Fitting a decay profile with one two and three exponential components 84 Data Analysis Fitting the data with only one component Fig 96 top delivers a large vy and clearly visible systematic variation in the residuals With some experience you may also spot systematic deviations between the decay data and the red curve calculated by the fitting procedure Fitting the data with two components delivers a perfect y and removes any systematic devia tions in the residuals This is an indication that the fit cannot be improved by adding more exponential components A attempt to fit the data with three components Fig 96 bottom indeed does not deliver any improvement Instead it delivers a third lifetime component almost identical with the second one This is a clear indication that the double exponential model is the right one Deriving an Apparent Lifetime from a Multi Exponential Decay Sometimes the fluorescence decay functions are clearly multi exponential but only the life time of a single exponential approximation
26. H 5V Hj 5v EN ETE O n 3 Cooling 5j 5v Ej 5V E 5v 2 mi sav may mOn ME 5v Hj 5 H 5y Curr Imt 100 E o7 100 Amps Mm sy o 5 E 5v 2 E b6 OVLD Bj o5 OVID aw z F100 H o7 100 Amps a b6 Laser Power Ub Detector Gain J O b3 OVLD af bs 1 01 4 dba 2 z Z b2 H n3 Reset i oo p2 Volts 49 32 a 94 00 a 5 00 o b1 0 o ain HV DigOut Gain HV Cooler 47 62 ai bo 85 71 5 00 Shutter Settings from auto set Enable Outputs Gain HV DigOut Gain HV Cooler Outputs disabled ettings from auto se Enable output Disable outputs Fig 71 DCC 100 detector and diode laser control panel Left Control of laser power shutter and detector gain 8 9 Right Overload shutdown of detector Check the count rates displayed in the lower left part of the SPCM software panel see Fig 72 Adjust the laser power to obtain a CFD and TAC count rate between 50 10 and 1 10 photons per second Higher count rates yield better signal to noise ratio for a given acquisi tion time However the high excitation power needed may cause excessive photobleaching If you have photobleaching you see it by a slow decrease of the CFD count rate In that case reduce the laser power In one photon systems you may compensate for the loss in count rate by increasing the pinhole size eo I Rate IFh si ler 1e6 i 7 56E 7 eas z z3 SED iea 8 E El SIRS Ene 1e3 1 45E 5 100 5 E E B
27. Im aging in cells using confocal fluorescence lifetime imaging microscopy Analytical Bio chemistry 227 302 308 1995 M Snippe J W Borst R Goldbach R Kormelik The use of fluorescence microscopy to visualise homotypic interactions of tomato spotted wilt virus nucleocapsid protein in living cells J Vir Meth 125 12 15 2005 P T C So K H Kim L Hsu P Kaplan T Hacewicz C Y Dong U Greuter N Schlumpf C Buehler Two photon microscopy of tissues in M A Mycek B W Pogue eds Handbook of Biomedical Fluorescence Marcel Dekker New York Basel 181 208 2003 P Theer M T Hasan W Denk Multi photon imaging using a Ti sapphire regenerative amplifier Proc SPIE 5139 1 6 2003 100 100 101 102 103 104 References U K Tirlapur K Konig Targeted transfection by femtosecond laser Nature 418 290 291 2002 B Treanor P M P Lanigan K Suhling T Schreiber I Munro M A A Neil D Phil lips D M Davis P M W French Imaging fluorescence lifetime heterogeneity applied to GFP tagged MHC protein at an immunological synapse J Microsc 217 36 43 2005 M A M J Van Zandvoort C J de Grauw H C Gerritsen J L V Broers M G A Eg brink F C S Ramaekers D W Slaaf Discrimination of DNA and RNA in cells by a vi tal fluorescent probe Lifetime imaging of SYTO13 in healthy and apoptotic cells Cy tometry 47 226 232 2002 J G White W B Amos M Fordham An evaluation of conf
28. Mode with the parameter settings shown in Fig 58 E SPC 830 2D Traces Attributes i TT amamnamnnmnmnh i Oh OP 24 22 OP a a ae ar ar lale ale lae ae EEEEEEE EEEEE EE nd Fig 58 Parameter settings recommended for startup System Parameters left Display Parameters upper right Trace Parameters lower right The parameters are shown for an SPC 830 module The panels for other modules may differ slightly but are not significantly different When you have set the parameters close the Sys tem Parameters panel but keep the display and trace parameter panels open Resize and place the panels as shown in Fig 59 Make sure that the Rate button in the main panel is switched on bi SPC 830 Oo Le E k E z E z E E E a v al E al E i I ar ar Laid Lah Laid Laid Lair 2 3 4 5 al 7 a IONII T A B EBEEEESE i Fig 59 Recommended configuration of the SPCM main panel for the first tests in the Oscilloscope mode Getting Started First Light 57 After having set the parameters save the setup in a file see Fig 45 page 44 Use the File Format option setup define a file name and a destination folder and click on Save The DCC 100 panel with the detector and in case of the one R E n b DCC 100 Simulation i 0 x photon system laser control parameters is shown in Fig 60 amp ee Pull down the detector gain regula
29. Pixel Clock 0 Delay ne 1 Routing chan s 1 Routing chan r Meas page Positive x Syne Polarity Positive Y Sync Polarity Positive a x Line Predivider Pixel Clock Divider Left border Upper border External Pixel Clock Data Format ADC Resolution Memon Offset Dither Range Count Increment Page Control Scan piels 4 Scan pisels r Pixel Clock Polarity Data Acquisition 37 Data Format ADC Resolution 256 Memor Offset oon Dither Range 1 16 Count Increment a 10 Fage Control Scan pimels Scan pels r x Syne Polarity S Positive Y Syne Polarity S Positive Pixel Clock Polarity Positive Line Predivider x Pixel Clock Divider e S Left border Upper border Pixel Clock External Delay nz T Routing chan s 1 Routing chan r 1 Meas page 1 l 256 256 Data Format ADC Resolution E E4 Memory Offset ooo Dither Range 1 16 Count Increment E 10 Fage Control Delay nz 0 Routing chan 5 2 Routing chan r Meas page 1 Scan pixels 3 256 Scan pixels Y E 256 More Parameters x Syne Polarity Positive Y Syne Polarity Positive Pixel Clock Polarity S Positive Line Predivider Pixel Clock Divider Left border Upper border Pixel Clock External Fig 36 Data format page control and scan parameters for a number of typical FLIM data formats 128 x 128 pixels 1 dete
30. SPCM Software ccccccsssssssssseeeeceeeeeeeeeeeeeaseeeeseseeeeeeeeeeeeeeeeeaaeeeeeseseeeeeeess 41 CmMIMe Depla yoia aa cinedGncw sich etuonatae Mabey cantata vowels uconawaln patie Ghats comanaiss 41 Display Parameters 23 severtucenas ane e aew tices eee eee ae ace am eee das 41 NWO W TINE EY al Saa batt cence E E sae baatstwntias vaca tet wamtoaeh A a E 42 Saving Setup and Measurement Data cccccccccccccccsceeesssesseseecceeeeeeeeeeeeaaeaessseeseseeeeeeeeeeeeeeeaaaaaagagags 44 Loading Setup and Measurement Daladier a a a as 45 Predenned SetipS alsa a a a E ET N 46 SY S O ni D PE A E TE E A T EE E E be caamn gaa caatentas tee 49 One Photon Confocal Microscopes esessssssssssessseeereesessssssssssseeerererrsssssssssssseecrereressssssssssseseeeeero 49 meilin the Diode Ease omoran iara a a rr N A Peano Go matantane 49 Alonmentof Che Laser enrennisrine nna e a avons a a aees 49 Mista the Dere OS a E E a chee taeue eo tatan sors 50 Multiphoton Microscopes x acoiisscecncwcastcaue sae eensandavecotntentebeud eased dar sandaasnatieeadasaxheeasigeitacsaniaetieediwsedcae 53 Installing Non Descanned DeteCtOrs cronicari E O r EEAO Ea 53 Installation of the SPCand DCC modules ereere hed ied Re ie a ee 53 SO E A E T EE TA teat ae E T TN J3 Pard A Steet tee eet tcc in E EAE E E E E O E 54 Gene stared First Leitast 56 System Setup Paramete S ovina ai p a E r E A es 56 Adjustine the CFO Parameter Sorena arn a A A aa a a a A A 60 R
31. The combination is possible because the photon count rate is considerably smaller than the repetition rate of the laser 21 see Fig 5 The timing pulse is sent through the time measurement block of the TCSPC device This block determines the time of the pho ton in the laser pulse period Along with the timing pulse the router delivers the number n of the PMT in which a photon was detected The detector number is stored in the channel register of the TCSPC device The detector number is used later to store the photons of the individual detectors in different memory blocks The routing technique can be used with several individual PMTs and with multi anode PMTs 22 The scanning interface of the TCSPC module receives the scan clock signals pixel line and frame clock from the scanning unit of the microscope For each photon the TCSPC module determines the location within the scanning area x and y The photon times t the detector channel number n and the spatial coordinates x and y are used to address a memory in which the detection events are accumulated Thus in the memory the distribution of the pho ton density over x y t and n builds up The result can be interpreted as a number of data sets for the individual detectors each containing a large number of images for consecutive times in 8 Introduction the fluorescence decay The individual data sets can also be considered images with a fluores cence decay curve stored
32. amp Dyes 7 zaoii 1TD LaserUnit1 458 E i a r Trans 515 a Lamp a 543 B f TDi Vv 7 2V 633 Specimen Objective Lens ae To FLIM FL Imaging detector DM DM DM UOO Glass Glass EN N Ny 422 _ 492 555 _655 nm nm nm nm BAG5OIF 4 CHS1 CHS2 CH3 80 37 591 SPD ExcitationDM oof um Dye Iv Dye Dye cA ax yY ECFP BD Fig 70 Beam path configuration for confocal FLIM detection 66 FLIM Measurements 7 Enable the outputs of the DCC 100 detector controller see Fig 71 Make sure that the op erating voltage buttons 12V 5V 5V are switched on For R38009U detectors or NDD detectors of multiphoton microscopes open the shutters of the detectors Activate the de tector or the detectors For the standard PMC 100 detector set the detector gain to 90 to 100 and turn on the cooling of the detector For the PMC 100 0 or 1 detectors moderate cooling 0 5 to 0 8A 5V is sufficient For the R3809U MCP PMT set a DCC gain of 88 corresponding to a voltage of 3000 V In extreme cases it may happen that the detec tor is overloaded and shuts down In that case reduce the laser power or the pinhole size Re activate the detector the detector by clicking on the Reset button and re open the shut ter SS iol Connector 1 Connector 2 Connector 3 Cooling Main Parameters Exit On gav may Wav On Connector 1 Connector 2 Connector 3
33. available Detectors for Confocal FLIM Detectors for the FV1000 confocal FLIM systems are shown in Fig 15 All PMT based detec tors are controlled via the DCC 100 detector controller card see Fig 22 Fig 15 Detectors for confocal FLIM systems Upper row left to right R3809U MCP PMT PMC 100 H7422P 40 id 100 50 Bottom PML SPEC multi wavelength setup R3809U The R3809U detector 55 is the detector for ultimate time resolution Its instrument response function IRF has a width of 30 ps FWHM 22 However if the R3809U is used in one photon excitation systems the pulse width of the diode laser limits the resolution The effec tive IRF is thus about 60 ps In other words the gain in time resolution over a PMC 100 de tector is only a factor of 2 5 Nevertheless the R3809U is highly recommendable for FRET experiments Quantitative FRET requires the decay components of the interacting and non interacting donor fractions to be separated This requires a detector response that is not only short but also free of tails or bumps after the main peak FRET measurements benefit there fore considerably from the clean response of the R3809U Other applications of the R3809U are autofluorescence measurements Autofluorescence of cells and tissue contains several fluorescence components with lifetimes as short as 100 ps The fast response of the R3809U makes it easier to resolve the complex decay profiles 16 The FV1000 FLIM Sys
34. compromise in resolution for the shortest lifetime components H7422P 40 The H7422P 40 detector 56 has an exceptionally high quantum efficiency It is recom mended for applications that require ultimate sensitivity In the wavelength range from 500 to 600 nm a sensitivity improvement of a factor of 2 to 3 over the R3809U and the PMC 100 is obtained The IRF width of the H7422P 40 is 250 to 350 ps The large IRF width makes the H7422P 40 less useful for FRET and autofluorescence imaging MW FLIM The MW FLIM detection system 6 detects the fluorescence simultaneously in 16 wavelength channels The fluorescence light leaving the back aperture of the microscope objective lens is projected at the input of a fibre bundle The fibre bundle transfers this light into the input slit of a poly chromator Fig 24 left shows the input of the polychromator with the holder of the fibre bundle The fibre bundle is shown in the middle Fig 24 right shows the shutter assembly The shutter assembly contains also the projection lens and a laser blocking filter The polychromator splits the light spectrally and projects the spectrum on the photocathode of a PML 16 detector This detector contains a 16 channel PMT and the associated routing elec tronics Thus 16 lifetime images are simultaneously recorded in a single SPC 810 TCSPC module Typical applications of the MW FLIM detector are FRET experiments 33 and auto fluorescence imaging 22 SS
35. curves are then multi exponential see Fig 4 page 4 The fitting procedure delivers lifetimes and amplitude coefficients for the individual exponen tial components For example a double exponential decay function is described by N th a e Caa 1 a a l 2 Fitting the decay curves with this model delivers the lifetimes 7 and 7 and the amplitudes a OF az It is of course impossible to display three independent decay parameters and the fluorescence intensity simultaneously in one image The analysis software therefore provides a number of options for the display of the parameters You may assign either 7 a Or a2 to the colour of the display or you may use ratios such as 7 T or a a2 An example is given in Fig 83 It shows the same cell as Fig 82 but analysed by a double exponential model Fig 83 Double exponential decay analysis Left to right Images showing the decay time of the fast lifetime component tl the decay time of the slow lifetime component t2 and the ratio of the intensity coefficients al a2 of the lifetime components Mouse kidney sample stained with Alexa Fluor 488 wheat germ agglutinin and Alexa Fluor 568 phalloidin Two photon excitation at 860 nm Frequently used functions of the SPCImage FLIM data analysis software are described in the following paragraphs A comprehensive description is given in 8 Analysing Fluorescence Lifetime Images 71 Analysing Fluorescence Lifetime Image
36. id 100 50 detector is overload proof up to light intensities far above the daylight level An overload shutdown is therefore not necessary R3S09U Systems The connecting diagram of an R3809U FLIM system is shown in Fig 18 A BDL 405SMC or BDL 473 SMC diode laser is used for excitation The optical output of the laser is coupled to an input fibre of the FV1000 scan head The power of the laser is controlled via connector 1 of the DCC 100 detector controller To provide maximum safety against detector damage all R3909 systems use electro mechanical shutters in the beam path The shutter is controlled via connector 2 of the DCC 100 card The operating voltage of the R3809U detectors is provided by an FuG HCN 14 3500 A high voltage power supply The high voltage is controlled by a signal from connector 3 of the DCC card One Photon Confocal FLIM Detection Systems 19 Laser Power Control Power Supply 80 50 cont_ _ O Power amp Control BDL 405 SMC A mi Single Mode Sel Fibre into Laser Sync Output Microscope Rear Panel ae Polarity Wall Mounted Power Supply T7 12V DCC2 3CC1 3 VOEE Current P Box a gt 0 Shuti Shut2 Detect Analog ov HV a Mf oO J Digital FuG HCN 14 3500 High Voltage Power Supply Filter a 3 FLIM adapter OUT a SPC Board Scan HFAH 20 01 Clocks Preamplifier Fig 18 FLIM system with one R3809U detector The single photon pul
37. in each pixel The data acquisition runs at any scanning speed of the microscope As many frame scans as necessary to obtain an appropriate signal to noise ratio can be accumulated At the typical count rates obtained from living specimens the pixel rate is higher than the photon count rate This makes the recording process more or less random a photon is just stored in a memory location according to its time in the fluorescence decay its detector channel number and the location of the laser spot in the sample in the moment of detection It should be noted that multi dimensional TCSPC does not use any time gating wavelength scanning or detector multiplexing For count rates up to several MHz virtually all detected photons contribute to the result Consequently a near ideal signal to noise ratio for a given fluorescence intensity and acquisition time is obtained The time resolution is determined mainly by the transit time spread of the detectors With multichannel PMTs the width of the instrument response function IRF is about 30 ps full width at half maximum fwhm with the PMC 100 detectors an IRF about 150 ps fwhm is achieved The fluorescence decay curves in the individual pixels of the image are resolved into a large number typically 64 to 1024 time channels The large number of time channels in combina tion with the near ideal counting efficiency results in a near ideal standard deviation of the measured fluorescence lifetime
38. of the FV1000 use the connector system of Point Source Ltd UK The BDL 405SMC lasers for the FV1000 FLIM systems are therefore delivered with a Point Source compatible fibre adapter see Fig 12 right For alignment of the fibre coupler please see Fig 50 page 50 The BDL 405SMC is operated from a simple wall mounted 12 V power supply Fig 12 left A control box Fig 12 middle is inserted in the power and control cable to the laser This box contains the frequency selection switch and the mandatory safety key switch and emis sion indicator LEDs A timing synchronisation signal 1s provided at an SMA connector at the back of the laser The polarity of the pulses is positive therefore an A PPI pulse inverter has to be used in the SYNC line of the SPC 830 module Fig 12 BDL 405SMC picosecond diode laser Left Power supply Middle Control box Right Laser with beam profile corrector and coupler for single mode fibre The bh BDL lasers have an input for electronic control of the optical output power The con nector is located at the back of the control box In the bh FLIM systems the power control input is driven by a signal from the bh DCC 100 detector controller Moreover all bh BDL lasers have an input for fast on off control The on off input was im plemented for beam blanking during the line and frame flyback and for FRAP applications l Versions with 375 nm 440 nm and 473 nm wavelength are available However the a
39. or protonation states The fluorescence decay functions are therefore usually multi exponential A few typical decay profiles are shown in Fig 4 al a j JEE wple al JE Fig 4 Decay profiles of biological samples total length of time axis 10 ns logarithmic intensity scale Left to right CFP YFP FRET in HEK cell at emission wavelength of CFP autofluorescence of the stratum corneum of human skin plant tissue sample The curves show the fluorescence intensity versus the time in the fluorescence decay The total length of the time axis is 10 ns the intensity scale is logarithmic All curves were meas ured by TCSPC FLIM The blue dots are the photon numbers in the subsequent time channels of a selected image region the red curve is a fit by a double exponential or triple exponential model Even without data analysis it is clearly visible that the decay profiles are not single exponentials In the case of CFP YFP FRET the decay profile can be fitted well by a double exponential model The fit delivers a lifetime of 590 ps for the interacting donor component a lifetime of 2 4 ns for the non interacting donor component and the respective amplitudes of 51 and 49 The fluorescence decay of skin autofluorescence is fit by two components of 208 ps and 2 1 ns with amplitudes of 77 and 23 For the plant tissue even a double exponential fit is not
40. parameter but also yields direct information about the metabolic state and the microenvironment of the fluorophores 74 75 84 Moreover autofluorescence imaging has benefits in cases when the reaction of tissue to optical radiation is to be investigated such as tumor induction by UV irradiation Such experiments forbid the use of exogenous fluorophores because energy or electron transfer from the fluorophores to the proteins could induce additional photodamage The fluorescence decay profiles of tissue autofluorescence are multi exponential with decay components from about 100 ps to several ns The deviations from single exponential decay are substantial see Fig 105 Extracting meaningful decay parameters from the data therefore requires at least double exponential analysis 100000 aap 4 ips 083 3 AL 31 A H apos a 4 10000 1000 100 Fig 105 Typical decay curve of the autofluorescence of the stratum corneum of human skin two photon excita tion at 800 nm The samples used for tissue imaging are considerably thicker than samples containing single cells Often tissue imaging is even performed on living animals Therefore optical sectioning and a large penetration depth is required The method of choice is therefore two photon exci tation with non descanned detection Two photon autofluorescence lifetime image obtained with the bh FLIM technique are shown in 19 23 71 90 Two photon multi spectral auto fluore
41. pream plifier for R3809U and H7422P 40 detectors HRT 41 router for simultaneous operation of several detectors at one SPC 830 module The detectors of the bh FLIM systems are controlled via a DCC 100 detector controller card The card supplies the power to the detectors controls the shutters of NDD FLIM systems provides software control of the detector gain and shuts down the detectors in case of over load The DCC 100 card is shown in Fig 9 2 h Be E NMXD051280 Fig 9 DCC 100 detector controller The SPC 830 module and the DCC 100 detector shutter controller may be operated in a stan dard PC or the Simple Tau lap top based system 22 may be used The Simple Tau has the SPC 830 and the DCC 100 module readily installed in an extension box of the lap top computer This makes the data acquisition system compact and portable see Fig 10 12 The FV 1000 FLIM Systems Fig 10 Simple Tau compact data acquisition system The Simple Tau can be used for all configurations that require no more than one TCSPC module and one DCC 100 detector controller card and To increase the acquisition speed of TCSPC FLIM systems several detector and TCSPC chan nels can be operated in parallel 17 Systems with up to four channels are supported by the data acquisition software Any set of similar TCSPC FLIM modules SPC 830 SPC 140 or SPC 150 can be used However size and cost considerations normally lead to s
42. proves the quality of the lifetime parameter histogram see Lifetime Parameter Histo gram page 86 Pos X and Y shows the position of the blue crosshair in the intensity and the lifetime im age The field far right shows the fluorescence lifetime in the hot spot or in the pixel selected by the blue crosshair Default is the mean lifetime Tm defined by N N C Jar Xa 3 Ei For a single exponential decay Tn 1s identical with the fluorescence lifetime 7 Please note that 7 of a multi exponential fit is not identical with the lifetime obtained from a single exponential fit of the same data Binning in the Lifetime Analysis When an image is taken by a scanning microscope the point spread function of the micro scope lens is usually oversampled to obtain best spatial resolution As a rule of thumb the diameter of the central part of the Airy disc should be sampled by 5x5 pixels see Fig 90 left In practice even higher oversampling factors are often used unintentionally Under these con ditions lifetime data should be calculated from several binned pixels When the binning func tion of SPCImage is used the lifetime images are built up from the unbinned intensity pixels and the binned lifetime pixels This yields substantially improved lifetime accuracy without noticeable loss in spatial resolution Sampling artefacts are largely avoided by overlapping binning see Fig 90 right Analysing Fluorescence Life
43. rate from the donor to the acceptor decreases with the sixth power of the distance Therefore it is noticeable only at distances shorter than 10 nm 75 FRET is used as a tool to investigate protein protein interaction Different proteins are labelled with the donor 4 Introduction and the acceptor and FRET is used as an indicator of the binding state of these proteins Dis tances on the nm scale can be determined by measuring the FRET efficiency quantitatively Requirements to a FLIM Technique Time Resolution It is sometimes believed that FLIM in cells does not require a particularly high time resolu tion It is certainly correct that the fluorescence lifetimes of most fluorophores used in cell imaging are on the order of a few ns However the lifetime of autofluorescence components and of the quenched donor fraction in FRET experiments can be as short as 100 ps Lifetimes of dye aggregates in cells have been found as short as 50 ps 63 The lifetime of fluorophores bound to metallic nano particles 51 78 can be 100 ps and shorter A good FLIM technique should therefore by able to record lifetimes down to less than 100 ps Moreover it should be able to resolve the components of complex decay profiles see below Decay Profiles of Biological Samples The fluorophore populations in biological specimens are normally not homogeneous Several fluorophores may be present in the same pixel or one fluorophore may exist in different bind ing
44. satisfactory A triple exponential fit delivers 150 ps 313 ps and 1716 ps with amplitudes of 53 39 and 8 8 respectively There are certainly cases when fitting multi exponential decay profiles by a single exponential decay is feasible These may be pH imaging oxygen quenching experiments or ion concentra Requirements to a FLIM Technique 5 tion measurements Even in FRET experiments a single exponential fit is acceptable if only the locations of protein interaction in a cell not the quantitative values are required In most cases however describing the fluorescence of the sample by a single Fluorescence Lifetime means discarding useful information or even leads to wrong conclusions Efficiency Under ideal conditions the lifetime of a single exponential decay can be obtained from the recorded data with the same accuracy as the intensity 4 52 65 In both cases the standard deviation is VN with N being the number of photons in the pixel considered One might therefore conclude that FLIM does not set special requirements to the photon economy Un fortunately this is not so An intensity image with a standard deviation of 10 may look pleas ing and show the spatial structure of a sample very well However lifetime changes investi gated by FLIM may be on the order of a few Thus a standard deviation on the order of 1 is required Thus except for a few relatively trivial cases FLIM experiments need to record a la
45. that has a space the working dis tance between the lens and the cover slip see Fig 80 right This gap is filled with the im mersion fluid In the commonly used setup of Fig 80 left however the working distance is above the cover slip i e in the dye solution The dye is normally dissolved in water or a sol vent of similar index of refraction The setup of Fig 80 left therefore works only for a water immersion lens For an oil immersion lens the mismatch of the refractive index is so severe that a reasonably small focal volume is not obtained Consequently a TCSPC FIFO measure ment shows a fluorescence decay curve but no fluorescence correlation If you want to run an FCS test measurement with an oil immersion lens you may use the setup shown in Fig 80 right However because of the low fluorescence intensity of a highly diluted dye solution it is difficult to find the right focal plane It is therefore easier to use the setup shown in Fig 80 left and fill the working distance with cover slips use immersion oil be tween them This reduces the index mismatch and will enable you to recorded at least some correlation function Or you may use immersion oil as a test solution Low fluorescence grade immersion oil usually contains enough fluorescent contamination the fluorescence decay and FCS functions of which can be recorded Data Analysis 75 Data Analysis Introduction A measurement in the FLIM mode of the SPC 830 de
46. the SPCM software should dis play occasional dark pulses of the detector If the system does not record any photons at this stage make sure that the signal cables are connected correctly that the power supply cable to the preamplifier is connected and that the power supply and Enable Output buttons in the DCC panel are activated 58 System Setup First Detection of Fluorescence Signals Turn the detector gain down Put a sample into the microscope Use a sample that delivers fluorescence over a large image area not only from a few spots The sample should have high photostability therefore do not use living cells The most suitable specimens are stained tissue samples or plant tissue samples like the one shown in Fig 92 page 81 Focus into the sample by using an internal confocal detector of the FV1000 Use a 512 X 512 pixel scan For multiphoton NDD systems turn the laser power down to a few per cent Configure the NDD beam path as to send the light to the FLIM detector For confocal systems send the light to the fibre output of the scan head see Fig 70 page 65 If not already running start a con tinuous scan in the FV1000 The FV1000 sends laser light to the sample only when the scan is running If you have an R3809U detector or a multiphoton NDD system open the shutter bO at connector 2 of the DCC panel Turn up the gain of the detector or 1f you have several of one detector Watch the CFD count rate and the curv
47. the appropriate number of exponential components Select a characteristic spot of the sample Increase the binning factor until you see a clear fluorescence decay function Then change the number of components and check the displayed and the curve of the residuals A good fit is characterised by a y close to one and residuals showing no noticeable systematic variations Often you see a poor fit already by comparing the fitted curve red with the photon data blue in the decay window In most cases your decay curves will be fitted adequately by a single or double exponential model If you define more exponential components than needed to fit the data you normally obtain two components of almost identical lifetime or an extremely long lifetime component of very low amplitude An example is given in Fig 96 counts 11 2433 T2248 TMax 40 Bins f3 Thid 45 Pos 37 x 64 y 21 I0 Multiexponential Decay Components i aijo tips 2174 I Fix arjo tipso Fik axo HY tps 0 Fin 100000 10000 4 f 1000 4 100 0g AT Ne A crept A Naina aaa Shit 1 4 Fi 5 i I i f 1 I 1 l 1 I 1 l 1 i I Scatter 0 053 i Fix i i i i j i Offset 14 H Fix counts T E T2 248 TMax 41 Bin Em Thid aad 5 Pos rs 37 x 64 y tet 3 69 Multiexponential Decay 100000 tee at ee 0 ee ee a bae a Components 2 42 1 00 aps ___ ti ps 6943 4 I Fix a2 61 t2lps 25588 I Fix anp H psj F
48. wrong SYNC rate you may also attempt to change SYNC Threshold In general we discourage changing these parameters unless you are famil iar with the TCSPC hardware Please see 22 for details Data Format and Page Control ADC Resolution is the number of time channels in the decay curves recorded For FLIM re cording there is a conjunction between the available number of pixels and the available ADC resolution The maximum ADC resolution is 4096 the minimum one ADC resolution 1 does of course not yield any time resolution It can however be used to obtain life display of images for adjusting a sample the focus or the region of interest of the scan Unless you want to run a life display or a page stepping sequence we recommend to use the highest ADC reso lution available Scan Pixels X and Scan Pixels Y are the number of pixels of the FLIM Image Please note that several adjacent pixels of the FV 1000 scan may be binned into one pixel of the FLIM data see Fig 36 and Fig 37 Scan Pixels X and Scan Pixels Y can therefore be smaller than the pixel numbers of the scan defined in the FV1000 software There is a conjunction between the available number of pixels and the available ADC resolution Routing Channels X and Routing Channels Y are the number of detector channels Two rout ing channel parameters are provided to define a two dimensional detector array For FLIM systems set Routing Channels X to the number of detector
49. 0 Hb 100 he Hibs 0 E 2 ovo d 1e8 H M N Rate Pns wo oi J im E a ee ee ee eee Measurement ui ba 1e7 E Device state PTS 1e6 i SYNC Scan Clocks Z b2 volts a a e 0 00E 0 bi A P mn l ee tee 0 00E 0 Displaying data a 3m m mwm sm Gain 7 HV Dig Out Gain HV Cooler 1e3 0 00E 0 Dig Zoom _ Selings tom aw l 100 C Ty E ollection 4 Range al Limit Low i 8 1 1 a l il TAC CRE Syl SYNC CFD TAC ADC Me of 3 50 04E 9 39 22 4 Fig 32 SPCM main panel for a dual detector system Images of both channels displayed display parameters open detector control panel placed lower right Fig 33 shows the main panel configuration of a multi wavelength system Images in eight wavelength intervals are displayed the detector control panel is placed in the lower right cor ner of the screen Data Acquisition 33 Ibi SPC 830 E la Main Parameters Display Start nterupt al Exit W1 Scan Sync In Mode m32a bt W2 Scan Sync In Mode m32a b W3 Scan Sync In Mode m32a bt W4 Scan Sync In Mode m32a bt bt_ W7 Scan Sync In Mode m32a bF_ W8 Scan Sync In Mode m32a bF 51 x CC Main Parameters Exit is Eo E Jl Measurement le7 Device state 1e6 a ra SYNC Scan Clocks e e Taie 1 08E 5 Displaying data b 183 1 06E6 5 Dig Zom _ E 100 4 59E 4 D Gre coer 7 Collecti
50. 0 depends on the coun try you are living in This handbook should be considered a supplement to the bh TCSPC Handbook 22 and the user manual of the FV1000 Moreover we recommend 21 as supplementary literature Why Use FLIM Since their broad introduction in the early 90s confocal and two photon laser scanning micro scopes have initiated a breakthrough in biomedical imaging 44 67 85 103 The applicabil ity of multi photon excitation the optical sectioning capability and the superior contrast of these instruments make them an ideal choice for fluorescence imaging of biological samples However the fluorescence of organic molecules is not only characterised by the emission in tensity and the emission spectrum it has also a characteristic lifetime In the simplest case the fluorescence lifetime can be used to distinguish between different fluorophores Moreover the fluorescence lifetime is not only different for different fluorophores it also depends on the molecular environment of the fluorophore molecules Any interaction between an excited molecule and its environment in a predictable way changes the fluorescence lifetime Since the lifetime does not depend on the concentration of the fluorophore fluorescence lifetime imaging is a direct approach to the mapping of cell parameters like pH ion concentrations or oxygen saturation protein interaction and other effects on the molecular scale The Fluorescence Decay Function When
51. 00 are provided at connector 3 of the DCC 100 The cooling circuit of the DCC 100 is configured for one detector see 7 DCC 100 manual The detector pulses are fed directly into the CFD input of an SPC 830 TCSPC FLIM module The timing reference signal SYNC signal of the TCSPC module is obtained from the laser Most Ti Sapphire lasers deliver positive synchronisation pulses Therefore an A PPI pulse inverter is inserted in the SYNC line The scan clock signals from the User IO connector of the microscope controller are connected to the upper sub D connector of the SPC 830 module Wall Mounted Power Suppl DCC 100 Board mi Scan Clocks Laser Sync Output Detector from PMC 100 Microscope ooh Photodiode Power Suppl Output N4 amp Control Fig 25 NDD FLIM system with one PMC 100 detector Single R3S09U system A system with a single R3809U detector is shown in Fig 26 The R3809U is connected to the side port of the microscope via its shutter assembly The shutter is controlled via connector 2 of the DCC 100 card The operating voltage of the R3809U detectors is provided by an FuG HCN 14 3500 A high voltage power supply The high voltage is controlled by a signal from connector 1 of the DCC card The single photon pulses delivered by the R3809U are amplified by an HFAH 26 preamplifier and connected into the CFD input of the SPC 830 module The timing reference signal SYNC signal of the TC
52. 01 P S Dittrich P Schwille Photobleaching and stabilization of fluorophores used for single molecule analysis with one and two photon excitation Appl Phys B 73 829 837 2001 R R Duncan A Bergmann M A Cousin D K Apps M J Shipston Multi dimensional time correlated single photon counting TCSPC fluorescence lifetime 1m aging microscopy FLIM to detect FRET in cells J Microsc 215 1 12 2004 C Eggeling S Berger L Brand J R Fries J Schaffer A Volkmer C A Seidel Data registration and selective single molecule analysis using multi parameter fluorescence detection J Biotechnol 86 163 2001 K W Eliceiri C H Fan G E Lyons J G White Analysis of histology specimens using lifetime multiphoton microscopy J Biomed Opt 8 376 380 2003 S Felekyan R K hnemuth V Kudryavtsev C Sandhagen W Becker C A M Seidel Full correlation from picoseconds to seconds by time resolved and time correlated sin gle photon detection Rev Sci Instrum 76 083104 2005 Th Forster Zwischenmolekulare Energiewanderung und Fluoreszenz Ann Phys Serie 6 2 55 75 1948 C D Geddes H Cao I Gryczynski J Fang J R Lakowicz Metal enhanced fluores cence MEF due to silver colloids on a planar surface Potential applications of indo cyanine green to in vivo imaging J Phys Chem A 107 3443 3449 2003 H C Gerritsen M A H Asselbergs A V Agronskaia W G J H M van Sark Fluores cence lifetime imag
53. 2 44 67 68 85 86 98 99 The problem of deep tissue imaging is however that the fluorescence photons are strongly scattered on their way out of the sample and emerge from a relatively large area of the sample surface Moreover the surface is not in the focus of the objective lens No matter which optical system is used it is impossible to focus this light into a pinhole The solution to deep tissue imaging is direct or non descanned detection The fluorescence light is diverted by a dichroic mirror directly behind the microscope lens and transferred into a detector see Fig 7 right Thus photons leaving the sample from a large area are collected and fed into the detector Unfortunately the large detection area of a direct detection system has also a drawback It in creases the detection efficiency for scattered photons and for photons of background light similarly Any direct detection system with TCSPC has therefore to be operated in absolute darkness The FV 1000 FLIM Systems 11 The FV1000 FLIM Systems The bh FLIM systems are modular 22 Consequently a large number of system configura tions are available The excitation light source can be a picosecond diode laser attached to a confocal microscope or the Ti Sapphire laser of a multiphoton microscope The detection light path may the a fibre output from the scan head of the FV1000 a direct output form the FV1000 scan head or a non descanned direct output of an F
54. 4 99996e 07 Max 4 99996e 07 CFO rate Min 263736 blas 391936 TAC rate Min 249552 Mas 364450 goe rate Min 239008 Max 350784 Accumulate E Collection Time 60 0000 4 Stop on time al Stop on Overflow 5 CFO Limit Low 49 02 m CFD ZC Level 5 29 m m Fig 47 Block info window of the load panel Load Options Under What to Load the options All data blocks amp setup Selected data blocks without setup or Setup only are available The default setting is All data blocks amp setup which loads the complete information from a previously saved data file Except for special cases see below we recommend to use the All data blocks amp setup option Loading Files from older Software Versions Older software versions may contain less system parameters than newer ones Therefore load ing older files into a newer software or vice versa can cause warnings of missing or un known parameters To load the file click on the Continue button until the file is loaded Un known parameters are ignored and missing parameters are replaced with default values To avoid further problems with such a file we recommend to save it in the current software ver sion Use option All used data blocks see Saving Setup and Measurement Data Predefined Setups Setups of frequently used system configurations can be added to a list of predefined setups Changing between these setu
55. B2 for maximum image intensity Do not turn the screws by more than 1 2 turn Once the manipulator is totally misaligned you need to go through the complete alignment procedure Beam Profile Fibre Manipulator Corrector Alignment Screws m B2 Input Collimator of Fibre Alignment Screws Alignment Tool Fig 49 Front end of the BDL 405SM laser Beam profile corrector fibre manipulator with alignment screws input adapter of the single mode fibre and alignment tool The complete alignment procedure is illustrated in Fig 50 For the first steps an alignment tool is required see Fig 49 The tool is a tube which has a pinhole in the optical axis 50 System Setup Step 1 adjust A1 and B1 insert alignment tool this side first Step 5 adjust A1 and A2 turn screws in same direction insert alignment tool this side first Step 3 Repeat step 1 Step 6 adjust B1 and B2 turn screws in same direction adjust A1 and B1 insert alignment tool this side first Fig 50 Steps of the alignment procedure To align the fibre coupler proceed as follows Step 1 Insert the alignment tool as indicated in Fig 50 and adjust Al and B1 for maximum throughput Step 2 Reverse the alignment tool and adjust A2 and B2 for maximum throughput Step 3 Repeat step 1 After step 3 the optical axis of the fibre manipulator is aligned with the axis of the laser beam Step 4 Insert the fibre Adjust Al and B1 for maximum image intensit
56. C board Display of Images in the SPCM Software 4 Display of Images in the SPCM Software Online Display Images can be displayed at the end of a FLIM measurement or on line in regular intervals within the measurement On line display is achieved by defining a large number of measure ment cycles and activating accumulate and display each cycles in the system parameters of the SPCM software see Fig 42 Fig 42 System control parameters for on line display The setting shown runs 100 cycles of the specified collection time accumulates the data and displays the accumulated data after each cycle The display of the data itself is controlled by the Display Parameters and Window Parameters see paragraphs below Display Parameters The data recorded by the FLIM system are multi dimensional There is a two dimensional array of pixels and each pixel contains a decay curve 1 e photon numbers in a large number of time channels within the laser period Each pixel may even contain such data for a number of different wavelength channels or for different times from the start of the experiment The SPCM software is able to display such data in various display modes The display is con trolled by the Display Parameters The display parameters recommended for FLIM acquisi tion with a single detector are shown in Fig 43 Tacs J me Solid Square Fig 43 Display parameter panel of the S
57. FD threshold and detector gain the count rate and thus the intensity of the image recorded becomes almost independent of the detector gain The setup of the detector gain and the CFD threshold is described under Adjusting the CFD Parameters page 60 Please see also 6 21 22 for details Please note The detector gain should not be used to control the intensity of the recording or to avoid overload shutdown at high intensity Reducing the detector gain results is loss of pho tons 1 e decrease of the signal to noise ratio of the lifetime images Power of Picosecond Diode Laser In the confocal one photon systems a picosecond diode laser is used for excitation The power of this laser is controlled via the slider under Connector 1 Shutters The shutters of the NDD detectors are controlled by the Dig Out buttons bO and b1 In case of overload shutdown the shutters close automatically After an overload shutdown the shut ters must be opened by clicking on the bO and b1 buttons Overload Shutdown If the light intensity at one or both detectors is too high the DCC 100 shuts down the gain and the 12 V supply voltage and closes the shutters In extreme cases this may happen at a gain far below the single photon detection level 1 e before the SPC module displays a CFD count rate The DCC 100 panel after an overload shutdown is shown in Fig 40 If an overload shut down has occurred first remove the source of the overl
58. GE l Details of FLIM Data Acquisition 69 Details of FLIM Data Acquisition Synchronisation and Count Rates The count rates are displayed in the status window of main panel of the SPCM data acquisi tion software see Fig 75 z i Rate Ph fs teig E Measurement g F Device state 18 i SYNC e Scan Clocks 9 95E 6 9 es 2 S Bs 14 0 E 22E e a a U u a Ese 1e3 2 Bs as 1 66E 5 Time TAC CFD a miend 10 0 E F Collection Collection Range _ Range I Limit Low e 7 BSE 4 TL 10 ee 100 0 E 50 04E 4 100 00 Fig 75 Count rates left status information upper middle SYNCCFD TAC ADC The SYNC rate is the repetition rate of the laser The Ti Sapphire laser of a multiphoton microscope has a repetition rate between 78 and 92 MHz If the SYNC rate differs from the nominal repetition rate of the laser either the mode locking is not running properly or the SYNC threshold is set too high or too low The repetition rate of the BDL SMC picosecond diode laser is 20 50 or 80 MHz With the correct SYNC threshold being set the SYNC rate should correspond to the selected laser repe tition rate An exception are lasers that are operated with beam blanking Beam blanking switches the laser off during the beam flyback The indicated SYNC rate is therefore lower than the laser repetition rate The CFD TAC and ADC rates i
59. If you don t have the installation disk available you can download the driver file from the bh web site Open www becker hickl com and click on Software On the Software page click on TCSPC Modules Operating Software for Windows 95 98 NT4 2k XP see Fig 56 Then click on BH Device Drivers and download the drivers After the software and the hardware have been installed start both the SPCM and the DCC application Both applications should come up with startup panels indicating Hardware Mode see Fig 57 Installation of the SPC and DCC modules 55 SPC 830 S N 3D00T3 B2 addr 1400 hex slot 10 Module 1 S N 810025 address 1440 hex Slot 11 Notfound Module 2 Not found Not found Module 3 Not found Notfound Module 4 Not found Hardware mode ___ Hardwaremode Fig 57 Initialisation panels for SPC left and DCC module right With the modules correctly installed the software comes up in the Hardware Mode When the initialisation panels have come up without any error messages click on OK to start the main panels of the SPCM and the DCC software 56 System Setup Getting Started First Light System Setup Parameters TCSPC Parameters If you have a minimum of experience with optical detectors it should be no problem for you to put a TCSPC FLIM system into operation For the first steps we recommend to use the SPC module in the Oscilloscope
60. M systems For details of the DCC 100 panel please see Fig 39 page 38 32 SPCM Software Main Parameters Display Start nter Exit W1 Scan Sync In Mode convalaria sdt AES Io x Main Parameters Exit Cooling ee div Maw mU Hi Hw Hia cumimi lav aj a may j2 i 10 dn 107 f Hb rae re si 101 EE 1e8 m O n r aj 2 Volts BELA Measurement _ D Hh n 5 CRE E E Device state Poss Mi sij d l 1e6 E E SYNC W Scan Clocks Gna TT oe 8 98E 7 s n gou Gant HY Cooler 1e5 E E Sine Se lings tom aux E e n 2 21E 6 isplaying data 7 aos ae 1e4 Disable culpus bast 1e3 E 0 E 1 09e 6 Dig Zom _ rf 7 100 8 1 08E 6 s Collection J R Range Limit Low Freq Div Disp P age E 1 10 S E E Time 4 TAC GEO a SYNG 3 Meas P SYNC CFD TAC ADC E 10 00 E 50 00E 9 3 78 43 E 1 eas r age 1 Fig 31 SPCM main panel recommended for a single detector system Display parameter panel open detector control panel placed in lower right corner The recommended main panel configuration for a dual detector system is shown in Fig 32 The intensity images of both detector channels are displayed simultaneously and the display parameters and the detector control panel are kept open lol Main Parameters Display Start erupt Stop Exit W2 Scan Sync In Mode hekO a sdt Hazy Hazy WE 2v Hev Hev Mss Curr Imt a sv sv W 5y 2 10
61. Measurement Data The Load menu is shown in Fig 46 It contains fields to select different file types to specify a file to display information about the file selected and to select different load options File format a SPC Data File namne autoskin g 33x edt ne File Info eibhispcmanualidata ty pical setups Select file F9 rj Title autoskine 3 3 Version 1 850 Fl Rewsion 10 bits ADC Load F10 i Return Esc Fig 46 Load panel File Format You can chose between SPC Data and SPC Setup The selection refers to different file types With SPC Data sdt files are loaded These files contain both measurement data and system parameters Thus the load operation restores the complete system state as it was in the moment when the file was saved If you chose SPC Setup set files are loaded These files contain the system parameters only The load operation sets the system parameters but the actual measurement data are not influ enced Note Measurements in the FIFO time tag mode deliver an spc file that contains the micro time the macro time and the detector channel for each individual photon These files are loaded by using the Convert routines see 22 File Name Select File The file to be loaded can is selected in File Name field Select File opens a dialog box that displays the available files These are sdt files or set files dep
62. Microscopes Installing the Diode Laser Upgrading one photon laser scanning microscopes for FLIM requires a laser input fibre for the BDL SMC diode laser to be available The BDL SMC lasers come in different wavelength versions 405 nm 440 nm and 473 nm Inside the FV1000 scan head the laser radiation is coupled into the beam path via dichroic mirrors Consequently the characteristics of the inter nal dichroics must correspond to the laser wavelength used Please make sure with your Olympus representative that the correct fibres and dichroics are installed Connecting the input fibre to the BDL diode laser is simple Just push the ferrule of the input fibre into the fibre manipulator of the laser The principle is shown in Fig 49 For electrical connection of the laser please refer to the wiring diagrams Fig 16 to Fig 20 Alignment of the Laser The front end of the laser is shown in Fig 49 The laser ends in a fibre manipulator that ac cepts the cylindrical input adapter of the single mode fibre The fibre manipulator has four adjustment screws Al A2 Bl and B2 Inside the manipulator the fibre input adapter is pressed against the alignment screws by a spring loaded counter bearing Thus the fibre adapter can both be shifted and tilted by turning the adjustment screws Under normal use e g after unplugging the fibre from the manipulator only fine adjustments are required It is then sufficient to adjust the front screws A2 and
63. PCM software A detailed description of the display parameters and their influence on the display of multi dimensional TCSPC data is given in 22 The parameters important for the standard configu rations of the bh FLIM systems are briefly described below Please note that the setups com ing with you FLIM system contain reasonable display parameters The following paragraphs should therefore be considered supplementary information for advanced users 42 SPCM Software Scale Section Linear Logarithmic Defines a linear or logarithmic intensity scale For displaying FLIM images we recommend linear Max Count Baseline Display range in photon counts for linear display Log Baseline Lower display threshold for logarithmic display Autoscale Sets Max count automatically according to maximum number of photons in the displayed data For FLIM we recommend Autoscale ON Reverse Scale Images can be reversed both horizontally and vertically 3D Display Section 3D Curves Colour Intensity OGL Plot Displays data as a sequence of curves as an im age or as a curved surface For FLIM use Colour Intensity Colour Bar Assigns colours to the photon numbers in the pixels of the Colour Intensity display Interpolate Colours Pixels Switch on for FLIM HiColour Colour assigned to pixels with photon numbers out of the display range Please note that HiColour only indicates that these pixels cannot be displ
64. S E Sao C e SS Fig 24 Details of the NDD MW FLIM system Left Polychromator with adapter for fibre bundle Middle Fibre bundle polychromator end and shutter end Right Shutter assembly Wiring Diagrams of Typical NDD FLIM Systems Typical wiring diagrams for the most frequently used NDD FLIM systems are described be low Please note that the diagrams given are just one way to connect the system components Often there are alternative wiring options which can be used to satisfy special requirements of a FLIM setup Please see 22 for details Single PMC 100 system The wiring diagram of a FLIM system with a single PMC 100 detector is shown in Fig 25 The PMC 100 is connected to the side port of the microscope via its shutter assembly Both the detector and the shutter are controlled via the DCC 100 detector controller card A shutter Multiphoton NDD FLIM Systems 25 control signal is provided at connector 2 of the DCC 100 The P box power saving box reduces the current in the shutter coils when the shutter is open and thus avoids heating of the detector by the shutter Moreover the box combines the overload signal of the PMC 100 de tector with an overload signal from a photodiode in front of the shutter This prevents uninten tional opening of the shutter when strong light is present e g when the microscope lamp is on The 12V power supply the cooling current and the gain control signal for the PMC 1
65. SPC module is obtained from the laser Normally this signal is posi tive therefore an A PPI pulse inverter is inserted in the SYNC line The scan clock signals of the microscope controller are connected to the upper sub D connector of the SPC 830 In case of detector overload the preamplifier delivers an overload signal A second overload signal comes from a photodiode in front of the shutter Both signals are combined in the p box and connected to the overload input of the DCC 100 26 The FV1000 FLIM Systems DCC 100 Board Wall Mounted Power Supply Shut1 Shut2 Detect Voltage Current Shutter R3809U Digital FuG HCN 14 3500 High Voltage Power Supply IGEN u C Laser Sync Output HFAH 20 01 SPC Board Preamplifier Fig 26 NDD FLIM system with one R3809U detector Dual PMC 100 System A beamsplitter is connected to the side port of the microscope via a shutter assembly PMC 100 detectors are attached to both outputs of the beamsplitter As in the setups described above the detectors and shutters are controlled via the DCC 100 detector controller card and the p box The wiring diagram is shown in Fig 27 Wall Mounted DCC 100 Board etter DCC1 Scan Clocks from Microscope mf WBE m Laser Sync Output
66. Ulfhake Fluorescence lifetime imaging measurements in confocal mi croscopy of neurons labeled with multiple fluorophores Nature Biotech 15 373 377 1997 I Bugiel K K nig H Wabnitz Investigations of cells by fluorescence laser scanning microscopy with subnanosecond time resolution Lasers in the Life Sciences 3 1 47 53 1989 38 39 40 4l 42 43 44 45 46 47 48 49 50 51 22 59 54 55 56 IN 58 References 97 V Calleja S Ameer Beg B Vojnovic R Woscholski J Downwards B Larijani Monitoring conformational changes of proteins in cells by fluorescence lifetime imaging microscopy Biochem J 372 33 40 2003 Y Chen J D M ller P T C So E Gratton The Photon Counting Histogram in Fluo rescence Fluctuation Spectroscopy Biophys J 77 553 567 1999 Y Chen A Periasamy Characterization of two photon excitation fluorescence lifetime imaging microscopy for protein localization Microsc Res Tech 63 72 80 2004 Y Chen A Periasamy Two photon FIM FRET microscopy for protein localization Proc SPIE 5323 431 439 2004 W Denk J H Strickler W W W Webb Two photon laser scanning fluorescence mi croscopy Science 248 73 76 1990 W Denk J Strickler W Webb Two photon laser scanning microscopy Patent WO 91 07651 1991 Diaspro A ed Confocal and two photon microscopy Foundations applications and advances Wiley Liss 20
67. V1000 that is operated as a multiphoton microscope The fluorescence light may be detected by one or two detectors 15 or in 16 channels of a multi wavelength system 6 14 21 29 33 95 Depending on the par ticular application different detectors may be used 22 Moreover the systems may use a sin gle TCSPC channel or several TCSPC channels may be used in parallel to increase the acqui sition speed 17 21 22 Data Acquisition System Most confocal and NDD FLIM systems use the bh SPC 830 module for data acquisition In systems that use a single bh DCC 100 detector or the bh MW FLIM setup the detector signal is connected directly to the stop input of the SPC 830 module The R3809U and H7422P 40 detectors 55 56 are connected via preamplifiers Dual detector systems require an HRT 41 router 5 22 to connect both detectors to a single SPC 830 module Alternatively two SPC 140 or SPC 150 modules can be used with the detectors connected directly to the TCSPC modules The components of the data acquisition system are shown in Fig 8 A babbbanaesanesd SHUNebnddddtde rsedensesensans coacsoenineneing AAHANRELtOLLAd dttasitarsiibid b hiddibtideissd dtesbidtdbididis bhdeibhtahibiaid ddbhstdietbita bHALIHIIDHIAA bhbtbbsdldbiati 3 leon au E 4 a _ manm i poli oe rm e Bis bes tom ork L J j Era kecu Fig 8 Components of the FLIM data acquisition systems Left to right SPC 830 TCSPC FLIM module
68. Y zs 10 SYMC CFO TAC ADC Fig 72 Count rate display of the SPCM software If not already running start a repetitive scan in the FV1000 see Fig 73 left Start the measurement in the SPCM software by clicking on the Start button If you have the soft ware configured for online display cycle and accumulate functions see Fig 42 you will see intermediate results in intervals of Collection Time Let the measurement run until you are satisfied by the obtained signal to noise ratio or until it stops by reaching a speci fied number of accumulation cycles The image you see is either the intensity accumulated over all time channels of the pixels or the intensity within a selected time gate see Display Parameters page 41 Keep in mind that reasonable FLIM in general requires a higher signal to noise ratio than pure intensity imaging Steps of a FLIM Measurement 67 gt 99 F Bleach start stop by Key m ImageAcquisitionControl Focus x2 lt P Focus x4 xY Repeat m ImageAcquisitionControl Stop SU CA a Enj EA A belt Auto Fig 73 Starting left and stopping right a repetitive scan in the FV 1000 10 If not stopped yet stop the measurement by clicking on the stop button of the SPCM software Stop the scan of the FV1000 see Fig 73 right Save the data by using the Save function of the SPCM software see page 44 Saving the data into a
69. Zoom m j o CH3 e or Dm Q _ cA mah CET aa 25um 0 Sa 6 Enable the DCC outputs Set the detector gain to 90 gt One photon FLIM Set the laser power to 50 For other EEE s gt 5 j Bate Phd Measurement ee ER ies I gt 2 Start the DCC 100 and the SPCM application parameters use the settings as shown below ome Oe eat te Connector 1 Connector 2 Connector 3 Sealne i a ee a E _ amp E E 12 E 12v E 12V Mon iat I H Lee i Erie oo TE L ERD Je svc aa l il i X O sv Ej 5V E sv l Curr Imt pcc 100 SPCM n E n 9 Stop the measurement Stop the scan in the FV1000 Save 100 H b7 ae ae the FLIM data by using Main Save Send the data to the b5 3 One photon FLIM Turn on the BDL 405 SM diode laser eee cl Detector Gain SPCImage lifetime data analysis Multiphoton FLIM Activate the Ti Sapphire laser a Main Parameters Display i a R N j BO 4 54 00 4 5 00 Load ali D ea see asel Load Predifined Setups z Shutter Settings from auto set Enable Outputs Save E Enstie gist Send Data to SPC_Image i Clear SPC mer or 7 Adjust the laser power to obtain a CFD count rate between a 5 10 and 5 10 High power yields higher count rate and Sleep Policy E eni i About 4 Load a setup file a predefined setup or a data file of a shorter acquisition t
70. a a a a a a a seats ieee ies 86 SpPecAlCOMMand Sernene n a a E N atten 86 APP HCINONS kena a E a digideadnseamoieinced ante dea dace 89 Measurement of Local Environment Parameters ccccccccccceceecceeceeseesseeseeeeeceeeeeeeeeeeeeeeaaaaaaeesnees 89 Fluorescence Resonance Energy Transfer FRET cccccccccccccccceessssseeeeeeeeeeeeeeeeeeeaeeaaesseseeeeeeeeeees 89 Autotlworescence Microscopy of J issue cavities ie ites EA E dena as 93 FRETS CNICE S sae dats tens ssiraa Sasa EE AE aacelediseealeacadeed as avaaien cian eaeedaaien ase 95 Introduction 1 Introduction This handbook describes the bh TCSPC FLIM systems for the Olympus Fluo View FV 1000 laser scanning microscopes The systems are based on bh s proprietary multi dimensional TCSPC technique The systems are modular and a large number of system configurations are available The fluorescence light may be detected by one two or four detectors or in 16 channels of a multi wavelength system Moreover the systems may use a single TCSPC channel or several TCSPC channels may be used in parallel to increase the acquisition speed The bh FLIM systems work both for microscopes with multiphoton excitation and for micro scopes with one photon excitation It should however be noted here that multiphoton excita tion is covered by patents owned by Zeiss 43 and Leica 57 The patent situation and thus the availability of the multiphoton technique for the Fluo View FV100
71. a large number of decay curves The memory may even hold data of several measurements containing a large number of decay curves each In particular there may be enough space to store the data of a large number of images with moderate numbers of pixels and time channels The individual memory blocks are termed pages By defining a number of steps greater than one a sequence of recordings can be defined that automatically steps through subsequent pages A measurement sequence may also be defined with several cycles The results of the indi vidual cycles can either be accumulated accumulate button or read from the device mem ory and automatically saved into subsequent data files autosave The functions can be used for on line display during a FLIM measurement see Fig 42 page 41 Moreover they can be used to record and save a number of subsequent recordings taken at the same sample Repeat Function By activating the repeat button the complete measurement cycle is repeated until it is stopped by user interaction The repeat function can be used to create a Life Mode of TCSPC imaging The image is de fined with a moderate number of scan pixels X and scan pixels Y and an ADC resolution of one With one ADC channel the recorded image is a pure intensity image of moderate data size This keeps the time for the data readout on a negligible level With a fast scan rate and a collection tim
72. a second DCC 100 card The output pulses of the detectors are fed into two inputs of an HRT 41 router The routing signal generated by the router and the combined single photon pulses of the detectors are con nected to into the routing input and the CFD input of the SPC 830 module mportant In or der to maintain correct timing between the routing signals and the photon pulses the CFD ca ble must not be longer than the routing cable 20 The FV1000 FLIM Systems Power Supply Laser Power Control lt cont S _ Single Mode Fibre into Microscope DCC 100 Board BDL 405 SM DCC 100 Board PMC 100 F Filter FLIM adapter Scan Clocks from Microscope j c i Routing SPC Board Filter Poua Beamsplitter Fig 19 Dual detector confocal FLIM system with PMC 100 detectors Multi Wavelength System The BDL 405SM laser is connected as described for the standard PMC 100 FLIM systems The PML SPEC multi wavelength detector can be coupled to the FV1000 scan head via a single optical fibre or via a fibre bundle The fibre bundle adapter is compatible with the di rect coupling adapters of the PMC 100 and R3809U systems The fibre bundle is therefore recommended for systems that are to be used both with the PML SPEC and the PMC 100 or R3809U It is recommended that a longpass laser blocking filter be inserted in the fibre or fibre bundle adapter of the PML SPEC Laser Power Control
73. ad Default List Nickname Scanning256x256x1 Load with data File name c spcpack spem defaultsetups spc 330 zeiss ism 510 scann w A File Info Title scanning256x256x1 4 Version 1 790M Revision 0 bits ADC Date 07 04 2003 Time 13 55 02 Author BH Company BH Contents LSM510 Scanning 256x256 pixels ADC Res 1 F rH Fig 48 Editing the list of predefined setups To create your own predefined setups first save a setup file of the system configuration you want to add the list Use the Save panel option setup as described under Save Then add the file to the setup list as described above You can also add an sdt file to the setup list The sdt file contains not only the system set tings but also measurement data You can define whether the file is loaded with or without the data by clicking on the load with data marker Please note that loading files with data can take a longer time than without especially for data recorded in the FLIM mode of the SPC 830 System Setup 49 System Setup For all FLIM systems described in this handbook in site setup by bh is available However experienced TCSPC users may prefer to do the setup on their own Moreover modifications in the microscope system may require the system to be modified or partly re installed There fore the following paragraphs describe the general steps of FLIM system setup One Photon Confocal
74. alculate Mask Conditions Calculating Decay Matrix Fiz F3 Progress Improve Matris Sistem Response F6 Unlock Fig 97 Calculation of the lifetime image Especially for double and triple exponential decay models large pixel numbers and for large binning factors the calculation can take several minutes If the calculation is extraordinarily slow we recommend Check the Sleep Policy of the SPCM software Click into Main Sleep Policy The set ting should be Never be put to sleep during measurement and Be put to sleep for a longer period outside measurement Check whether your image contains large dark areas Set the threshold appropriately to exclude these areas from calculation You may also set a Region of Interest to calculate lifetime data only in the area where you really need them Display of Lifetime Images The display of the lifetime image is controlled by the parameters in the Colour and Inten sity panel The panels are shown in Fig 98 Mode Intensity Image Lifetime Image f Continuous Discrete Brightness ol Brightness 63 Range Ranges B ERR i TI Min Max Min Max gone E3 Eye o E E o paman TTT Green 4 5 Direction AGB BGA Blue 6 4 Ta M Autoscaling Max Intensity 3710 photons per pixel Other Cod f oding o C Reverse x Scale MW Interpolate pixels Value alue t1 ps Reverse y Scale Time gating Fig
75. an organic dye is excited by light of appropriate wavelength a part of the light is ab sorbed A fraction of the absorbed light is converted into heat the rest 1s emitted at a wave length longer than the excitation wavelength The effect 1s known as fluorescence The fluo rescence light not only has a characteristic spectrum it 1s also emitted with a characteristic time constant the fluorescence lifetime or fluorescence decay time 75 The fluorescence lifetime becomes apparent if a sample is excited by light pulses shorter than a few nanosec onds see Fig 1 2 Introduction Intensity Fluorescence a t T 3 Time ns 4 Fig 1 Decay of the fluorescence red after excitation with a short light pulse blue In the simplest case the fluorescence lifetime can be used as an additional parameter to sepa rate or identify the emission of different fluorophores The application of the lifetime as a separation parameter is particularly useful to distinguish the autofluorescence components in tissues These components often have poorly defined fluorescence spectra but can be distin guished by their fluorescence lifetime 71 FLIM has also been used to verify the laser based transfection of cells with GFP 100 Fluorescence Quenching An excited molecule can also dissipate the absorbed energy by interaction with other mole cules The effect is called fluorescence quenching The fluorescence lifetime 7 becomes shorter than the normal
76. an that of single molecules on the other hand the fluorescence may be almost entirely quenched Aggregation is influenced by the local environment the associated lifetime changes can be used as a probe function Aggrega tion has also been used to observe the internalisation of dyes into cells 63 However in most applications aggregation is to be avoided by keeping the dye concentration at a reasonable level FRET A particularly efficient energy transfer process between an excited and a non excited molecule is fluorescence resonance energy transfer or FRET The effect was found by Theodor Forster in 1946 50 The effect is also called Forster resonance energy transfer or simply resonance energy transfer RET Fluorescence resonance energy transfer is an interaction of two mole cules in which the emission band of one molecule overlaps the absorption band of the other In this case the energy from the first dye the donor transfers immediately into the second one the acceptor The energy transfer itself does not involve any light emission and absorption FRET can result in an extremely efficient quenching of the donor fluorescence and conse quently in a considerable decrease of the donor lifetime see Fig 3 Intensity Intensity Donor Donor Acceptor Acceptor Absorption Emission Absorption Emission Exci tation Emission unquenched donor Wavelength Fig 3 Fluorescence Resonance Energy Transfer FRET The energy transfer
77. aser can be operated both in CW mode and picosecond pulsed mode In the pulsed mode the repetition rate can be switched between 20 MHz 50 MHz and 80 MHz The pulse width is about 60 ps Higher repetition rate gives higher average power However the fluorescence may not completely decay between the pulses Incomplete decay can in principle be taken into account in the data analysis but results in less than optimal standard deviation of the lifetime For most of the fluorophores including the GFPs 50 MHz repetition rate is the best compromise The BDL 405SMC laser 9 is shown in Fig 12 The laser head shown right contains the complete driving electronics Confocal laser scanning microscopes use single mode fibres to transfer the light of the excitation lasers into the scan head It is therefore essential to couple the light of the laser diode efficiently into the fibre For laser diodes this is difficult because the beam generated by a laser diode has a high divergence non circular shape and substantial astigmatism The BDL 405SMC laser therefore has a beam profile corrector that corrects both for beam shape and astigmatism As a result more than 60 of the laser power are coupled into the single mode fibre The power available from the single mode fibre of a 405 nm laser is about mW and 30 mW for pulsed and CW operation respectively The optical output of the laser is coupled to the 405 nm input fibre of the FV1000 The input fibres
78. ath Different microscopes may have different optical path lengths from the laser into the micro scope inside the microscope and from the sample to the detector Also the cable length from the detector and from the laser reference output to the TCSPC module may be different Light needs 1 ns to travel 30 cm in air and about 20 cm in an optical fibre electrical signals about 1 ns to travel 20 cm in a cable Thus signal transit time differences of several ns are common Therefore the decay curves recorded may appear shifted in time It may also happen that the time measurement in the SPC module see Fig 6 page 7 1s not stopped by the laser pulse the Getting Started First Light 59 photon originates from but by a pulse from a period earlier or later For a Ti Sapphire laser this is no problem because the laser pulse period is constant and highly stable The diode laser however can be switched between different pulse periods Stopping with the right pulse is therefore mandatory Because there is considerable transit time in the optical fibres and in optical path of the microscope about 8 m cable are required in the SYNC path of a one photon system To adjust the delay set SYNC Frequency Divider 1 and TAC gain 5 see Fig 62 With these settings only one signal period is recorded and stretched over the recording range Fig 62 CFD TAC and SYNC parameters for adjusting the signal delay Start a measurement in the oscilloscope mode
79. ator should turn green It may cycle between red and green if the frame rate is slower than one frame per second Put the mouse cursor on the indicator to see all three scan clocks If one or several clocks are missing check whether you have the right scan clock cable for your microscope and whether it is connected correctly At the SPC 730 and 830 modules it has to be connected to the upper sub D connector Recording Images 63 Measurement Repeat SYNC Scan Clocks EE Siop a Y g m e iw Craven YF o et tee et 96 0sec k TAC CFD Frame RYNC Range Limit Low Line feq Div Digg Page So 1 50 04E 9 lt foo 00 Pixel OFT Meas Page 30 74 Fig 68 Scan clock indicator If the scan clocks are there activate the detector Start the measurement in the SPCM soft ware With the system parameters shown in Fig 64 the measurement runs for 10 seconds stops displays an image and restarts The measurement continues until 100 such cycles are accumulated You can stop the recording at any time Please note that the autoscale function of the display routine is switched on The increase of image intensity is therefore not visible only the increase in signal to noise ratio The image you get may be shifted in x or the x or y axis may be reversed A shift is normal and easily corrected by changing left boarder in More Parameters Reversion of the x or y axis can be corrected in the
80. ayed within the display scale used The corresponding pixels are usually not saturated Display page A measurement can contain several smaller images recorded consecutively These images are contained in different pages of the device memory Selection of subsets of multidimensional data T Window TCSPC data can be multi dimensional data cubes but only one plane through the cube can be displayed at a time For FLIM measurements with a single detector images in selectable time windows are displayed The time window are defined in the Window Intervals see below Mode Defines the plane through the multidimensional data cube For FLIM images use f x y Window Intervals The display routines of the SPCM software display subsets of multi dimensional data arrays These can be images within specified time windows or ranges of detector channels decay curves along one coordinate within a spatial interval of the other coordinate time controlled sequences of waveforms within a range of detector channels or intensity values along a one dimensional scan within specified time windows The required window definitions are pro vided by the Window Intervals see Fig 44 a ao js io jon le joo ro PEREDE l afl lalla oO w io jon le joo ro Ab abla bia blabla blabla oo lw oD jon le joo ro PREEEEER i KIOKCINKCIDEIDKCIDKIOKIAKEN Fig 44 Window parameters One detector system therefore no routing window
81. ble to backup all these settings by using the Con ditions gt Store command This is especially useful if two images or traces 1 e acquired in different routing channels have to be analysed with exactly the same settings Conditions Load F Loads all settings which were saved with the Conditions Store command Applications 89 Applications Measurement of Local Environment Parameters Microscopic pH imaging can be achieved by staining tissue with a pH sensitive fluorescent probe 59 These probes usually have a protonated and a deprotonated form There is an equi librium between both forms that depends on the pH of the local environment If both forms have different fluorescence lifetimes the average lifetime is a direct indicator of the pH 75 96 A typical representative of the pH sensitive dyes is 2 7 bis 2 carboxyethyl 5 and 6 carboxyfluorescein BCECF 59 In aqueous solution the lifetimes of the protonated form and the deprotonated form have been found 2 75 ns and 3 90 ns respectively 58 In the pH range from 4 5 to 8 5 both forms exist and the fluorescence decay function is a mixture of both decay components Thus the lifetime of a single exponential fit can be used as an indica tor of the pH The measurement of the concentration of intracellular Cl in neurones by TCSPC FLIM was described in 62 MQAE was used as a fluorescent probe MQAE is quenched by CI and the concentration can be calculated from the li
82. cay profiles encountered in FRET and autofluorescence measurements have a much more favourable composition see Fig 4 page 4 Usually the lifetime components are 72 FLIM Measurements separated by a factor of 5 to 10 and the amplitude of the fast component is 50 to 90 Under such conditions double or even triple exponential analysis is feasible on no more than a few 1000 photons per pixel and very satisfactory results are obtained from 10 000 photon per pixel Nevertheless the required photon numbers are difficult to obtain especially for image sizes of 512 x 512 pixels and samples of poor photostability If the images size cannot be re duced the solution is binning of the lifetime data see Binning page 80 It should be noted here that long acquisition time is not a feature of FLIM in general or TCSPC FLIM in particular As shown above the lifetime accuracy is comparable to the accu racy of intensity images The difference is that typical TCSPC FLIM applications are aiming at effects not or not fully accessible by steady state imaging The lifetime changes caused by these effects are usually small Consequently the accuracy requirements and the expecta tions to FLIM results are higher than to steady state images Inexperienced FLIM users often stop the acquisition once they see an intensity image of satis factory signal to noise ratio The pitfall is however that an image with 10 intensity noise looks very pleasing while a l
83. channels used and Routing Chan nels Y 1 Please note If you have several detectors and set Routing Channels X 1 the sig nals of all detectors are combined into a single FLIM image A number of frequently used combinations of data format and page control parameters are shown in Fig 36 and Fig 37 For details please see 22 Scan Parameters The image acquisition in the SPC 830 module is synchronised with the scan in the microscope via the scan clock pulses see Fig 6 page 7 The details of the synchronisation are defined under More parameters More Parameters are hardware settings specific for the selected operation mode In the Scan Sync In FLIM mode the more parameters panel contains the scan control and pixel binning parameters see Fig 36 and Fig 37 You may change these parameters to create FLIM image sizes different from the sizes defined in the setup files deliv ered with the system It should however be noted that changing these parameters requires some knowledge about the scanning and data acquisition hardware Please refer to 22 and save a Setup file of the current settings before you make any changes Data Format Memor Offset Dither Range Count Increment Page Control Delay ns Routing chan FAouting chan r Meas page Scan pixels Scan pixels r x Syne Polarity Y Syne Polarity Pixel Clock Polarity Line Predivider Pixel Clack Divider Left border Upper border
84. complete set of system control and configuration parameters Thus all you have to do is to load the right setup files created during the setup of your FLIM system Moreover measurement data files contain the full set of hardware and software setup parameters You may therefore also load the data file of a successful measurement and run a new measurement with exactly the same parameter set Please see Loading Setup and Measurement Data page 45 Data Acquisition Configuration of the Main Panel As mentioned above the main panel of the SPCM software is configurable by the user see 22 Configuration of the SPC Main Panel A number configurations are shown in Fig 31 to Fig 34 Fig 31 shows the main panel of a single detector system An intensity image or an image in a defined time window is displayed on the left The Display parameter panel is kept open on the right It allows the user to define the display scale the colours of the display and the time window within the fluorescence decay in which the image is displayed Moreover the display style can be changed in order to display fluorescence decay curves along selected horizontal or vertical stripes of the image We recommend to keep also the main panel of the DCC 100 software open see lower right corner of Fig 31 The DCC 100 panel is used to control the detectors the shutters of the NDD FLIM systems and the power of the diode laser of the one photon confocal FLI
85. control parameters 38 for confocal FLIM 15 50 for NDD FLIM 23 53 gain 38 39 60 H7422P 40 16 24 id 100 50 SMC 16 insertion of filters 50 53 MW FLIM detector 17 24 Index 101 number of definition 36 overload shutdown 22 39 PMC 100 16 17 23 24 26 power supply 38 R3809U 15 23 Diode laser alignment procedure 49 BDL 405SM 13 installation 49 picosecond 12 power control 39 Direct detection 10 22 Display 3D display modes 42 autoscale 42 display colours 42 display of gated images 42 display parameters 41 display scale 42 reverse axis 42 routing windows 43 saturation of pixels 42 scan windows 43 time windows 42 43 Donor of FRET 3 89 Dual module FLIM systems 12 28 Efficiency of FLIM systems 5 FCS 8 35 73 File formats data and setup 44 45 File Info 44 45 Files loading of 45 saving of 44 Filters 50 53 Fit procedure 75 Fit selection parameters 79 FLIM adjusting of images 63 autofluorescence of tissue 93 data acquisition components 11 data acquisition software 31 efficiency 5 excitation source 8 fluorescence quenching 89 FRET measurements 89 ion concentrations 89 pH imaging 89 scan rate 8 TCSPC parameters 33 time resolution 4 8 FLIM measurement 65 acquisition time 70 72 count rates 69 photobleaching 69 70 Fluorescence decay functions 1 4 lifetime 1 of aggregates 3 of complexes 2 of protonated and deprotonated forms 2 quenching 2 89 resonance energy transfer 3 89 Forster resonance energy t
86. crement Columns Select data Routing Channel fi z Page Number W a i E Module Humber W T W Cancel Fig 86 Information and data selection panel of the SPCImage import function Import as f Scan Image Single Curve Instrumental Response Hot Spot and Region of Interest Selection After the FLIM data have been imported SPCImage displays an intensity image of the loaded FLIM data see Fig 87 Intensity Image T Region of Interest upper right corner al Hot spot Defines a pixel from which the current Region of Interest i decay trace is shown lower left corner Fig 87 Intensity image after import of FLIM data After loading the data the software chooses the brightest pixel of the image as a Hot spot The location is indicated by the blue crosshair It is also given as a numerical position x y in the decay window see Fig 89 The Hot Spot is used to calculate an IRF for the fitting proce dure see below Instrument Response Function The same spot is used to display a fluores cence decay curve see Fig 89 If necessary the pixel selection can be changed by moving the blue crosshair Two white crosshairs are located in the upper right and lower left corner of the image They define a region of interest ROI which will be used during the data analysis They can be changed by clicking on the white dots and moving them to a different location Defining ROIs
87. ct or specify a file to display information about existing file and to select between different save options File format 2f SPC Data Filename autofluorl sdt v e bh spc manual data typical setups s Select file F9 ferrssresconseee y What to save sj All used data sets File Info Selected file doesn t exist Save F10 Return Esc 4 r Version f 850 M Author E ond James Company Mark all F1 Unmark all F2 duvvcuvvevecevevsusuveveeuvevevseerevsuervevevere Contents Autofluorescence of skin BDL 405 laser PML SPEC E r fe Fig 45 Save panel File Format You can chose between SPC Data and SPC Setup The selection refers to different file types With SPC Data files are created which contain both measurement data and system parameters When this file is loaded not only the measurement data are restored but also the complete system setup With SPC Setup files are created that contain the system parameters only When such files are loaded the system setup is restored but no data are loaded Files created by SPC Data have the extension sdt files created by SPC Setup have the exten sion set File Name Select File A file name can be written into the File Name field Select File opens a dialog box that allows you to change or create directories Moreover it shows the names of existing files These are sdt files or set
88. cting diagrams of some typical one photon confocal FLIM systems are shown in Fig 16 through Fig 20 PMC 100 System The wiring diagram of a confocal FLIM system with a single PMC 100 detector 1s shown in Fig 16 A BDL 405 SMC or BDL 473 SMC diode laser is used for excitation The optical output of the laser is coupled to an input fibre of the FV1000 scan head The power of the la ser is controlled via connector 1 of the DCC 100 detector controller The fluorescence light is fed into a PMC 100 detector via a direct output from the confocal scan head of the FV1000 To separate the fluorescence light from scattered excitation light it is important that a suitable longpass or bandpass filter be inserted in front of the PMC 100 input For optical configuration please see Fig 51 page 51 Power Supply Laser Power Control DCC 100 Board Single Mode Fibre into Microscope Scan Clocks from Microscope FLIM adapter Power Supply SPC Board amp Control Fig 16 One photon confocal FLIM system with PMC 100 The 12V power supply the cooling current and the gain control signal for the PMC 100 are provided at connector 3 of the DCC 100 The cooling circuit of the DCC 100 must be config ured for one detector see DCC 100 manual 7 The connecting cable has also a line for the overload signal of the PMC 100 In case of overload the DCC 100 shuts dow
89. ctor FV1000 scan is 512 x 512 pixels Data Format ADC Resolution a E4 Memon Offset ooo Dither Range 1716 Count Increment E 10 Fage Control Delay ng g Routing chan 2 1 Ronting chan w 1 Meas page 1 Scan pels A Scan pels r E Al x Syne Polarity SJ Positive Y Syne Polarity Positive Pixel Clock Polarity Positive Line Predivider 5 1 x Pixel Clock Divider E 1 Left border Upper border Pixel Clock External 128 x12 pixels 2 detectors 256 x 256 pixels 1 detector Data Format ADC Resolution b4 Memory Offset 0 00 Dither Range 1 16 Count Increment 10 Page Control Delay rs 0 Routing chan s 16 Routing chan r 1 Meas page 1 x Syne Polarity Positive Y Syne Polarity E Positive Pixel Clock Polarity Positive Line Predivider a 4 x Pixel Clock Divider sj 4 Left border 8 Upper border E 0 Pixel Clock 3 External Fig 37 Data format page control and scan parameters for 512 x 512 pixels 1 detector FV1000 scan is 512 x 512 pixels 128 x 128 pixels Scan pels a 128 Scan pels a 128 More Parameters 256 x256 pixels 2 detectors 16 channel multi wavelength detector Access of System Parameters from the Main Panel To facilitate on line adjustments the essential hardware and measurement control parameters are accessible directly from the main panel see Fig 38 38 SPCM Software
90. d is a relatively unlikely event The detection of several photons in one signal period is even less likely The idea behind TCSPC is that only one photon per signal period needs to be considered If only one photon needs to be detected per signal period the build up of a photon distribution over the time in the signal period and in case of multi dimensional TCSPC over additional parameters is a straightforward task Of course the neglecting of a possible second photon and the resulting pile up effect are subject of never ending discussion It can however be shown that the effect on the recorded lifetime under practical conditions is negligible 21 The architecture of a multi dimensional TCSPC device operated in the FLIM mode is shown in Fig 6 n Channel Wavelength Time Measurement ADC Time within decay curve from Laser Stop Frame Sync Counter Y Line Sync Scanning Interface Pixel Clock Counter X X Location within scanning area from Microscope Fig 6 Multidimensional TCSPC in the FLIM mode At the input of the detection system are a number of photomultipliers PMTs detecting the fluorescence light from the excited spot of the sample in different wavelength intervals In the subsequent router the single photon pulses of the PMTs are combined into a common timing pulse line
91. e a a Fig 34 SPCM main panel for the oscilloscope mode Fluorescence decay curves are measured in short inter vals and displayed The oscilloscope mode is used to adjust the CFD and TAC parameters an the signal delays in the detector and synchronisation path System Parameters The System Parameters contain the complete set of hardware and measurement control pa rameters of the TCSPC module If your system has been set up by a bh engineer you need not change any of the setup parameters For users who like to setup a FLIM system on their own bh deliver a number of setup files for different FLIM configurations If you start from these you need only adapt the TAC parameters and signal delays to the special requirements of your 34 SPCM Software microscope see Getting Started First Light page 56 The following paragraph should therefore be considered supplementary information for advanced users The system parameters are accessible by clicking into Parameters System Parameters The System parameter panel is shown in Fig 35 CFD Parameters TAC Parameters ba 5 Each start of sequence cycle Add only Fig 35 System parameter panel A detailed description of the system parameters is given in 22 The following paragraphs give an Overview about the available operation modes and the system parameters controlling the FLIM acquisition Measurement Control Parameters Operation Mode
92. e command Multi dimensional TCSPC is described in detail in 21 22 TCSPC makes use of the special properties of high repetition rate optical signals detected by a high gain detector Understanding these signals is the key to the understanding of TCSPC The situation is illustrated in Fig 5 Excitation pulse sequence repetition rate 80 MHz 1 100ns Fluorescence signal expected AAA UU AAPA Detector signal oscillocope trace Fig 5 Detector signal for fluorescence detection at a pulse repetition rate of 80 MHz Fluorescence of a sample is excited by a laser of 80 MHz pulse repetition rate a The ex pected fluorescence waveform is b However the detector signal measured by an oscillo scope has no similarity with the expected fluorescence waveform Instead it consists of a few pulses randomly spread over the time axis c The pulses represent the detection of single photons of the fluorescence signal The photon detection rate c is about 10 s This is on the order of the maximum possible detection rate of most detectors and far above the count rates available from a living speci men in a scanning microscope Thus the fluorescence waveform c has to be considered a probability distribution of the photons not anything like a directly observable signal wave form Moreover Fig 5 shows clearly that the detection of a photon in a particular signal pe Multi Dimensional Time Correlated Single Photon Counting 7 rio
93. e display At a detector gain higher than 80 to 90 the first photons should be detected Eventually the fluorescence decay curves should show up With the settings of Fig 58 decay curves from several signal periods should become visible see Fig 61 left If the result looks like Fig 61 middle no fluorescence light arrives at the detector Only dark counts are detected In this case check that the beam path in the microscope is correctly con figured that the shutter is open and that the excitation laser is activated Fig 61 right shows the test result if daylight or other continuous light is detected In NDD systems turn off the room lights and make sure that all microscope lamps are turned off In the worst case it may happen that the detector shuts down by overload before any reason able signals are recorded In this case the light intensity at the detector is far too high The reason my be an unusually bright sample or more likely a missing laser blocking filter pickup of daylight or leakage from a microscope lamp Fig 61 Signals recorded with the settings of Fig 58 Left Fluorescence signal Middle No fluorescence signal arriving at the detector only dark counts detected Right Daylight detected Adjusting the Signal Delay When you managed to record a reasonable fluorescence signal you can start to adjust the sig nal delay in the synchronisation and detection p
94. e on the order of one second a sufficiently fast update rate for adjusting the focus or selecting an image area of the sample is obtained Trigger The start of a measurement the steps of a page stepping sequence or the cycles of a meas urement sequence can be triggered For microscopes that deliver a pulse at the transition at the next z plane the trigger function can be used to record z stacks of FLIM images Please see 22 Collection Time Collection time is the acquisition time for the measurement or 1f page stepping or cycling is used for each step or cycle of a measurement With the parameters shown in Fig 35 100 cy cles of 10 seconds are performed with the results being accumulated and displayed after each 36 SPCM Software cycle It is not required that you run a FLIM measurement over the full collection time or the full number of cycles If you are satisfied by the signal to noise ration you can stop the meas urement at any time After an operator stop command the internal scanning machine com pletes the current frame so that artefacts by accumulating incomplete frames are avoided CFD SYNC and TAC Parameters These parameters control the constant fraction discriminators at the inputs of the detector and laser synchronisation signal and the time conversion circuitry in the TAC You may possibly change the conversion range of the TAC by changing TAC gain and TAC offset In case of synchronisation problems indicated by a
95. eaching during confocal microscopy of fluorescent probes bound to chromatin role of anoxia and pho ton flux J Microsc 215 281 296 2004 D K Bird K W Eliceiri C H Fan J G White Simultaneous two photon spectral and lifetime fluorescence microscopy Appl Opt 43 5173 5182 2004 C Biskup A B hmer R Pusch L Kelbauskas A Gorshkov I Majoul J Lindenau K Benndorf F D B hmer Visualization of SHP 1 target interaction J Cell Sci 117 5155 5178 2004 C Biskup T Zimmer K Benndorf FRET between cardiac Na channel subunits meas ured with a confocal microscope and a streak camera Nature Biotechnology 22 2 220 224 2004 C Biskup L Kelbauskas T Zimmer K Benndorf A Bergmann W Becker J P Ruppersberg C Stockklausner N Kl cker Interaction of PSD 95 with potassium channels visualized by fluorescence lifetime based resonance energy transfer imaging J Biomed Opt 9 735 759 2004 C Biskup L Kelbauskas Th Zimmer S Dietrich K Benndorf W Becker A Berg mann N Kl cker Spectrally resolved fluorescence lifetime and FRET measurements Proc SPIE 5700 188 196 2005 J W Borst M A Hink A van Hoek A J W G Visser Multiphoton spectroscopy in living plant cells Proc SPIE 4963 231 238 2003 S Y Breusegem In vivo investigation of protein interactions in C Elegans by F rster resonance energy transfer microscopy University of Illinois at Urbana Champaign 2002 H Brismar B
96. eaeeseeseeeeeeeeeeeeeeeseeeaaaaseeeeees 12 Picosecond Diode East rasons inoino oid ikon snes dskwodendncn AOE eO ES 12 Optical Interas Eeee ar a a a ater os saceekes saad seonaeiecantnee 14 Detectors FOr C ontoca l FLIM eaae T E TE 15 Wiring Diagrams of Confocal FLIM Systemss cccccccccccccccccsessssseeeeeeeeceeeeeeeeeeeaasaaaessneseeeeeeeees 17 MultiphotoniN DD FLIM S ystems aieiaa a a n a a r A 22 Synchronisation with the Laser scssi ornp en e eevee agony a A aS 22 Ope all INEA CC earan a a a A 22 Detectors for NDD PLIM seara e E E E 23 Wiring Diagrams of Typical NDD FLIM Systems nnnnneeessesssssssssseerrreessssssssssssscererresssssssssssse 24 SPCM SOMWA ioina sat anss saesanudssuuanunsateesodsie ses satucsadaae aa swsaatuetossaueesossatuesedanedasesastece ee AT 31 Datt ACUS ON naoa e Memmatedtieats sotunine A T a mass 31 Conficuratiom ofthe Main Panel ssiis icq cwsvdasdeveatauncwcer hades eE E E GEE aK 31 SY eni Paranee e ara A a a A a T S 33 Measurement Control Parameters fccssveienteoatitacs nv eatoniey n a a 34 CFD SYNC amd TA PO a a rra 36 Data Format and Page Control iccsiicoucledenvaesendevanstiaadindsn ieaahadeedensandccsnetseoddnsih ea sideateosaniacnsduseadeveds 36 Scan Paramet T senie EE E OEE Oig 36 Access of System Parameters from the Main Panel 1 0 0 0 cccccccccsssssssssseeececceeeeeeeesaesaaeeseseeeeeeeeees 37 Detector ontot Paramete S te ios scat sects ca son ptadesesadnsi TE E A 38 Display of Images in the
97. ecay time with negligible background of environment light and detector dark counts or detector af terpulsing The equation given above can be used to estimate the number of photons and the acquisition time needed to record a fluorescence lifetime image The relative lifetime accuracy for a given number of photons per pixel is shown in Fig 76 left The diagram shows that the standard deviation improves only slowly with the number of photons A lifetime accuracy of 10 can ideally be obtained from only 100 photons However 10 000 photons are required to obtain a lifetime accuracy of 1 1 10 000 106 photons second 1 000 512x512 256x256 Rel stand deviation of T 2 0 1 100 128x128 Acquisition time seconds 10 64x64 0 01 1 1 10 100 1 000 10 000 0 01 0 1 Photons per pixel Rel stand deviation oft 1 N Fig 76 Left Relative standard deviation versus the average number of photon per pixel Right Acquisition time as a function of the desired lifetime accuracy for different image sizes Count rate 10 s The acquisition time as a function of the desired standard deviation for a count rate of 10 s is shown in Fig 76 right E
98. ecordin MTNA SS coreene E E E 61 FEIM Measure men ao rE e E EE E E E Oat 65 Steps Of 4 ELIM Measurement ssrseiniseimie a e e E 65 Details of FLIM Data Acquisition usses5scannss ceotonrnsducedagsa waded neatsonondoemoednyaldesiass sedelneceonarsomnontnedventascsedseed 69 Synchronisation and Count RATS spins dec cansuntoaduvouecunocndnadsvadedeuetenveadhvaeniwenunndsedaioa Iedensewlacdsvenenuneneedents 69 POLO DIC ACIS oar seve crahs a aaah a sh yawmasassateasee a Gaateneet pacoses tena Ouh cena iss 70 Acquisition Lime of FLIM esnie nenna a a isi a tide 70 MAYS 917 AAE E E E A E E E E AE E EE A E A E A 12 Single Point Fluorescence Decay and FCS Measurements ccccccceeecceeeeeecceeeeeeaaeaeeaseesesseesenseeeeeeees 73 Waa AMAL SIS e E ceennaats senate newest O E E E O 75 troduction E T E T O R 75 Analysing Fluorescence Ile time lima Ses irer enera E sdeumbeiescusionssuenteie est Ta M IV Contents Loadne ot FEIM Dildaar re perme cement rer ir E A ene en nee ree ear ad Hot Spotand Region of Interest Selectionner atau euler cactih wien E ct eeu 78 Mstiment Response Fanchon nerenin ri oa qataidesouen saipavedetedaxedannnduyeiaan tadoNarineeiskacanneelats 79 PICS ClECIION Pardines saranen a n a a Sen eee eee eee 79 Binning inthe Liietime Analysis cdir a a a a e a 80 Moget Selec Homa a a E E O O EE ete 82 Calculation oF the kit ime Mate aein a E a a ewes 85 Display OF Pile time Tia Ce Soa r a r R A 85 iketime Parameter HIStOS tamin
99. ect software component nT Please select which features you would like to install M SPCM v 7 6 application M DCC v 1 2 application M SPCimage v 2 2 License number required I SPCM DLL 1 8 License number required fo with Labview Library r DCC DLL v 1 0 License number required I STP DLLv 2 0 Please contact info becker hickl de to get license number lt Back Next gt Cancel Fig 55 Installation panel Software components required for FLIM are marked 54 System Setup If you have purchased the FLIM boards in a Simple Tau system you get the software readily installed You may however update your software from time to time Updates are free and can be downloaded from www becker hickl com at any time Click on the Software button see Fig 56 On the Software page click on TCSPC Modules Operating Software for Windows 95 98 NT4 2k XP Then click on Setup for SPC 830 SPC 134 SPC 730 SPC 630 Download TCSPC_setup_web exe and start it It works the same way as the instal lation from the CD High Performance Operating Software for Windows 95 98 NT4 2K XP Photon Counting ea Modules Name Version Setup Notes Manual Size I kb up n SPC 830 SPC 134 spcm 76 p2 TR 8210 SPC 7x0 SPC 6x0 i Phot SPC 5x0 SPC 4x0 spc 725 i 5 3288 SPC 3x0 Za Integrated into self extracting TCSPC Package 1 4 Installation requires Windows Installer 2 0 Bh Device Driv
100. elect the best location in the image and the best bin ning factor In any case you should switch on Display Instrumental Response to make sure that your data analysis is based on a reasonable IRF Fit Selection Parameters The decay curve in the Hot Spot of the image is displayed beneath the intensity image see Fig 89 It shows the photon decay data in the subsequent time channels of the selected pixel blue dots the convolution of the model function and the IRF fitted to the decay data red curve and the instrument response function green curve The deviations between the photon data and the fit trace are shown at the bottom The values shown are weighted residu als T1 Start of fit channel num Threshold Position of T2 End of fit channel num selected Pixel TMax Maximum photon K number channel num BP counts 2S fe Bin Sf Thi 45 Pos 93 a2 tm 2059 72 100000 Selected fit parameter 10000 Measured decay trace Response function dotted and fit curve Weighted Residuals Reduced Chi Square line Fig 89 Decay curve window 80 Data Analysis The time interval in which the fitting is done is selected by two vertical cursor lines The cur sor positions Tl and T2 are shown numerically in the status line above the decay curve win dow The setting of the cursors have considerable influence on the fit quality The left cursor should be set at the beginning of the rise of the f
101. em The two PMC 100 detectors are connected to individual TCSPC mod ules With additional beamsplitters in front of the detectors FLIM systems with four fully parallel can be built A demonstration of a four channel system was given in 17 SPCM Software 31 SPCM Software The TCSPC FLIM system is controlled by the SPCM software of the SPC 830 module and by the software of the DCC 100 detector controller The SPCM software allows the user access the full functionality of all Becker amp Hickl TCSPC modules 21 22 There are operation modes for recording single decay curves time controlled sequences of decay curves fluorescence correlation FCS curves and photon counting histograms PCH FLIM images in several detector channels multi wavelength FLIM images or sequences of FLIM images Although not tested in details most of these features can be used in conjunction with the Olympus Fluo View FV1000 scanning micro scopes The SPCM software therefore contains by far more system and measurement control parameters than you need for recording a simple FLIM image in a single detector channel Depending on the application different often multi dimensional results are to be displayed Therefore the SPCM software contains a number of one dimensional and multi dimensional display modes The main panel of the SPCM software can be configured by the user Please do not get confused by the variety of options The SPCM software stores the
102. end to set the parameter to 10 mV If you want to optimise it please refer to 21 or 22 Recording Images 61 Recording Images TCSPC Parameters for Imaging Change the operation mode to Scan Sync In Use the system parameters and the display pa rameters shown in Fig 64 but retain the CFD SYNC and TAC parameters found in the pro cedure described above Acx La Display Parameters W1 E A Solid Square EA 1 Fig 64 Startup System and Display parameters for Scan Sync In mode Set More Parameters as shown in Fig 65 The parameters refer to the standard a 512 X 512 pixel scan of the FV1000 therefore make sure that your scan resolution is 512 X 512 see Fig 69 Sync Polarity Positive Y Sune Polarity Positive Pixel Clock Polarity Positive Line Predivider Pitel Clock Divider Left border Upper border Fisel Clack External Fig 65 Scan Control Parameters for recording a 256 x 256 pixel image with a 512 X 512 pixels FV1000 scan Define the Window Parameters as shown in Fig 66 The time windows define time gates in which the intensity is displayed Window 1 covers the complete ADC range the other win dows define subsequent time gates over the ADC range 62 System Setup fh SPC 830 Simulation Window Intervals E fei Time Windows Rout Windows Rout Y Windows Scan Windows Scan Y Windows BE Equidistant BE Equidistant BB Eguidistant BE Equidi
103. ending on the selected file format A history of previously loaded files is available by clicking on the button File Info The file info window displays information about the file selected The first three lines of the file info are inserted automatically when a file is saved The last three items can be typed in by the operator see Saving Setup and Measurement Data 46 SPCM Software Block Info Activating a data block in the Block Number in File field enables a Block Info Button Clicking on this button opens a list that contains the device number of the SPC modules by which the data were recorded the time and data of the recording and all system parameters see Fig 47 At the end of the block information the minimum and maximum count rates of the corresponding measurement are shown see Fig 47 right The block info often helps to re cover the exact recording conditions of an older measurement Pinel Time gt 1 0000e 01 Piel Clock Source O Internal Scan Size Scan Size Y ae Scan AoutChan amp 1 Scan AoutChan w 1 Est Pie Clock Divider 1 Module Identification SPC F30 360033 Creation date amp time 2004 11 08 18 43 51 Measurement mode FIX Noof meas steps 1 Repeat Time gt 0 0000 s Stepping Motor Not Used Information collected when the measurement was finished Status Ox000c Flags 8000 Stop Time 62 1838 Curent step 1 Current cycle 1 Current page Sync rate Min
104. erey aT TTT ii 4 4 w B 23h D G NMXD0512S0 A j Bo SS e525 25 Sa 4 terres RTTE g g ot g Gz g S l iiiiiiiji F f j e A c c an 3 za amp sf h as a amp gt A a c one By 2 Soa ES z mn H EEE E eq bar Pob ga TE 7 20 a gt w Ci A c j 4 Frm Fig 22 The detectors of non descanned FLIM systems are protected by shutters left The detectors and shutters are controlled via the DCC 100 detector controller card middle The box right reduces the power dissipation in the shutter coils Detectors for NDD FLIM Different detectors available for the non descanned FLIM systems are shown in Fig 23 The detectors are shown with the shutter assemblies All FLIM detectors are controlled via the DCC 100 detector controller card see Fig 22 Fig 23 Detectors of the bh non descanned FLIM systems Left to right R3809U MCP PMT PMC 100 H7422P 40 bh MW FLIM detector R3809U The R3809U detector 55 is the detector for ultimate time resolution Its instrument response function IRF has a width of 30 ps FWHM 22 55 The R3809U is used when lifetimes or lifetime components shorter than 150 ps are to be resolved Typical applications are FRET experiments with resolution of the interacting and non interacting donor fraction 15 16 32 46 tissue autofluorescence 70 71 72 and fluorescence of dyes attached to metallic nano particles
105. ers 1 5 must be installed prior to this installation under Service Pack 3 for Windows 2000 SPC830 High Resolution TCSPC Imaging and FCS Module Nahmitzer Damm MM TCSPC Modules D 12277 Berlin r phone 49 30 787 56 32 W Gated Photon Counters Multiscalers fax 49 30 787 57 34 mail Nahmitzer Damm 30 i www becker hickl com a Experiment Control Delay Generators Tokyo Instruments Fig 56 Updating the TCSPC package from www becker hickl com Hardware To install the SPC and the DCC module switch off the computer and insert the modules into a free slot To avoid damage due to electrostatic discharge we recommend first to touch a metal lic part of the computer with one hand and then to grasp the module at the metallic back shield with the other hand This will drain any potentially dangerous charge off your body and the module Then insert the module into a free slot of the computer Keep the SPC module as far as possible apart from loose cables or other computer modules to avoid noise pick up When the computer is started the first time with an SPC or DCC module the operating system detects the modules and attempts to update its list of hardware components Therefore it may ask for driver information from a disk When this happens put the installation CD into the drive and select between Win9x for Windows 95 98 and Win2kNT for Windows 2000 Windows NT or Windows XP
106. exponential FRET experiments the diffusion tail makes it difficult to accurately resolve the decay components of the interacting and non interacting donor fractions gt The R3809U can be used at count rates up to 3 MHz 21 22 However the output current at this count rate is beyond the permissible maximum specified by Hamamatsu In FLIM applications high count rates normally oc cur only in a few pixels of the image Immediate damage under these conditions appears therefore unlikely nev ertheless life cannot be guaranteed One Photon Confocal FLIM Detection Systems 17 MW FLIM The MW FLIM detection system 6 detects the fluorescence simultaneously in 16 wavelength channels A polychromator splits the light spectrally and projects the spectrum on the photo cathode of a PML 16 detector This detector contains a 16 channel PMT and the associated routing electronics 6 Thus 16 lifetime images are recorded in a single SPC 830 TCSPC module For confocal detection the MW FLIM system can be coupled both to the fibre output of the FV1000 scanner or to the bh FLIM adapter In the second case the light is transferred into the polychromator via a fibre bundle Despite of the non ideal filling factor of the bundle the efficiency for the fibre bundle is slightly higher than for the single fibre Typical applica tions of the MW FLIM detector are FRET experiments 33 and autofluorescence imaging 22 Wiring Diagrams of Confocal FLIM Systems Conne
107. fetime change via the Stern Volmer relation Be cause 2 photon excitation does not cause photobleaching and photodamage outside the focal plane the authors were able to obtain z stacks of the Cl concentration in dendrites over depth intervals up to 150 um Fluorescence Resonance Energy Transfer FRET FRET is an interaction of two fluorophore molecules with the emission band of one dye over lapping the absorption band of the other see FRET page 3 In this case the energy from the first dye the donor can be transferred immediately to the second one the acceptor The en ergy transfer itself does not involve any light emission and absorption 50 75 Forster reso nance energy transfer or resonance energy transfer RET are synonyms of the same effect The energy transfer rate from the donor to the acceptor decreases with the sixth power of the distance Therefore it is noticeable only at distances shorter than 10 nm 75 FRET results in an extremely efficient quenching of the donor fluorescence and consequently decrease of the donor lifetime Because of its dependence on the distance FRET has become an important tool of cell biology 86 Different proteins are labelled with the donor and the acceptor FRET is then used to verify whether the proteins are physically linked and to determine distances on the nm scale The problem of steady state FRET techniques is that the concentration of the donor and the acceptor changes throughout
108. ged Fig 92 Lifetime images obtained from 256 x 256 pixel data Left to right Binning 0 1 and 2 82 Data Analysis Fig 93 Lifetime images obtained from 256 x 256 pixel data Left to right Binning 4 8 and 10 Model Selection The model parameters are selected in a panel right of the decay curve window see Fig 94 Multiexponential Decay Components Components Number of exponential components used by the ates model eps E43 H I Fis tl t2 t3 Lifetimes of the exponential components aa fer al a2 a3 Amplitudes of the exponential components E O Fis Shift Shift between the calculated or loaded IRF and the actu aao yH ally used IRF ios 0 STF Scatter Amount of scattered excitation light detected or shto HT F amount of other prompt emission Can be used to extract Scatter 0 018 H Fi second harmonic generation O id 2 Fis Offset Baseline offset of the decay curve Offset is not really a fitting parameter It is determined from the time channels left Fig 94 Model selection of the left cursor see Fig 89 parameters More model parameters are available under Options Model The corresponding panel is shown in Fig 95 Fit Model f Multiexponential Decay Incomplete Multiexponentials Repetition Time 0 000 he Parameter Constraints Algorithmic Settings ltterations mas 10 Delta Chi min 0 010 Allow negative amplitudes Minimu
109. guration for 56 recording the first image 61 scan mode 61 shutters 57 signal cable length 58 synchronisation with laser 57 synchronisation with scan 62 system parameters for oscilloscope mode 56 system parameters for scan mode 61 Steps of a measurement 35 SYNC parameters 36 SYNC rate 69 Synchronisation with laser 13 17 22 25 57 with laser cable length 58 with scan 62 System Parameters 33 37 System setup 49 TAC parameters 36 TAC rate 69 TCSPC classic TCSPC 6 detector signals 6 FIFO mode 8 73 multi dimensional 7 multi wavelength 5 pile up effect 7 router 7 time calibration 8 time channels 8 time resolution 8 time tag mode 8 73 Threshold CFD 60 in lifetime analysis 80 Time channels 36 Time gating of lifetime images 86 Time windows 42 43 Trigger of experiment 35 Two photon absorption 9 Two photon excitation 8 9 Unlock data analysis of pixels 87 Window parameters 42 61 Wiring diagrams Confocal FLIM systems 17 Confocal single id 100 50 18 Confocal single PMC 100 17 NDD FLIM systems 24 NDD dual PMC 100 26 NDD dual R3809U 27 NDD single PMC 100 24 NDD single R3809U 25 Zero cross level CFD 60 Zoom 72
110. he Laser In a multiphoton system the fluorescence of the sample is excited by femtosecond pulses of a titanium sapphire laser Different lasers may be used with pulse repetition rate ranging from 78 to 90 MHz In terms of TCSPC data acquisition there 1s little difference between the la sers The only requirement is that a timing reference signal for the TCSPC card be provided Most of the lasers deliver a reference signal at a BNC connector at the back panel of the laser housing The output pulses are usually positive The polarity has then to be reversed by a pas sive pulse inverter see Fig 21 left For wiring diagrams of the complete FLIM systems please see Fig 25 to Fig 29 Some of the older titanium sapphire lasers do not deliver a reference signal In these cases the timing reference signal for the SPC card must be generated by a photodiode module see Fig 21 right A reflex of the laser beam from a glass plate is normally sufficient to obtain a useful reference signal A PP D Putse invertor Fig 21 Left A PPI pulse inverter to reverse the pulse polarity of the reference pulses of the Ti Sapphire laser Right PHD 400 N photodiode module used to generate reference pulses for lasers without reference output Optical Interface In general the FLIM detectors of multiphoton systems can be attached to the microscope as described for the one photon systems The pinhole is opened wide and only used to suppress daylight leak
111. hoton FLIM quantification of the EGFP mRFP1 FRET pair for lo calization of membrane receptor kinase interactions Biophys J 88 1224 1237 2005 M Prummer B Sick A Renn U P Wild Multiparameter microscopy and spectros copy for single molecule analysis Anal Chem 76 1633 1640 2004 I Riemann P Fischer M Kaatz T W Fischer P Elsner E Dimitrov A Reif K Konig Optical tomography of pigmented human skin biopies Proc SPIE 5312 24 34 2004 R Rigler E S Elson eds Fluorescence Correlation Spectroscopy Springer Verlag Berlin Heidelberg New York 2001 R Richards Kortum R Drezek K Sokolov I Pavlova M Follen Survey of endoge nous biological fluorophores In M A Mycek B W Pogue eds Handbook of Bio medical Fluorescence Marcel Dekker Inc New York Basel 237 264 2003 B Riquelme D Dumas J Valverde R Rasia J F Stoltz Analysis of the 3D structure of agglutinated erythrocyte using CellScan and Confocal microscopy Characterisation by FLIM FRET Proc SPIE 5139 190 198 2003 A R ck F Dolp C Happ R Steiner M Beil Time resolved microspectrofluorometry and fluorescence lifetime imaging using ps pulsed laser diodes in laser scanning micro scopes Proc SPIE 5139 166 172 2003 A R ck F Dolp C Hiilshoff C Hauser C Scalfi Happ FLIM and SLIM for molecu lar imaging in PDT Proc SPIE 5700 2005 R Sanders A Draaijer H C Gerritsen P M Houpt Y K Levine Quantitative pH
112. iency It 1s recom mended for applications that require ultimate sensitivity In the wavelength range of 500 to 600 nm a sensitivity improvement of a factor of 2 to 3 over the R3809U and the PMC 100 is obtained The IRF width of the H7422P 40 is 250 to 350 ps 22 The large IRF width makes the H7422P 40 less useful for FRET and autofluorescence imaging The H7422P 40 is cur rently the best compromise for systems that are to be used both for FLIM and FCS 19 23 49 id 100 50 SMC Single Photon Avalanche Photodiode The id 100 50 SMC is a single photon avalanche photodiode The instrument response width of the id 100 50 itself 1s about 45 ps However in one photon systems the effective IRF is a convolution of the laser pulse shape and the detector IRF Thus the typical IRF width of the complete system is on the order of 80 ps The id 100 50 is overload proof an overload shut down function is not required Because the id 100 50 SMC has a fibre input it is connected to the standard fibre output of the FV 1000 scan head Due to its fast response the 1d 100 50 is an alternative to the R3809U MCP PMT Some care is however recommended if the 1d 10 50 is used at wavelengths longer than 500 nm All sin gle photon APDs show a diffusion tail in their response 21 22 The relative amplitude of the tail increases with increasing wavelength The diffusion tail can therefore easily be mis taken for a fluorescence component Especially in double
113. ifetime accuracy of 10 is not sufficient for the majority of FLIM applications Therefore the general advice is Run the FLIM acquisition for a time as long and at a count rate as high as the photostability of the sample allows you Image Size The acquisition time increases linearly with the number of pixels recorded This statement may be considered trivial Nevertheless it is often ignored by microscope users A laser scan ning microscope scans the image area with a constant pixel dwell time regardless whether or not the pixels contain useful information Thus you can decrease the acquisition time by ex cluding useless pixels from being recorded The simplest way of achieving this is to zoom into the right area of the image see Fig 77 Fig 77 Image of a single cell Left Without zoom the image contains only 15 pixels with useful information Right After zooming into the correct region of interest 75 of the pixels are used The acquisition time is re duced by a factor of 5 The cell shown in the left image fills only 15 of the total scan area In other words only 15 of the acquisition time are spent on recording the image of the cell 85 of the time are spent on recording dark pixels After zooming into the an appropriate region of interest the cell fills 75 of the scan area and 75 of the acquisition time are used to image the cell This is an improvement of a factor of 5 over the recording shown left You may object that
114. ime but may cause increased photo Exi i bleaching successful measurement Place the DCC panel in a conven 8 7 _ 10 Set the cursors as shown in the SPCImage panel Click on o i ient area of screen Man Boze Eo O Calculate system response then Calculate decay matrix ET Eav HHz see ete aaa ae a ae Main Parameters E Interrupt Stop Exit p E 5v BEN mE es miera Mon bamm can 2 Sync In Mode mouseintestineS 1 sdt _ T 5 5V Curr Imt I F es j E 5v mv H H 5 E 5v F2 100 100 i b7 7 100 amps E OLD bs ono ts ie Ti b4 i Hj b4 me 188 0 1 A N Rate Pns abs eset je2 tezama Ea e Hj b2 volts Hbi aE U U Yrer l 0 e TEREE i H0 a w E E pre e gen dea C Laser e DigOut 1e 7 E 14565 ms oe anti cc power en Ef inves xas S A 2 CFD TAC ADC _Dicable outputs counts NENE 1224257 MaS on tha fo Poe fra e fi y m a If the detector shuts down by overload right panel de O om i a2 10 1 a crease the laser power or reduce the pinhole size Then eae Scan Clocks Beljo Fix click on the Reset button of the DCC panel to re activate the detector Shift 0 4 H N Fix Scatter 0 005 H M Fix Offset 5 6 ml 145E 5 Dig Zoom _ 137E 5 5 Time E Collection TE JR aan Fl Limit Low IE 10 CFD TAC ADC E J 50 150 98 E Ph s Measurement Device state SYNC 7 56E6 7 z EEN Displaying da data from file e bhispcmanualdataitypical ete pS OU eA el CED 1 sdt
115. in front of the detector Filter Holder FV 1000 Scan Head PMH 100 Detector C Mount Adapter C Mount Adapter Bracket Barrel i Rin Filter Ring Filter 9 optional Support Post N Fig 51 Direct coupling of detectors to the FV 1000 scan head PMC 100 detector The optical principle for the R3809U MCP PMT is shown in Fig 52 The fluorescence light passes the removable emission filter The next element is a negative lens that expands the beam diameter over the full cathode area of the R3809U MCP PMT The expansions is neces sary to achieve a peak count rates in the MHz range without degradation of the detector re sponse 21 22 Moreover a shutter is used to protect the cathode of the MCP PMT against excessively high light intensities A second filter normally a laser blocking filter can be in serted between the shutter and the MCP PMT 52 System Setup Shutter 7 Holder Filter Holder Rard Beam Expander can rea C Mount C Mount Barrel x PE Adapter E Adapter Lm Camo T Balj i U HE Seis iee ghey ia R3809U MCP PMT optional C Mount Negative Lens Filter Adjust Screws Ring n Pi Fig 52 Direct coupling of detectors to the FV 1000 scan head R3809U MCP PMT For electrical installation of the laser and the detectors please refer to the wiring diagrams given in Fig 16 t
116. in the micro scope Two photon imaging requires careful blocking of the excitation laser wavelength All bh de tector shutter assemblies for multiphoton microscopes are therefore delivered with an addi tional laser blocking filter see Fig 54 Please do not remove the filter from the shutter assem bly even if there is a blocking filter in the microscope Shutter Shutter R3809U C Mount thread Barrel va Detector inserts into side port i of microscope and Filter Ring microscope Ring Filter Ring Fig 54 Location of the blocking filter in the detector shutter assemblies Left PMC 100 and H7422P 40 Right R3809U For electrical installation of the detectors please refer to the wiring diagrams given in Fig 25 to Fig 29 If your system differs from the configurations shown there and you are not sure how to connect the components please see 22 or contact bh Installation of the SPC and DCC modules Software The SPC 830 FLIM module uses the SPCM software a comfortable software package that allows you to access all functions of all bh TCSPC devices The installation package contains also the operating software of the DCC 100 detector controller and the SPCImage FLIM data analysis software see Fig 55 The DLL libraries and the step motor control software included in the package are not required for FLIM For details of the installation procedure please refer to 22 i TCSPC Package v 1 4 Setup Sel
117. ing in scanning microscopes acquisition speed photon economy and lifetime resolution J Microsc 206 218 224 2002 M Goppert Mayer Uber Elementarakte mit zwei Quantenspriingen Ann Phys 9 273 294 1931 A Habenicht J Hjelm E Mukhtar F Bergstrom L B A Johansson Two photon exci tation and time resolved fluorescence I The proper response function for analysing sin gle photon counting experiments Chem Phys Lett 345 367 375 2002 Hamamatsu Photonics K K R3809U 50 series Microchannel plate photomultiplier tube MCP PMTs 2001 Hamamatsu Photonics K K H7422 series Metal package PMT with cooler photosen sor modules 2003 P H nninen S Hell Luminescence scanning microscopy process and luminescence scanning microscope Patent WO 95 30166 1995 K M Hanson M J Behne N P Barry T M Mauro E Gratton Two photon fluores cence imaging of the skin stratum corneum pH gradient Biophys J 83 1682 1690 2002 98 59 60 6l 62 63 64 65 66 67 68 69 70 71 72 EDs 74 75 76 T 78 79 References R P Haugland Handbook of fluorescent probes and research chemicals 7th Ed Mo lecular Probes Inc 1999 A Hopt E Neher Highly nonlinear Photodamage in two photon fluorescence micros copy Biophys J 80 2029 2036 2002 M K Y Hughes S Ameer Beg M Peter T Ng Use of acceptor fluorescence for de termining FRET lifetimes Proc
118. ing into the detection light path As long as the two photon microscope is used for imaging single cells or thin cell layers using the confocal detection path is not objectionable For imaging deep tissue layers however the efficiency of the confocal beam path degrades rapidly with increasing depth in the sample Two photon microscopes therefore often use non descanned detection see Fig 7 page 9 The FLIM detector are attached to the side port of the microscope body Two FLIM detectors can be attached via a beamsplitter unit see Fig 14 page 15 A non descanned system detects light from a relatively large area The detectors can therefore easily be overloaded by daylight Worse if the microscope lamp in the transmission light path is switched on extremely strong light is sent directly into the detection light path The micro scope lamps are therefore a potential thread to the detectors To avoid detector damage all non descanned FLIM detectors are equipped with electro mechanical shutters The shutters are closed when the safe limit of the detector current is exceeded Moreover a photodiode in Multiphoton NDD FLIM Systems 23 front of the shutter prevents the shutter from being opened as long as the light intensity 1s high 16 22 The shutters are controlled via the DCC 100 detector controller 7 22 and the p box which reduces the power dissipation in the shutter coils The system components are shown in Fig 22 22h P
119. ing process Especially the shift and the scatter can often be fixed either to zero or to values close to zero To find the best values do some tries with large binning in different spots of the image If you find that the scatter and the shift remain constant fix them to the indicated values Fixing the offset 1s not recommended The offset contains a signal dependent component caused by the detector afterpulsing Thus the offset can only be fixed for detectors of low afterpulsing MCP PMTs and for samples with small relative variation in the pixel intensities You may also fix one of the lifetime components A typical example is the donor fluorescence in FRET experiments Theoretically the slow lifetime component comes from non interacting donor molecules In first approximation it should therefore be constant throughout the image You should however be very careful when you use such a priori information For all lifetime components there are usually subtle lifetime variations induced by variation in the local envi ronment or the refractive index 23 101 If the lifetime you fixed is not really constant the fitting procedure attempts to compensate for the variations by changing the lifetimes of other components This can result in large systematic errors Display of Lifetime Images 85 Calculation of the Lifetime Image The calculation of the lifetime data is started by clicking into Calculate Decay matrix see Fig 97 C
120. is needed A typical example is FRET measure ments and pH measurements The FRET donor decay functions normally show the decay components of interacting and non interacting donor molecules For calculating distances these components must be resolved However if the task is only to determine where FRET occurs in a cell single exponential analysis is sufficient In pH measurements often the life times of a protonated and a deprotonated form of the fluorophore are discernible but the pH can better be determined from a single exponential approximation In these cases do not hesi tate to use a single exponential fit even if it delivers a large X Because the number of model parameters is smaller see below the variance of the lifetime will usually be smaller than for an average or mean lifetime calculated from a double exponential fit Which Model Parameters are Best In general the accuracy of a fit procedure is the better the lower the number of model parame ters 1s In particular the fitting routine has a hard time to determine parameters which have an almost identical influence on the modelled curves These are in particular the shift parameter and an extremely short lifetime component the scatter and an extremely short lifetime component the offset and a slow lifetime component two components with lifetimes close together Therefore most of the parameters can be set to fixed values They remain then unchanged dur ing the fitt
121. it is only important that for al most all dyes the fluorescence lifetime depends more or less on the binding to proteins DNA or lipids 64 74 75 94 102 The lifetime can therefore be used to probe the local environ ment of dye molecules on the molecular scale independently of the concentration of the fluo rescing molecules Extremely strong effects on the decay rates must also be expected if dye molecules are bound to metal surfaces especially to metallic nano particles 51 78 The fluorescence behaviour of a fluorophore is also influenced by the solvent especially the solvent polarity 75 Moreover when a molecule is in the excited state the solvent molecules around it re arrange Consequently energy is transferred to the solvent with the result that the emission spectrum is red shifted Solvent or spectral relaxation in water happens on the time scale of a few ps However the relaxation times in viscous solvents and in dye protein con structs can be of the same order as the fluorescence lifetime The effects can be measured by TCSPC 89 applications to cell imaging have not been reported yet Aggregation The radiative and non radiative decay rates depend also on possible aggregation of the dye molecules The electron systems of the individual molecules in aggregates are strongly cou pled Therefore the fluorescence behaviour of aggregates differs from that of the free mole cules The lifetime of aggregates can be longer th
122. l parameters 82 84 Multi module FLIM systems 12 28 MW FLIM system 28 MW FLIM detector 17 24 Nano particles 3 4 NDD 22 Non descanned detection 10 22 Offset parameter 82 One photon excitation 8 Online display 35 41 Operation mode 34 accumulate function 35 autosave function 35 f t T mode 34 FIFO mode 35 oscilloscope mode 34 repeat function 35 scan sync in mode 34 scan sync out mode 35 single mode 34 steps and cycles 35 Out of focus suppression 9 Overload protection of detctors 22 Overload shutdown 39 p box 22 25 26 27 28 Parallel Acquisition FLIM systems 12 28 Parameter constraints 83 PCH 8 35 Photobleaching 5 69 70 Photon counting histogram 8 35 Pile up effect 7 Pinhole 9 Pixel clock 7 62 Pixels binning of 36 Pixels number of 36 PMC 100 16 17 23 24 26 PMT operating voltage 60 Polygone definition 87 Preamplifiers 11 19 25 27 Predefined setups 46 63 Protein interaction 3 89 91 Pulse inverter 22 R3809U 15 23 25 27 Region of interest 78 Repeat function 35 Repetition time 83 Residuals 79 Router 7 27 Routing channels number of 36 Routing windows 43 Saturation of pixels 42 Save 44 data files 44 file formats 44 setup files 44 Scan number of pixels 36 scan parameters 36 61 scan rate 8 scan windows 43 Scan clock indicators 63 Scan control signals 7 62 Scatter parameter 82 Selection of fit model 82 83 Setup of FLIM system adjusting of images 63 adjusting the signal delay 58 CFD thresh
123. le photon pulses of the detectors are connected to into the routing input and the CFD input of the SPC 830 module mportant In order to maintain correct timing between the routing signals and the photon pulses the CFD cable must not be longer than the routing cable DCC 100 Board Wall Mounted Power Suppl aa a Zom DCC2 DCC1 3 P Box Zom Shut2 Detect High Voltage Control J A L 2 Rear Panel Shutter kV R3809U R3809U _ O mA negative ojii E 5 Eo Voltage Current C 7 O C s0 Photodiode ea if Analog V L Off Digital o FuG HCN 14 3500 LI High Voltage Power Supply 12V F 12V OVLD OVLD om WAH wi Laser Sync Output S ar HFAH 20 01 HFAH 20 01 SPC Board Fig 28 Dual R3809U NDD FLIM system 28 The FV1000 FLIM Systems NDD MW FLIM systems A wiring diagram of a multi wavelength FLIM system is shown in Fig 29 The MW FLIM detector assembly is connected to the side port of the microscope via its shutter and fibre bun dle please see also Fig 24 The shutter is controlled in the usual way 1 e via the p box and connector 2 of the DCC 100 card The power supply of the PML 16 detector comes from con nector of the DCC 100 The connecting cable contains also a line for the overload signal The cable is therefore fed through the p box which combines the overload signals of the de tector and the photodiode in front of the shutter The routing signals and the photon pulses of the PML 16C are c
124. light signal the measurement of the detection times and the reconstruction of the waveform from the individual time meas urements The technique in its classic form is successfully used since the early 70s 82 104 Due to the low intensity and low repetition rate of the light sources and the limited speed of the electronics of the 70s and 80s the acquisition times of classic TCSPC applications were extremely long More important classic TCSPC was intrinsically one dimensional 1 e limited to the recording of the waveform of a periodic light signal For many years TCSPC was there fore used primarily to record fluorescence decay curves of organic dyes in solution A few attempts were made to use TCSPC in combination with scanning microscopes 37 However the classic TCSPC technique was limited not only to relatively low count rates but also to slow scanning 36 The situation changed with the introduction of the multi dimensional TCSPC technique of Becker amp Hickl The new technique not only increased the recording speed by two orders of magnitude 10 13 it also added additional dimensions to the recording process The photon distribution is recorded not only versus the time in the fluorescence decay but also versus the coordinates of a scanning area 16 the wavelength 14 or the time from the start of the ex periment 10 18 The technique is extremely flexible and the configuration of the hardware can be changed by a simple softwar
125. livers the photon distribution over the coordinates of the scan the time within the fluorescence decay and if several detector in dif ferent spectral channels are used the wavelength see Fig 6 page 7 The data can be consid ered an array of pixels each containing a large number of time channels spread over the fluo rescence decay In other words the FLIM measurement delivers images with a decay curve in each pixel see Fig 81 left Several such arrays may exist if several detectors are used To obtain fluorescence lifetimes the decay curves in the individual pixels must be fitted with an appropriate model However the time resolution of the measurement system is finite Therefore the fitting routine has to take the instrument response function IRF into account The IRF is the pulse shape the FLIM system records for an infinitely short fluorescence life time The fitting procedure convolutes the model decay function with the IRF and compares the result with the photon numbers in the subsequent time channels of the current pixel Then it varies the model parameters until the best fit between the convoluted model function and the measured decay data is obtained Typical models are single exponentials or a sum of exponential terms The models are nor mally characterised by several parameters e g the fluorescence lifetimes of the exponential terms and their amplitudes The fitting procedure delivers these parameters for all pixels of the
126. luorescence curve or a few time channels left of it A few time channels should remain left of the left cursor These channels are used to calculate the baseline offset The right cursor should be set at the last reasonable time channel of the fluorescence decay curve There may be a few time channels at the right end of the curve that drop visibly below the expected level of the curve The drop results from the ADC error correction technique used in the SPC 830 module 21 22 the channels should be ex cluded from the fitting Parameters frequently used to control the fit procedure are displayed above the decay curve window TI and T2 are the positions of the cursors in the decay curve window Only the data points between the cursors are used for the fit Max is the time channel in which the fluorescence is at maximum Binning defines an area around the current pixel the photon data of which are combined for lifetime analysis The binning factor is adjusted according the number of photons in the raw data the spatial oversampling factor used when the image was recorded and the com plexity of the decay model used Please see below Binning in the Lifetime Analysis Threshold defines a minimum number of photons in the peak of a fluorescence curve Pix els with lower photon numbers are not analysed by the fitting procedure Threshold is used to suppress dark pixels This not only accelerates the calculation process but also 1m
127. ly in typical FRET systems especially CFP YFP the acceptor decay cannot be observed directly because of the strong overlap of the donor fluorescence with the acceptor fluorescence spectrum An attempt was made in 17 to subtract the donor bleedthrough from the acceptor decay and to build up an a b image In any case using the acceptor fluorescence requires simultaneous de tection of both the donor and acceptor images to reject photobleaching artefacts from the re sults A promising approach to the exploitation of the acceptor fluorescence is multi wavelength FLIM Reference spectra of the donor and the acceptor are recorded and the FRET fluores cence is fit by a model containing these spectra and the unknown intensity coefficients and lifetimes of the donor and the acceptor Thus multi wavelength FLIM may lead to a combina tion of FLIM based and sensitised emission FRET techniques A demonstration of the tech nique has been given in 33 Applications 93 Autofluorescence Microscopy of Tissue Biological tissue contains a wide variety of endogenous fluorophores 71 92 However the fluorescence spectra of endogenous chromophores are often broad variable and poorly de fined Moreover absorbers present in the tissue may change the apparent fluorescence spectra It is therefore difficult to disentangle the fluorescence components by their emission spectra alone Autofluorescence lifetime detection not only adds an additional separation
128. ly observed fluorescence lifetime 7 see Fig 2 Typical quenchers are oxygen halogens and heavy metal ions and a variety of organic molecules The fluorescence lifetime of most fluorophores depends more or less on the concentration of ions in the local environment and on the oxygen concentration For fluorescence lifetime microscopy it is 1m portant that the rate constant of fluorescence quenching depends linearly on the concentration of the quencher The concentration of the quencher can therefore be directly be obtained from the decrease in the fluorescence lifetime 75 Intensity Unquenched Fluorescence we et T9 Quenching a t ITq 3 Time ns 4 Fig 2 Fluorescence quenching Protonation Many fluorescent molecules have a protonated and a deprotonated form The equilibrium be tween both depends on the pH It the protonated and deprotonated form have different life times the apparent lifetime is an indicator of the pH A typical representative of the pH sensitive dyes is 2 7 bis 2 carboxyethyl 5 and 6 carboxyfluorescein BCECF 58 59 75 Complexes Many fluorescent molecules including endogenous fluorophores form complexes with other molecules in particular proteins The fluorescence spectra of these different conformations Why Use FLIM 3 can be virtually identical but the fluorescence lifetimes are often different The exact mecha nism of the lifetime changes is not always clear In practice
129. m Lifetime 1100 ps Maxiniurn Lifetime 30000 ps Minimum A atio am Offset Correction Manual Selection Fig 95 Model parameter options Moulti exponential decay The model is a sum of exponential terms Incomplete multi exponentials The model is a sum of exponential terms It takes into ac count that the fluorescence does not fully decay within a singe laser pulse period Based on Analysing Fluorescence Lifetime Images 83 Repetition Time the fluorescence left over from all previous excitation pulses is included in the model Repetition Time Time between the laser pulse for Incomplete multi exponentials Parameter constraints Minimum lifetime maximum lifetime and minimum ratio of the lifetimes of two exponential terms used to fit the data Algorithmic Settings Maximum number of iterations and minimum difference in the y between subsequent iterations Negative amplitudes of lifetime components may or may not be allowed for Negative amplitudes may occur in the fluorescence decay of the accep tor of a FRET system in the fluorescence of excimers and in fluorescence depolarisation measurements Which Model is the Right One It is often not clear which model in particular which number of exponential components should be used to fit the data If there is no a priori knowledge about the shape of the fluores cence decay the model can only be found by try and error It is normally not difficult to find
130. may be fluorescent themselves Photobleaching is clearly nonlinear for two photon excitation 83 and a nonlinear component probably also exists for one photon excitation 28 Therefore photobleaching can be reduced by reducing the excitation power and compensating for the lower count rate by an increased acquisition time For confocal FLIM you may also increase the pinhole size The increased detection volume allows you to obtain the same count rate at a lower laser power A slightly impaired depth resolution may be better than unpredictable lifetime changes by photobleaching It is often believed that photobleaching is lower for two photon excitation than for one photon excitation This is not generally correct Of course two photon excitation does not yield exci tation outside the focal plane and consequently no photobleaching above and below the im age plane However for the same number of emitted fluorescence photons photobleaching within the scanned plane is stronger 45 for 2p excitation If photobleaching is a problem and your system contains both an Titanium Sapphire laser and a diode laser a comparison between both excitation principles may be useful Giving general recommendations for adjusting the count rate is difficult On the one hand the concentration of the fluorophore and the photostability may vary by orders of magnitude on the other hand the tolerance of the users to possible artefacts may differ considerably In pub
131. mmend to switch Interpolate on to avoid aliasing of the scan pixels with the pixels of the screen Time gating Normally the intensity is taken from all time channels of the FLIM data With Time Gating switched on the intensity is taken from the range defined by the cur sors in the decay window Time gating can be useful to exaggerate image details emitting a fast lifetime component on a background of a slow component or vice versa Lifetime Parameter Histogram SPCImage shows a histogram of the displayed decay parameter calculated over the whole im age or over a region of interest see Fig 99 left The parameter may be the lifetime of a fluo rescence component an amplitude factor or another parameter obtained in the fitting proce dure Depending on the settings in Preferences the histogram either displays the pure pixel frequency or the pixel frequency weighted with the pixel intensities see Fig 99 left The his togram window has two black cursors that can be used to select a lifetime interval The se lected interval automatically changes the parameter range of the lifetime image and vice versa B zooms the distribution into the selected parameter range zooms out All sets the parameter range automatically Distribution Window Histogram Pixel Intensity weighted 500 3000 3500 4000 f Pisel Frequency Let suse o not weighted Crass 3603 09 0 Right 4515 6 oO AMS
132. mouse models of Alzheimer s disease Proc SPIE 5323 71 76 2004 R M Ballew J N Demas An error analysis of the rapid lifetime determination method for the evaluation of single exponential decays Anal Chem 61 30 1989 Becker amp Hickl GmbH Routing modules for time correlated single photon counting manual available on www becker hickl com Becker amp Hickl GmbH 16 channel detector head for time correlated single photon counting user handbook available on www becker hickl com 2006 Becker amp Hickl GmbH DCC 100 detector control module manual available on www becker hickl com Becker amp Hickl GmbH SPCImage Data Analysis Software for Fluorescence Lifetime Imaging Microscopy available on www becker hickl com Becker amp Hickl GmbH BDL 375 BDL 405 BDL 475 Ultraviolet and Blue Picosecond Diode Laser Modules www becker hickl com W Becker H Hickl C Zander K H Drexhage M Sauer S Siebert J Wolfrum Time resolved detection and identification of single analyte molecules in microcapillar ies by time correlated single photon counting Rev Sci Instrum 70 1835 1841 1999 W Becker K Benndorf A Bergmann C Biskup K Konig U Tirlapur T Zimmer FRET measurements by TCSPC laser scanning microscopy Proc SPIE 4431 94 98 2001 W Becker A Bergmann K Konig U Tirlapur Picosecond fluorescence lifetime mi croscopy by TCSPC imaging Proc SPIE 4262 414 419 2001 W Becker A Bergmann H Wabnit
133. n sdt file of the SPCM software is important because only these files contain the complete set of photon data and setup data Only by saving an sdt file the measurement can be reproduced After having saved the measurement data send them to the SPCImage data analysis software Man Parameters Display Load Load Predifined Setups Save Corry art Send Data to SPC_Image Clear SPC men ory Print Sleep Policy About E xt Fig 74 Saving of the measurement data and starting a decay analysis 11 Run a FLIM data analysis in SPCImage See Data Analysis page 75 The steps of recording a FLIM image are summarised on the next page Steps of a FLIM Measurement 68 1 Turn on the FLIM system First the extension box then the 5 Configure the beam path in the FV1000 and define an 8 Start the measurement in the FLIM system With the pre laptop computer image size of 512 x 512 pixels Start a Continuous Scan defined settings the TCSPC module displays intermediate ai results in intervals of about 20 seconds Let the measure Da Ni ment run until you are satisfied by the signal to noise ratio lt lt Fast 10 0us Pixel Slow gt gt A re aaa P 10 0us L 6 240ms F 3 264s S 3 264s Torin jo Ratio 1 1 C4 3 arbitrary mas x El T H swf z if a2 a 555 a a BASSON la EE tho
134. n the gain con trol signal and the 12V power supply of the PMC 100 The detector pulses are fed into the CFD input of an SPC 830 TCSPC FLIM module The timing reference signal SYNC signal of the TCSPC module is obtained directly from the 18 The FV1000 FLIM Systems BDL 405SMC laser The BDL 405SMC delivers positive synchronisation pulses therefore an A PPI pulse inverter is inserted in the SYNC line The scan clock signals of the microscope controller are connected to the upper sub D connector of the SPC 830 or SPC 730 module id 100 50 System Fig 17 shows a FLIM system that uses an id 100 50 single photon avalanche photodiode as a detector The excitation part is the same as for the PMC 100 system described above The light signal from the scan head is fed through a filter adapter into the optical input of the id 100 50 module The id 100 50 gets its power supply voltage from the DCC 100 detector controller The single photon pulses of the id 100 50 are inverted by an A PPI pulse inverter and fed into the CFD input of the SPC 830 TCSPC board Power Supply Laser Power Control DCC 100 Board Single Mode Fibre into Microscope Scan Clocks from Microscope Fibre from Scan Head Filter id 100 50 Fibre SPC Board Fig 17 One photon confocal FLIM system with id 100 50 detector The
135. ndicate the rate of the detected converted and stored pho tons respectively 22 The rates are thus direct indicators of the progress of a FLIM meas urement The count rates may fluctuate due to inhomogeneous intensity in the scan area due to the beam blanking during the beam flyback and due to suppression of photons outside the useful scan area When a FLIM measurement is running from time to time take a look at the CFD and TAC count rate A gradual decrease in the count rates indicates photobleaching Photobleaching can change the recorded lifetimes It is not only that different fluorophores or fluorophore in dif ferent binding states bleach at different rate the photobleaching products may also fluoresce themselves As long as the total decrease does not exceed 20 the effect on the lifetimes may still be negligible If the drop is larger you either have to reduce the laser power or to increase the detection volume see below Photobleaching Both the SPC 830 module and the PMC 100 detector work well up to a detected CFD count rate of about 8 MHz Although there is about 50 loss of photons at a count rate this high Systematic errors in the recorded lifetimes are still small 21 22 You should however take into regard that the displayed count rates are averages over the whole scanning cycle includ ing the beam flyback With the sample reasonably filling the image area you can use CFD count rates up to a few million photons pe
136. nner Scanner Detector Detector Dichroic Mirror Objective Objective Lens Lens Sample Sample One Photon Excitation Two Photon Excitation Fig 7 One photon FLIM left and multi photon FLIM right One Photon Excitation With Confocal Detection The laser is fed into the optical path via a dichroic mirror and focused into the sample by the microscope objective lens Scanning is achieved by deflecting the beam by two galvanometer driven mirrors The excitation light excites fluorescence within a double cone throughout the whole depth of the sample The fluorescence light from the sample goes back through the ob jective lens and through the scanner After travelling back though the scanner the beam of fluorescence light is stationary or descanned The light is focused into a confocal pinhole in an image plane conjugate with the image plane in the sample Light from outside the focal plane is not focused into the pinhole and therefore substantially suppressed 44 85 103 Out of focus suppression is the basis of the superior image quality and the optical sectioning capa bility of scanning systems For FLIM out of focus suppression is even more important Any mixing of the possibly different fluorescence lifetimes of different focal planes adds addi tional lifetime components to the apparent decay functions However the difficulties of un mixing lifetime components increase dramatically with the number of components Out
137. o Fig 20 If your system differs from the configurations shown there and you are not sure how to connect the components please see 22 or contact bh Fibre Coupled Confocal Detectors In the direct coupled FLIM systems the fibre output of the FV1000 is replaced with the bracket that holds the detector assembly If the fibre output has to be maintained for whatever reasons the FLIM detectors can also be attached to the fibre The fibre is coupled to the detec tor by an SMA 905 fibre adapter see Fig 53 No special alignment is required Please make sure that a bandpass filter or a longpass laser blocking filter is placed in front of the detector The filter is inserted inside a threaded barrel between the detector and the fibre adapter C Mount Thread Barrel SMA 905 Fibre Connector a Detector Fibre from Scan Head Fig 53 Connection of the FLIM detector to the output fibre from the FV1000 scan head Left Principle Right Example for PMH 100 detector Experiments with fibre coupled FLIM detectors have shown that the sensitivity is by a factor of two to three lower than for directly coupled FLIM detectors Direct coupling should there fore be preferred Installation of the SPC and DCC modules 53 Multiphoton Microscopes Installing Non Descanned Detectors Non descanned FLIM detectors for multiphoton microscopes are installed at the side port of the microscope Please make sure that the correct beamsplitters are installed
138. oad Then click on the Reset button and open the shutters The detector then resumes normal operation Please do not attempt to avoid overload by decreasing the detector gain This may result in poor counting efficiency and in a multi wavelength system poor channel uniformity 6 TS 1o Main Parameters Exit Connector 1 Connector 2 Connector 3 i Cooling MEiN mav may Mon E s Hj sv E s Curr Imt E 5y H 5v H 5v 2 100 H b7 F 100 Amps J b6 0 ow yes ETE b4 5 J b3 Reset Bf b2 Volts 0 aj 1 0 0 jbo 47 62 85 71 5 00 Gain HV DigOut Gaini HY Cooler Settings from auto set Disable outputs Fig 40 DCC 100 panel after an overload shutdown Maximum Values of Detector Gain The maximum safe gain may differ for different detectors We recommend not to exceed 40 SPCM Software 86 for MCP PMTs operated via an HCN 14 3500 power supply 95 for PMC 100 detectors 90 for H7422P 40 detectors 100 for PML 16C detectors Maximum values of the gain can be defined in the Adjust Parameters panel of the DCC 100 The panel is shown in Fig 41 ie Production amp Adjust Data Reload from EEPROM F9 5197380001 17 10 2001 Retum Esc Fig 41 DCC 100 Production and adjust parameters panel setting maximum values of the detector gain The values are defined under Cl Gain HV Limit and Cl Gain HV Limit They are auto matically saved in a non volatile memory on the DC
139. ocal versus conventional imaging of biological structures by fluorescence light microscopy J Cell Biol 105 41 48 1987 J Yguerabide Nanosecond fluorescence spectroscopy of macromolecules Meth En zymol 26 498 578 1972 Index Acceptor of FRET 3 89 Accumulate function 35 Acquisition time 35 70 72 ADC rate 69 ADC resolution 36 Aggregates 3 4 Amplitudes of decay components 82 Apparent lifetime 84 A PPI pulse inverter 13 17 22 25 Autofluorescence decay profiles 93 lifetime 93 of tissue 93 Autosave function 35 Autoscale fucntion 42 BDL 405SM ps diode laser 13 Binning pixels in data acquisition 36 pixels in lifetime analysis 80 Block Info 46 CFD parameters 36 rate 69 threshold 60 zero cross level 60 Chi square 83 Clock signals from scanner 7 Collection time 35 Colours of display 42 Complexes 2 Components of decay function 82 Confocal detection 9 14 Confocal FLIM systems 12 Count Rates 69 Cycles of a measurement 35 Data analysis 75 Data and Setup File Formats 44 45 DCC 100 detector gain 38 detector gain maximum 39 detector overload shutdown 39 detector power supply 38 initialisation panel 54 installation 54 laser power 39 overload shutdown 22 parameters for startup 57 shutter control 16 17 20 22 24 25 26 27 28 39 57 software panel 31 38 Decay components 82 Decay curve window 79 Depth resolution by confocal pinhole 9 by two photon excitation 9 Descanned detection 9 14 Detectors
140. of focus suppression is therefore mandatory to obtain good FLIM results Two Photon Excitation With Descanned Detection With a fs Ti Sa laser the sample can be excited by two photon absorption 42 53 The effi ciency of two photon excitation increases with the square of the excitation power density Noticeable excitation is therefore obtained only in the focus Thus two photon excitation is a second way to obtain depth resolution and suppression of out of focus fluorescence see Fig 7 right Different from one photon excitation and confocal detection which avoids out of focus detection two photon excitation avoids out of focus excitation Therefore detection through a confocal pinhole is not required to obtain a good image quality Nevertheless feed ing the fluorescence light back through the scanner and the pinhole often has benefits The 10 Introduction accuracy of FLIM can be seriously impaired by detection of background light and by optical reflections in the beam path A pinhole even if wide enough to transmit virtually all fluores cence light yields substantial suppression of daylight and of optical reflections Two Photon Excitation With Direct Detection Since the scattering and the absorption coefficients at the wavelength of the two photon exci tation are small the laser beam penetrates through relatively thick tissue Two photon excita tion can therefore be used to excite fluorescence in tissue layers as deep as mm 4
141. old 60 CFD zero cross 60 DCC parameters 57 detctor gain 60 detectors 57 first fluorescence detection 58 main panel configuration 56 62 recording the first image 61 shutters 57 synchronisation waith scan 62 synchronisation with laser 58 system parameters for oscilloscope mode 56 system parameters for scan mode 61 wiring diagrams confocal systems 17 wiring diagrams NDD systems 24 Shift parameter 82 Shutters 16 17 20 22 24 25 26 27 28 39 57 Simple Tau system 11 SPC module installation 54 SPC 830 TCSPC FLIM module 11 SPCImage 75 SPCM software 31 accumulate function 35 autosave function 35 CFD SYNC TAC parameters 36 data format 36 display of images 41 display parameters 41 FIFO mode 35 initialisation panels 54 installation 53 life mode 35 41 Loading of files 45 main panel 31 56 62 online display 35 41 Operation mode 34 oscilloscope mode 34 page control 36 panel configuration for startup 56 predefined setups 46 repeat function 35 saving of files 44 scan sync in mode 34 scan sync out mode 35 sequential modes 34 single mode 34 steps and cycles 35 system parameters 33 system parameters for startup 56 61 upgrade 54 window parameters 42 61 Special commands in data analysis 86 Startup adjusting of images 63 adjusting the signal delay 58 CFD threshold 60 CFD zero cross level 60 DCC parameters 57 detector gain 60 detectors 57 first fluorescence detection 58 Index 103 oscilloscope mode 56 panel confi
142. on Range E Limit Low Freg E a a A p a TAC GEDSTI SYNC SYNC CFD Tac ape Penne 50 00E 9 227 45 E 1 Fig 33 SPCM main panel for a multi wavelength system Images in eight wavelength intervals displayed detec tor control panel open For setup purpose the SPC 830 can be configures as an optical oscilloscope A single fluores cence decay curve or several curves recorded in different detector channels are displayed In FLIM systems the oscilloscope mode is used mainly to adjust the CFD and TAC parameters and the signal delays in the detector and synchronisation path Please see page 56 for details A suitable main panel setup is shown in Fig 34 ip sPc 830 Main Parameters Display Start nterupt Stop Exit i SPC 830 OSCI Mode bookoscill1 sdt f iol x Main Parameters Exit Comani Conmecee2 Conmecier3 a Cooling Hax aden Win mun Hw Hw H 2 Curim aj av da E 2 100 y 10 Amps Hee z OWLD 24 bd OVLD tm jhe z TE 4w 3 2 Ydi o Hwi o o 4 W no d gt Sto 531m gt m z Gani iv bigoul Gain Recker Rate E is Measurement Selings tom abs EN T Device state Outputs disabled E l SYNC Bree ouput IE Stopt 1 00E 8 l Te 2 42E 6 42E 6 Displaying data vil Control 2 03E 6 Dig Zoom _ 2 2 02E 6 02E 6 Ti 3 Collection aie JR Range E E Limit Low EON FreqDW isp Pace Joa aA ime fOe cro Tac wc rt are Sane S 0 250 50 04E 9 7843 Epo i o
143. onnected to the lower 15 pin connector and the CFD input of the SPC 830 module respectively Important In order to maintain correct timing between the routing signals and the photon pulses the CFD cables must not be longer than the routing cable Wall Mounted P S l aaa ower SUPP IY 12V DCC2 DCC1 P Box Shut1 Shut2 Detect DCC 100 Board Power Supply amp Control PML 16 C Photodiode Fig 29 Multi wavelength NDD FLIM system High Speed FLIM Systems A high speed FLIM system is shown in Fig 30 The detector part is the same as for the dual PMC 100 system shown in Fig 27 However the signals of the two detectors are connected to individual TCSPC modules Thus the counting capability is doubled without increasing counting loss and pile up effects Any pair of similar TCSPC FLIM modules can be used However for cost reasons high speed FLIM systems are normally built from SPC 140 or SPC 150 modules Multiphoton NDD FLIM Systems 29 Wall Mounted P o ower Supply tov DCC2 DCC1 Shut1 Shut2 Detect ih DCC 100 Board Scan Clocks from A PPI i uicrosconl E i fen oe T Laser Sync Output f i Power Splitter Detector Detector PMC 100 PMC 100 SPC 140 or SPC 150 TCSPC Board Power Suppl amp Control SPC 140 or SPC 150 TCSPC Board Scan Clocks Fig 30 High Speed NDD FLIM syst
144. over a wide range of lifetimes 65 Moreover standard multi exponential lifetime analysis techniques can be used to resolve complex decay profiles into their lifetime components and intensity coefficients It should also be mentioned that multi dimensional TCSPC FLIM does not require any cali bration by a lifetime standard Lifetime standards are difficult to use because the effective fluorescence lifetime depends on the pH and the possible presence of fluorescence quenchers see Fluorescence Quenching page 2 For the bh FLIM systems the time scale of the TCSPC module is factory calibrated and the fluorescence decay is recorded into a large number of time channels thus data analysis automatically delivers absolute lifetimes By a simple change of the operation mode see System Parameters page 33 the TCSPC modules can be configured for a number of different signal recording procedures In particular the FIFO or Time Tag can be used to simultaneously obtain fluorescence correlation FCS 27 49 77 91 photon counting histograms PCHs 39 81 and fluores cence decay curves in selected spots of a sample 19 21 23 The technique can even be used to identify the photon bursts of individual molecules and run a lifetime and anisotropy analy sis with the bursts The technique is termed Burst integrated fluorescence lifetime analysis or BIFL 21 47 89 The sequential recording modes can be used to acquire fast
145. performed simultaneously This not only reduces the sample exposure and the associated photobleaching it also avoids that the photobleaching of the first recording changes the results of the second one Only if both recordings are done simultaneously the results are comparable Compatibility with the Scanning Microscope Laser scanning microscopes with standard scanners scan the sample at pixel dwell times down to about us systems with resonance scanners even faster Photon rates obtained from typical samples are usually an order of magnitude smaller It is thus impossible to obtain enough pho tons for lifetime analysis within the time the beam is on one pixel Consequently the FLIM system must be able to acquire the photons from a large number of frames scanned at a pixel rate higher than the photon detection rate Another important issue is lateral resolution and depth resolution Mixing the fluorescence lifetimes of different sample planes or different locations of the sample must be strictly avoided Thus the FLIM technique must make full use of the depth resolution of confocal and two photon laser scanning microscopes 21 6 Introduction Multi Dimensional Time Correlated Single Photon Counting Time correlated single photon counting TCSPC is an amazingly sensitive technique for re cording low level light signals with picosecond resolution and extremely high precision TCSPC is based on the detection of single photons of a periodic
146. ps then requires only a mouse click To use the predefined setup option click on Main Load Predefined Setups This opens the panel shown right A setup my x is loaded by clicking on the button left of the name of the oscilloscope 5 ns setup J 4J oscilloscope 12 5ns To add or delete setups to or from the list or to change the names of the setups click into one of the name fields with the right mouse key This opens the panel shown in Fig 48 J oscillascope 25ns _J scanning 512x512x654 d scanning 2bbx2bbx25b d acanning2 bbx2 5b To add a setup click on the disc symbol right of the File Name field and select a set file Default setups coming with the SPCM software are in the default setups folder of the working directory defined during the software installation Please note that there may by sub directories for different classes of applications Select the files you want to put into the list Predefined Setups 47 of predefined setups and click on the Add button Every setup has a user defined nick name The default nickname is the file name of the set file To change the nickname click into the nickname filed and edit the name Then click on Replace b Manage Setups List oscilloscope 50ns 4 ascilloscope 12 5ns Add oscilloscope 25ns Move up Scanning 512x512x64 Scanning 256x256x256 Move Down Scanning256x256x1 F Remove Relo
147. r second see Image Size page 72 In practice you will reach count rates in the MHz range only for strongly stained samples of exceptionally high photostability Except perhaps for a few pH or ion concentration imaging tasks the count rates obtained from typical FLIM samples are considerably lower The effects investigated by FLIM normally require the fluorophores to be located in highly specific sub units of the cells Or in case of autofluorescence imaging there is no endogenous fluorophore at all Consequently the fluorophore concentrations are low and so are the count rates The 70 FLIM Measurements theoretical limit toward lower count rates is set by the detector dark count rate With the PMC 100 detector you can record lifetime data at count rates as low as 1000 photons per sec ond Measurements at count rates this low do of course require extremely long acquisition times Photobleaching Typical FLIM samples are characterised by low fluorophore concentration Moreover most of the effects investigated by FLIM have to be measured at living cells or tissue The photostabil ity of these samples is low The attempt to obtain high count rates by increasing the excitation intensity usually results in excessive photobleaching or even photodamage Both effects can cause noticeable changes in the lifetimes 17 Usually slow lifetime components bleach faster and the lifetime distribution changes Moreover the photobleaching products
148. ransfer 3 89 Frame clock 7 62 FRET 102 Index basics 3 by double exponential lifetime 90 by multi wavelength FLIM 92 by single exponential lifetime 89 measurement 89 protein interaction 3 89 91 H7422P 40 16 24 Hardware Parameters 33 37 38 High voltage for PMT 60 High speed FLIM systems 28 Histogram of lifetimes 86 HRT 41 router 11 27 id 100 50 18 id 100 50 SMC 16 Image size 72 Import FLIM data into SPCImage 77 Incomplete decay 82 Installation 49 detectors 50 53 diode laser 49 driver information 54 initialisation panels 54 Software 53 SPC and DCC modules 53 Instrument response function 75 79 87 Intensity image 78 Ion concentrations 89 IRF 75 79 87 Laser ps diode laser 12 Ti Sapphire 22 Laser blocking filter 53 Life mode 35 41 Lifetime images 75 autoscale of intensity 86 calculation of 75 85 continuous colour 85 contrast and brightness 86 discrete colour 85 display 85 multi exponential 76 parameter range 85 parameter to be displayed 85 time gating 86 Lifetimes of decay components 82 Line clock 7 62 Load 45 data files 45 file formats 45 files from older software versions 46 load options 46 predefined setups 46 setup files 45 Loading of FLIM data into SPCImage 77 Lock data analysis of pixels 86 Main panel of SPCM software 31 56 configuration 31 dual detector FLIM 32 multi wavelength FLIM 32 oscilloscope mode 33 single detector FLIM 31 Main panel of SPCM software 62 Model function 82 83 Mode
149. rge number of photons An even larger number of photons is required for resolving the components multi exponential decay functions K llner and Wolfrum 65 calculate a number of 400 000 photons for resolv ing two decay components of 2 ns and 4 ns with amplitudes of 10 and 90 respectively Of course resolving such decay functions in FLIM images is almost impossible Fortunately in practice the lifetimes are wider apart and the amplitude of the fast component is larger Such decay profiles can be resolved by analysing some 1000 photons per pixel see Fig 4 Nevertheless a large number of photons must be recorded Recording many photons means either a high excitation intensity or a long acquisition time Therefore photobleaching 45 83 and photodamage 60 66 69 are important issues in pre cision FLIM experiments Both effects are more troublesome for FLIM than for intensity im aging because they are likely to change the lifetimes 17 Photobleaching and photodamage are clearly nonlinear for two photon excitation A nonlinear component seems to be present also for high intensities of one photon excitation 28 A good FLIM technique must therefore not only make best use of the detected photons it must also be able to work reliably at low intensity Multi Wavelength Detection In some cases FLIM images are taken in several emission wavelength intervals or under dif ferent angle of polarisation It is important that both recordings be
150. rget to save the raw data into an sdt file F izdapi img ih SPC 830 File Calculate Mask Conditions Main Parameters Display Start nterupt Stop New iia can Sync In ieee Load Predifined Setups oan Se Close Save n Convert 4 es Send Data to SPC_Image FS Clear SPC memory Import Esport kl Sleep Policy 1 E BH j2dapiimag About 2 default img Exit Exit Fig 85 Loading of FLIM data Left Import of an sdt file via File Import Right Sending data directly from the SPCM data acquisition software 78 Data Analysis Loading the FLIM data via the Import function of SPCImage opens an information and data selection panel see Fig 87 For FLIM data recorded by a single detector in a single memory page of the SPCM software there is nothing you can select Just click on OK and load the data For FLIM data recorded by several detectors or within several memory pages of the SPCM software you can select a window of routing channels and page numbers FLIM data may also be recorded in a multi module TCSPC system In this case you can select the TCSPC module or a range of modules Measurement Info Sizes Size DENTIFICATION ID SPC Setup amp Data Filel Title jzdapi Version 1 781M Revizion 6 bits ADC Date 10 23 2003 Time 175634 Author Unknown Company Unknown Contents END Time channels Routing channels Time Range na Count In
151. rs The pinhole must therefore be set to a diameter of approximately 1 airy unit Moreover FCS requires that the number of molecules in the detection volume be small Best results are obtained with less than 74 Single Point Fluorescence Decay and FCS Measurements 10 molecules The typical concentration is then on the order of 10 mol l The fluorophore concentration in cells is often much higher Although this has no direct influence on the corre lation time the correlation coefficient in cells can be much lower than in Fig 79 23 FCS measurements in solution are usually done in the setup shown in Fig 80 left A cover slip is placed directly on top of the microscope lens of an inverted microscope A droplet of a diluted dye solution is placed on the cover slip The same setup is often used for FCS test measurements Water with dye dissolved Focus at nmol concentration Focus Sample Slide Cover slip placed directly on lens Cover slip Immersion fluid Immersion fluid jective len bases Objective lens Objective lens Fig 80 FCS measurement of a dye solution Left Cover slip placed directly on microscope lens sample solution placed on cover slip A large part of the beam path is in the sample Right Correct optical configuration the beam path is in the immersion fluid The setup is simple and easy to use It does however pose a serious pitfall to inexperienced users The microscope lens is corrected for a beam path
152. s The main panel of the SPCImage data analysis software is shown in Fig 84 The panel shows an intensity image upper left a lifetime image upper middle a lifetime distribution over a region of interest upper right and the fluorescence decay curve in a selected spot of the 1m age 3000 3500 X s Left 2951 6 0 Cross 308846 0 Right 3273 0 wi P W counts T2 56 TMax 12 Bin ai Thld 50 Pos Ee x 4265 y tm 2721 29 Multiexponential Decay 100000 Components 2 O Em aix f7 24 tips 1425 6 I Fix 1000 ax H F 100 r a t2 ps 3856 9 lt 1 T Fix mE i i n gt ae Co a31 0 i iaa t3 ps 0 a4 T Fis Shift 1 2 7 Fix Scatter joo H Fix Offset 0 aT Fir 5 n 5 l ns 00 2 0 40 i 6 0 8 0 7 100 120 7 140 160 7 180 20 20 240 Fig 84 Main panel of SPCImage Loading of FLIM Data There are two ways to load FLIM data into SPCImage see Fig 85 You can open SPCImage and click on File Import The import routine of SPCImage loads the sdt files saved by the SPCM data analysis software The second way to load data is by using the Send data to SPCImage function of the SPCM software The function automatically opens SPCImage and transfers the data If several detectors are used SPCM sends the data within the Routing Win dow of the active display window If you use this option please do not fo
153. s are available Display of Images in the SPCM Software 43 The Time Windows are used for calculating integral photon numbers in selected time inter vals of decay curves or other waveforms The definitions shown in Fig 44 are for FLIM dis play of images recorded with an ADC resolution of 256 time channels Eight time windows are provided The first window covers all time channels An image displayed in this window is contains all photons i e is a pure intensity image The following windows are consecutive time gates within the laser period Images in these time windows are gated images as you can see by stepping through the T Windows of the Display Parameters Fig 43 The Scan X and San Y windows are used to display decay data over selectable stripes of an image Please see 22 The Routing X and Y windows are used to select data from an array of detectors in a multi detector setup In a FLIM having only one detector the routing windows are disabled In multi wavelength systems the routing windows are used to define wavelength intervals in which the images are displayed Please note that the Window parameters are different for different numbers of pixels or time channels When these parameters are changed the SPCM software automatically calculates new window parameters 44 SPCM Software Saving Setup and Measurement Data The Save panel is shown in Fig 45 It contains fields to select different file types to sele
154. scence FLIM of mouse kidney tissue was demonstrated in 20 Obtaining useful autofluorescence images from deep tissue by diode laser excitation is diffi cult The problem is not lack of excitation power or sensitivity but loss of contrast and spatial resolution due to scattering in the tissue Reasonable autofluorescence images can however be obtained from cells or thin tissue sections An example is shown in Fig 106 A BDL 405 SMC 405 nm laser was used for excitation the detector was an R3809U 52 MCP PMT A double exponential model was used for analysis the colour of the image shows the amplitude weighted mean lifetime tm The fluorescence decay curve at the cursor position is shown right 94 Applications aie 4 ies wf 1 es Fig 106 Autofluorescence lifetime image of a BHK cell Excitation at 405 nm detector R3809U 52 References 95 References I 2 10 11 12 13 14 15 16 17 18 19 20 S M Ameer Beg N Edme M Peter P R Barber T Ng B Vojnovic Imaging protein protein interactions by multiphoton FLIM Proc SPIE 5139 180 189 2003 B J Bacskai J Skoch G A Hickey R Allen B T Hyman Fluorescence resonance energy transfer determinations using multiphoton fluorescence lifetime imaging micros copy to characterize amyloid beta plaques J Biomed Opt 8 368 375 2003 B J Bacskai J Skoch G A Hickey O Berezovska B T Hyman Multiphoton imag ing in
155. sented by aj az in the lifetime images is shown right The distributions of To Tret and Neret No over the area of the cell are shown in Fig 104 The results show clearly that the variation in the fraction of interacting donor is much larger than the variation in the distance Consequently the variation in the single exponential life time Fig 100 is almost entirely caused by a variation in the fraction of interacting proteins not by a change in distance In other words interpreting variations in the single exponential lifetime as distance variations leads to wrong results Fig 103 FRET results obtained by double exponential lifetime analysis Left 7 7 Right Ngfre No Please note that the relative parameter range is the same for both images Data courtesy of Dr Harald Neumann Life and Brain K ln Germany 92 Applications 2 4 B 0 5 1 Fig 104 Distribution of W Tre left parameter range from 1 to 6 and N e No right parameter range from 0 2 to 1 2 over the area of the cell Similar double exponential decay behaviour is commonly found in FRET experiments based on multi dimensional TCSPC 2 11 15 16 32 38 46 87 Double exponential decay pro files have also been confirmed by streak camera measurements 30 31 A general characterisation of TCSPC FLIM FRET for monitoring protein interactions is given in 32 40 41 87 97 Applications to protein interaction related to Alzheimer s disease are described in
156. ses delivered by the R3809U are amplified by an HFAH 26 01 pream plifier and connected into the CFD input of the SPC 830 module The timing reference sig nal SYNC signal of the TCSPC module is obtained from the laser The signal from the BDL laser is positive therefore an A PPI pulse inverter is inserted in the SYNC line The scan clock signals of the microscope controller are connected to the upper sub D connector of the SPC 830 In case of detector overload the preamplifier delivers an overload signal A second overload signal comes from a photodiode in front of the shutter Both signals are combined in the p box and connected to the overload input of the DCC 100 Dual PMC 100 System Although only one output from the FV1000 scan head is available dual detector systems can be built by using standard optical components delivered by bh An example is the dual detector system shown in Fig 19 The excitation part of the system is the same as shown in Fig 16 The laser power is con trolled via output 1 of a DCC 100 card The fibre from the scan head is connected to a beamsplitter assembly The assembly contains a collimator lens and a dichroic mirror The dichroic mirror splits the light into a short wavelength and a long wavelength component The components are detected by two PMC 100 detectors Individual long pass or bandpass filters can be inserted in front of the detectors The detectors are controlled via the two outputs of
157. shutters open and close If you don t there is either light in front of the shutters or the shutter control cables or the power supply of the p box are not cor rectly connected Turn on the laser If you have a picosecond diode laser set the pulse repetition rate to 50 MHz If you have an older Ti Sapphire laser make sure that it is pulsing Check the SYNC rate display in the SPC main panel The frequency displayed must correspond to the repetition rate of the laser If there is no rate displayed or the rate is wrong change the SYNC threshold Click into SYNC field of the main panel and select Threshold You should achieve correct synchronisation with in threshold range of 40 to 100 mV possibly even be yond If there is no synchronisation check the signal at the SYNC input The pulse amplitude should be negative Close the shutters Start the measurement in the Oscilloscope mode Pull up the gain slider of the detector If you have two detectors pull up the slider for only one detector When the gain comes into the region of 80 to 100 of the maximum permitted for the detector you should see the first detected photons The CFD TAC and ADC rate bars start to display a rate and the first photons dark counts should show up in the curve display Please note that Count Increment was set to 10 therefore a single photon is displayed as a vertical line of 10 counts Even if there is absolutely now light at the detector
158. stant BB Equidistant Ae Nook Windows Fe Nook windows Ae Noof windows Ae Noot windows Fe Noot windows Wind From To Wind From To owon a wn gt a Return Esc Fig 66 Window parameters Time window 1 covers the photons of all time channels the other time windows define subsequent time gates in which the intensity can be displayed Click the System Parameters and Window Parameters off Turn the Display Parameters on and configure the main panel as shown in Fig 67 b SPC 830 Main Parameters Display Star Interrupt Stop Exit W1 Scan Sync In Mode scan firstlight zeiss set lb Display Parameters W1 z Fy b DCC 100 Main Parameters Exit per Juw Max H jt H v Ja Ja E F w jr F m 1e8 H Bel LE Measurement _ gt Ow jes ow et ft a Device state un a a8 Jw es SYNC d Scan Clocks re Set at 0 gu 0 eo 0 00E 0 User break cycle 1 of 100 m ho a B AAi ila Dig Zoom _ EEA w cll al eae f Collection c3 Range Limit Low E Div ho tt at oo o Time Eu SYNC CFD TAC ADC a 1000 AC al 50 00E 9 D y 49 02 ME aj K Fig 67 Main panel configuration recommended for FLIM The Display Parameters panel is placed upper right the DCC 100 detector control panel lower right Recording the First Image Start a continuous scan in the microscope Check the Scan Clocks indicator see Fig 68 If the scan clocks are present the indic
159. supply or unintentional activation of detectors Please note The DCC software can be configured to turn on the outputs automatically Option Enable Outputs on Startup This option is not intended for operation of PMTs and should not be used in con junction with the bh FLIM systems Power supply of detectors The power supply of the PMC 100 H7422P 40 and PML 16 detectors is provided by the DCC 100 To operate these detectors the corresponding power supply buttons have to be acti vated The PMC 100 and the H7422P 40 have internal coolers If you use these detectors activate the cooling by clicking on the Cooling On and set a cooling current of 0 5 to 1 A Set the cooling voltage to maximum 5 V Gain of Detectors The gain of the detectors is set by the sliders under Connector 1 and Connector 2 When changing the detector gain please remember that the detectors work in the photon counting Data Acquisition 39 mode In other words the detector delivers a pulse for every photon detected The data acqui sition system counts these pulses The light intensity is proportional to the number of pulses per time interval A change in detector gain changes the amplitude of the single photon pulses not their frequency The detector gain is therefore adjusted in order to obtain a single photon pulse amplitude well above the threshold of the input discriminator of the TCSPC card the CFD threshold With the right combination of C
160. synchronisation path from the laser to the SPC module 60 System Setup With the TAC settings recommended in Fig 62 the recorded time interval is slightly shorter than the signal period of the laser Within the recorded range you may therefore change the signal position also by changing TAC offset Details are described in 22 When you have made these adjustments save the setup parameters of the SPC module Adjusting the CFD Parameters CFD Threshold The detectors of the bh FLIM systems are operated in the photon counting mode That means the detector delivers an electrical pulse at the detection of nay individual photon Although the times of these pulses correlate tightly with the arrival times of the photons the pulse ampli tudes vary randomly see Fig 5 page 6 The average pulse amplitude increases with the detec tor gain To achieve good efficiency of a photon counting system a discriminator threshold CFD Threshold must be found that detects almost all of the photon pulses but suppresses noise from the environment from the preamplifiers and from the detector itself Finding a reasonable CFD threshold is simple Vary the CFD threshold and watch the CFD and TAC count rates With decreasing CFD threshold the count rate increases At some level the increase flattens and for good detectors turns into a plateau When you see something like a plateau it may be not very pronounced you detect the majority of the pulses
161. tems The maximum continuous count rate of the R3809U is about 1 10 photons per second Al though this is enough for the majority of applications it should be noted that the R3809U is not a solution to fast acquisition FLIM To achieve maximum safety against overload damage the R3809U is connected to the scan head via a shutter PMC 100 The PMC 100 PMT module is the standard detector for all bh FLIM systems It delivers an IFR of 150 ps FWHM 22 Lifetimes down to about 200 ps are easily resolved The PMC 100 features excellent timing stability at high count rates It can therefore be used up to the highest count rates applicable with the bh TCSPC boards without noticeable degradation in the IRF 22 Typical applications are pH imaging oxygen imaging and ion concentration measurements via fluorescence quenching The PMC 100 works well also for single exponential FRET measurements 1 e experiments that do not require separating of the inter acting and non interaction donor fraction Separating the different donor fractions of FRET is generally possible However the longer IRF makes the data analysis more difficult and less accurate than for the R3809U Autofluorescence imaging is possible as well though with some compromise in resolution for the shortest lifetime components All in all the PMC 100 is the most versatile detector for one photon FLIM systems H7422P 40 The H7422P 40 detector 56 has an exceptionally high quantum effic
162. the zoomed image of the cell has also 5 times more pixels and thus contains the same number of photons per pixels as Fig 77 left This is correct However with the zoom you may either use pixel binning during the FLIM acquisition see Scan Parameters page 36 or in the lifetime analysis see Binning in the Lifetime Analysis page 80 You then obtain an image that shows the cell with the same number of pixels as Fig 77 left but with a higher number of photons Binning in the lifetime analysis has the additional benefit that only the lifetimes are binned not the intensity information Details of FLIM Data Acquisition 73 Single Point Fluorescence Decay and FCS Measurements The bh SPC 830 SPC 140 and SPC 150 TCSPC modules can be used for combined fluores cence decay and fluorescence correlation FCS measurement in selected points of a sample 19 22 23 49 The measurement is performed in the FIFO mode The FIFO mode records the time in the laser pulse sequence the time from the start of the experiment and in case of a multi detector setup the detector channel number for each individual photon Because the photons are recorded with their detection times the FIFO mode is often called time tag mode System parameters suggested for combined decay and FCS measurement are shown in Fig 78 the recommended main panel configuration in Fig 79 Time Criari 1 04858E 7 SDE Resolution felernory Offset Decay
163. the R3809U but nevertheless unwelcome The second reason for not using fibre coupling is efficiency Coupling into the fibre inevitably causes loss in intensity in case of the FV 1000 fibre port up to 60 Except for the 1d 100 SPAD detector which requires a fibre connection the bh FLIM systems therefore use direct optical coupling The fibre adapter is removed or not installed altogether and an a direct coupling optics attached instead The FLIM adapter is shown in Fig 13 The FLIM beam pass contains a long pass or bandpass filter and in case of the R3809U detector an electronically controlled shutter Details are shown in Fig 51 and Fig 52 page 51 Filter gt gt k q Fig 13 Olympus Fluo View FV1000 scan head with bh FLIM adapter and FLIM detector attached The detector is an R3809U MCP PMT The shutter is used to protect the R3809U MCP PMT against high light intensities from the microscope lamp The other detectors are sufficiently protected by the overload shutdown function of the DCC 100 detector controller They are therefore operated without a shutter For dual detector operation bh provide suitable shutter beamsplitter assemblies Both dichroic and polarising beamsplitters are available An example is shown in Fig 14 One Photon Confocal FLIM Detection Systems 15 Beam R3809U Speed R3809U Fig 14 Dual detector assembly with two R3809U MCP PMTs Both dichroic and polarising beamsplitters are
164. the sample and that the donor bleedthrough into the acceptor fluorescence cannot be suppressed spectrally Steady state FRET techniques therefore require careful calibration including measurements of samples containing only the donor or the ac ceptor The calibration problems can partially be solved by the acceptor photobleaching tech nique An image of the donor is taken then the acceptor is destroyed by photobleaching and another donor image is taken The increase of the donor intensity is an indicator of FRET The drawback is that this technique is destructive and that it is difficult to use in living cells The use of FLIM for FRET has the obvious benefit that the FRET intensity is obtained from a single lifetime image of the donor No reference images or calibration measurements are nec essary Fig 100 shows a single exponential lifetime image of a cultured BHK baby hamster kidney cell that has different proteins labelled with GFP and CY3 The specimen was excited by a BDL 473 SMC laser 473 nm The detector was an R3809U 52 MCP PMT 90 Applications Fig 100 FRET between GFP donor and CY3 acceptor Single exponential lifetime image of the donor fluo rescence lifetime range 1 4 ns to 1 9 ns Data courtesy of Dr Harald Neumann Life and Brain K ln Germany Single exponential lifetime images as the one shown in Fig 100 are very useful to locate the areas in a cell where the labelled proteins interact They do however
165. time Images 81 ee EE hl THEE Le eT a SE _ JET gl Za oF gt a a o i if E o JE if A AE IN EE Mh JEF OIE EE NEEE BEN EEE FFF HEE OEE es sae Eee ___ ee Intensity Pixels Lifetime Pixels Oversampled Point spread function Binning of lifetime data Fig 90 Left Oversampling of the Airy disc in the intensity image and binned pixels for lifetime calculation Right Overlapping binning of pixels for lifetime calculation The binning is controlled by the Bin parameter above the decay curve window The function of the parameter is shown in Fig 91 Bin defines the number of pixels around the current pixel position Please note that the number of pixels of the lifetime image is not reduced Only the lifetimes are calculated from the combined pixels the intensities remain unbinned Binning 2n 1 x 2n 1 area n pea 0 1 2 3 Fig 91 Function of the binning parameter Fig 92 shows lifetime images obtained from 256 x 256 pixel raw data The binning factor used was 0 1 and 2 left to right It can be seen from these images that there is very little loss in lifetime detail due to the binning However the noise in the lifetime is considerably re duced Even larger binning is often acceptable Fig 93 left to right shows lifetime images obtained with binning 4 8 and 10 Even with these large binning factors the general struc tures in the lifetime remain unchan
166. tors and switch on the operat Semsstort Gonnector2 Connectors o iaing BAHN O ganv mav Won ing voltage buttons for all DCC connectors that control detec gi Jev mev cnn tors If you have a cooled detector PMC 100 or H7422 switch H5 MN 2 on the cooling and set it to 5V and a current between 0 5 and Je OS 1 A Then switch on the outputs by clicking on the Enable Out so mm Po puts button a Caution The gain sliders control the high voltage of the detec rf at r A 0 tors The DCC is used for a wide variety of detectors and high ae voltage power supplies It can happen that the gain or the high EEE voltage can be set higher than the permitted maximum of a 4 Pr given detector The DCC software therefore allows the user to Fig 60 DCC main panel set limits for the Gain HV The limits are accessible under Pro duction amp Adjust Data see Fig 41 page 40 Please make sure that the limits are reasonably set for your detector and high voltage power supply Although bh normally set correct limits in DCC modules delivered in conjunction with detectors this is not necessarily the case if the DCC and the detectors are purchased independently Shutters Detectors and Synchronisation Make sure that the detector gain regulators of the DCC panel are pulled down Enable the DCC outputs If you have a multiphoton NDD system check the shutters Click on the bO and b1 buttons You should hear the
167. triggered sequences of decay curves or even small images at high speed Typical applications are electro physiology ex periments or chlorophyll transients 6 21 22 It should however be noted that most of these techniques are not finally explored or even tested in conjunction with commercially available laser scanning microscopes One Photon FLIM versus Multi Photon FLIM As described above FLIM requires a pulsed excitation source of both high repetition rate and picosecond pulse duration This may be a picosecond diode laser a frequency doubled Ti Sapphire laser or the Ti Sapphire laser of a multiphoton microscope In terms of FLIM signal recording there is no difference between these systems There is however a difference One Photon FLIM versus Multi Photon FLIM 9 in the way the sample is excited and the fluorescence light is detected This results in a num ber of optical differences which are discussed below It should also be noted here that multiphoton excitation is covered by patents owned by Zeiss 43 and Leica 57 The patent situation and thus the availability of the multiphoton technique for the FV1000 depends on the country you are living in The general optical principle of a laser scanning microscope 44 79 85 is shown in Fig 7 One photon excitation is shown left two photon excitation right ps Diode Laser Pinhole Pinhole Ti Sa Laser Dichroic Dichroic Mirror Detector Mirror Confocal Confocal Sca
168. vailable laser input at the FV1000 is not necessarily compatible with these wavelengths due to the internal dichroic mirrors of the scan head The beam corrector is a development of LASOS GmbH Jena Germany 14 The FV 1000 FLIM Systems The standard FV1000 FLIM systems do not use the beam blanking capability of the BDL 405SMC laser Please contact bh if you need beam blanking Optical Interface One photon excitation requires confocal detection to achieve suppression of out of focus light see Fig 7 page 9 The scan mirrors the pinhole and the dichroic mirror that separates the fluorescence light from the excitation light are contained in the Olympus FV1000 scan head The scan head con tains several photomultipliers detecting the fluorescence signal in selectable wavelength inter vals Unfortunately these detectors and the associated preamplifiers are not fast enough for reasonable FLIM data acquisition Upgrading the FV1000 scanning microscope therefore re quires one or several fast detectors to be attached to an optical output from the FV1000 scan ner The designers of the Olympus Fluo View FV1000 have provided for such an output in form of a fibre port Although the fibre port can easily be used to transfer the light to a FLIM detector it 1s not the favoured FLIM solution for two reasons The first one is that the multi mode fibre used introduces pulse broadening The effect is no ticeable only for ultra fast detectors such as
169. ven for an image of 512 x 512 pixels a relative lifetime accuracy of 0 1 or 10 is obtained within less than 30 seconds for smaller pixel numbers the acquisi tion time is correspondingly shorter However an acquisition time of almost 1 hour would be required to record lifetime image of 512 x 512 pixels with a lifetime accuracy of The accuracy obtained in practice may differ from the values shown in Fig 76 In particular the background count rate in FLIM experiments is often not negligible and the fluorescence may not decay entirely within the laser pulse period Moreover a count rate of 10 s is a rela tively optimistic assumption The required number of photons increases if double exponential decay functions are to be resolved In 65 the number of photons required to resolve a double exponential decay was estimated to be N 400 000 A number of photon per pixel this high is of course entirely beyond the limits set by the photostability of a biological sample 65 is therefore often used as an argument that double exponential lifetime imaging is impossible Fortunately the prospects to separate two lifetime components improve dramatically with the ratio of the two lifetimes and with the amplitude factor of the short lifetime component The lifetime components assumed in 65 were 10 of 2 ns and 90 of 4ns This is an ex tremely unfavourable situation which indeed requires an extremely high number of photons Fortunately the de
170. with sub cellular resolution and picosecond time resolution using intense near infrared femtosec ond laser pulses Proc SPIE 4620 191 202 2002 K Konig I Riemann High resolution multiphoton tomography of human skin with subcellular spatial resolution and picosecond time resolution J Biom Opt 8 432 439 2003 K Konig I Riemann G Ehrlich V Ulrich P Fischer Multiphoton FLIM and spectral imaging of cells and tissue Proc SPIE 5323 240 251 2004 J R Lakowicz I Gryczynski W Wiczk J Kusba M Johnson Correction for incom plete labeling in the measurement of distance distributions by frequency domain fluo rometry Anal Biochem 195 243 254 1991 J R Lakowicz H Szmacinski K Nowaczyk M L Johnson Fluorescence lifetime 1m aging of free and protein bound NADH PNAS 89 1271 1275 1992 J R Lakowicz Principles of Fluorescence Spectroscopy 2nd edn Plenum Press New York 1999 A Lleo O Berezovska L Herl S Raju A Deng B J Bacskai M P Frosch M Iri zarry B T Hyman Nonsteroidal anti inflammatory drugs lower AB42 and change pre senilin 1 conformation Nature Medicine 10 1065 1066 2004 D Magde E Elson W W W Webb Thermodynamic fluctuations in a reacting system measurement by fluorescence correlation spectroscopy Phys Rev Lett 29 705 708 1972 J Malicka I Gryczynski C D Geddes J R Lakowicz Metal enhanced emission from indocyanine green a new approach to in vivo imaging J
171. y Use a uniform sam ple or a mirror in the image plane Run a repetitive scan of the FV1000 You may either check the images displayed by the FV1000 software or use the Oscilloscope Mode of the TCSPC module see Fig 58 You may need to take out the laser blocking filter of the detector see Fig 51 to Fig 53 Step 5 Adjust Al and A2 for maximum intensity This step is a lateral shift of the optical axis Therefore turn both screws in the same direction until you find the setting that yields maximum intensity Step 6 Adjust B1 and B2 for maximum intensity This step is a lateral shift of the optical axis Therefore turn both screws in the same direction until you find the setting that yields maxi mum intensity Installing the Detectors Direct coupled Confocal Detectors The standard solution for confocal FLIM is direct coupling of the FLIM detectors to the FV 1000 scan head The opto mechanical principle is shown in Fig 52 and Fig 51 In the FV1000 scan head a bracket is installed instead of the output fibre coupler The bracket holds the FLIM detector and the associated optical elements One Photon Confocal Microscopes 51 The optical principle for the PMC 100 detector is shown in Fig 51 The fluorescence light leaves the FV1000 scan head in a roughly collimated beam of light The light passes a filter holder that holds a removable emission filter An additional laser blocking filter can be in serted in a barrel directly
172. ystems con taining several SPC 140 or SPC 150 cards see Fig 11 ae x sju zH i S F oe Fig 11 Package of four parallel SPC 140 channels for high speed FLIM applications Two and four channel TCSPC systems require additional beamsplitters at the optical outputs of the FV1000 microscope Four channel TCSPC systems have a saturated count rate of 40 10 photons per second and can be reasonably operated at a recorded total count rates up to of 20 10 s Thus single exponential lifetime images can be obtained within acquisition times as shorter than a second 17 21 It should be noted however that most samples do not deliver count rates this high without substantial photobleaching Therefore please make sure that the photostability of the samples you are going to investigate is high enough to justify the cost of a two or four channel TCSPC system One Photon Confocal FLIM Detection Systems Picosecond Diode Laser Fluorescence lifetime imaging requires a pulsed excitation source This is no problem in multi photon microscope The titanium sapphire laser meets the requirements of TCSPC FLIM almost ideally Standard Fluo View FV1000 microscopes however use continuous lasers To upgrade these systems for FLIM a pulsed excitation source must be added The bh FLIM systems use a BDL 405SMC picosecond diode laser for excitation This laser has a One Photon Confocal FLIM Detection Systems 13 wavelength of 405 nm The l
173. z D Grosenick A Liebert High count rate mul tichannel TCSPC for optical tomography Proc SPIE 4431 249 245 2001 W Becker A Bergmann C Biskup T Zimmer N Klocker K Benndorf Multi wavelength TCSPC lifetime imaging Proc SPIE 4620 79 84 2002 W Becker A Bergmann C Biskup L Kelbauskas T Zimmer N Klocker K Benn dorf High resolution TCSPC lifetime imaging Proc SPIE 4963 175 184 2003 W Becker A Bergmann M A Hink K K nig K Benndorf C Biskup Fluorescence lifetime imaging by time correlated single photon counting Micr Res Techn 63 58 66 2004 W Becker A Bergmann G Biscotti K Koenig I Riemann L Kelbauskas C Biskup High speed FLIM data acquisition by time correlated single photon counting Proc SPIE 5323 27 35 2004 W Becker A Bergmann G Biscotti A R ck Advanced time correlated single photon counting technique for spectroscopy and imaging in biomedical systems Proc SPIE 5340 104 112 2004 W Becker A Bergmann E Haustein Z Petrasek P Schwille C Biskup T Anhut I Riemann K Koenig Fluorescence lifetime images and correlation spectra obtained by multi dimensional TCSPC Proc SPIE 5700 2005 W Becker A Bergmann C Biskup D Schweitzer M Hammer Time and Wave length Resolved Autofluorescence Detection by Multi Dimensional TCSPC Proc SPIE 5862 58620S 2005 96 21 22 23 24 25 26 21 28 29 30 31 32

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