SPC3 User Manual - Micro Photon Devices
Contents
1. SPC3 Single Photon Counting Camera 64x32 x IM PHOTON DEVICES Introduction SPC is a single photon counting camera based on a 2 D imaging array of 64 x 32 smart pixels Each pixel comprises a single photon avalanche diode SPAD detector an analogue front end and a digital processing electronics This on chip integrated device provides single photon sensitivity high electronic noise immunity and fast readout speed The imager can be operated at full resolution with a maximum frame rate of about 100 000 frames per second with negligible blind time dead time Inside each pixel three independent counters are integrated Each one of them can be independently gated through an external signal counter 1 can also be gated by an internally generated signal e g for FLIM mode acquisition Counters 2 and 3 instead can also count downward with their direction controlled through an external signal SPC pixels feature high photon detection efficiency PDE in the visible and near UV spectral region and low dark counting rates even at room temperature The imager is easily integrated into common optical setups thanks to a Thorlabs SM1 thread a C mount mechanical adapter and a high speed USB 3 0 computer interface The camera is shown in Figure 1 The camera differs from conventional Charge Coupled Devices CCD or CMOS sensors because it performs a fully digital acquisition of the light signal Each pixel effectively counts the number2. 350 300 ho un ao sg te Counts a u i i 150 A t oO 10 Time ns Figure 14 Screenshot of the FLIM window In this case the acquisition is the IRF of the system when Gate width is set to 2 ns and 800 gate steps spanning 16ns are acquired v 1 0 1 Micro Photon Devices S r l Italy All Rights Reserved Page 32 SPC3 Single Photon Counting Camera 64x32 x IM PHOTON DEVICES SPC3 SDK File Formats and ImageJ Plugin SPC is provided with a Software Development Kit SDK composed by a shared library and few example projects The SDK allows the user acquire data from the SPC and to change operating parameter directly from their own application It is available for Windows Mac OS X and Linux operating systems both in 32 and 64 bit versions More information on available functions can be found in the related documentation included in the USB key Acquired data from Snap acquisitions can be saved both in OME TIFF format and in a MPD proprietary binary file format Data from Continuous acquisition can only be saved in MPD proprietary format OME TIFF file could be opened with any image reader compatible with TIFF file since metadata are saved into the Image Description tag in XML format In order to decode OME TIFF metadata it is possible to use free OME TIFF readers such as OMERO or the Bio Formats plugin for ImageJ For more details see the OME TIFF web site http www openmicr
3. v 1 0 1 Micro Photon Devices S r l Italy All Rights Reserved Page 19 SPC3 Single Photon Counting Camera 64x32 x IM PHOTON DEVICES T 10 40 us 208 lt 511 PET 50 ns Nmax HIT thus any overflow is avoided since 511 is the maximum number that can be counted by a 9 bit counter before overflowing Instead when using all the three counters T is equal to 31 20 us and with the minimum Tgeag Of 50 ns the maximum number of photons that can be counted is T 31 20 ps Naar 624 gt 511 max HIT e 50 ns and the overflow may occur with very strong light As a consequence in order to minimise the probability of incurring in overflows or even to completely avoid them in some cases a clever strategy is to obtain long Frame Exposure Times by accumulating many Hardware Integration Times each equal to the Hardware Readout Time directly in the SPC3 Due to the short inter frame dead time smaller than 10 ns this operating mode induces only negligible signal losses and does not increase the noise since read out noise is not present in the SPC Camera In advanced mode both Hardware Integration Time and Frame Exposure Time are user adjustable thus particular care must be taken by the user in order to avoid artefacts due to the overflow of the integrated 9 bit counters which is no more avoided or minimized In addition also the ability to achieve shorter Equivalent Hardware Integration Time on Counter 1 by u
4. ap I PHOTON DEVICES MICRO PHOTON DEVICES SPC Single Photon Counting Camera Micro Photon Devices S r l Via Stradivari 4 39100 Bolzano BZ Italy email info micro photon devices com Phone 39 0471 051212 e Fax 39 0471 501524 SPC User Manual Version 1 0 1 October 2015 Micro Photon Devices S r l Italy All Rights Reserved SPC3 Single Photon Counting Camera 64x32 Table of Contents INTRODUCTION SINGLE PHOTON AVALANCHE DIODE Further readings SPC3 HARDWARE CHARACTERISTICS Electrical connections Camera mechanical dimensions SPC OPERATION Standard operation Gated operation FLIM Operation Further Readings Acquisition parameters Integration time Dead time time and dead time correction Background subtraction Additional information Calculation of the Snap size and the Continuous Acquisition Data rate Camera auto protection SPC3 SOFTWARE INTERFACE VISUALSPC WINDOWS ONLY Installation Software Interface Main window Control Panel Main Window Plot settings and Playback Control Main Window Control Buttons Main Window Acquisition Parameters Summary Counter Window FLIM Window SPC3 SDK FILE FORMATS AND IMAGEJ PLUGIN SYSTEM REQUIREMENTS COPYRIGHT AND DISCLAIMER v 1 0 1 Micro Photon Devices S r l Italy All Rights Reserved x I PHOTON DEVICES 10 11 11 12 14 18 18 18 20 23 23 23 24 25 25 25 26 28 30 31 31 a2 33 36 36 Page 2
5. e Import unsigned data as is and convert signed data to 32bit float e Import all data as is this means ImageJ will interpret signed data as unsigned e g the number 1 which in 8 bit binary is 11111111 will be shown as 255 If data is 16bit there are four options e Convert data to 32bit float e Import unsigned data as is and convert signed data to 32bit float e Import all data as is this means ImageJ will interpret signed data as unsigned e g the number 1 which in binary is 11111111 will be shown as 255 e Import all data as is and use an ImageJ calibration function to map signed data this means ImageJ will remap data to show signed values e g the number 1 which in 16 bit binary is 1111111111111111 and will read as 65535 without remapping will be properly shown as 1 If the selected file is properly formatted and no error occurred an image window for each SPC3 counter enabled opens Frames can be skimmed using the controller at the bottom of each window Selecting from the menu the command Image gt Show Info will show all camera parameters for the acquisition v 1 0 1 Micro Photon Devices S r l Italy All Rights Reserved Page 35 x IM PHOTON DEVICES SPC3 Single Photon Counting Camera 64x32 System requirements High speed USB 3 0 interface for full speed acquisition compatible with USB 2 0 Host computer minimum requirements o 2 GHz processor and 1 GB of RAM o SSD recommended for full soeed continuo
6. In order to import OME TIFF file generated by SPC3 camera simply select Load FLIM data from File menu For further support on FLIMfit please contact directly its developer SPCF file are binary files composed by a header with acquisition metadata followed by raw image data containing the 8 16 bit pixel values in row major order SPCF file can be read using the provided ImageJ Fiji plugin In addition since the format is fully documented in SPC3 SDK documentation users can directly read data from their own code v 1 0 1 Micro Photon Devices S r l Italy All Rights Reserved Page 17 SPC3 Single Photon Counting Camera 64x32 x IM PHOTON DEVICES Further Readings 1 Joseph R Lakowicz Principles of Fluorescence Spectroscopy 3rd edition Springer 2006 J Pawley Handbook of Biological Confocal Microscopy 3rd Edition New York Plenum Press 2006 T W J Gadella Ed FRET and FLIM Techniques Volume 33 Amsterdam Elsevier Science 2008 oes S P Chan Z J Fuller J N Demas and B A DeGraff Optimized gating scheme for rapid lifetime determinations of single exponential luminescence lifetimes Anal Chem vol 73 no 18 pp 4486 4490 2001 5 D D U Li S Ameer Beg J Arlt D Tyndall R Walker D R Matthews V Visitkul J Richardson and R K Henderson Time domain fluorescence lifetime imaging techniques suitable for solid state imaging sensor arrays Sensors vol 12 no 5 pp 5
7. Wait for extemal trigger Enable sync out Frame sync O Ref clock Playback Control onoono 000 N N p LE te de te O Figure 12 Screenshot of the main window of the graphical user interface v 1 0 1 Micro Photon Devices S r l Italy All Rights Reserved Page 25 SPC3 Single Photon Counting Camera 64x32 x IM PHOTON DEVICES Main window Control Panel This panel allows setting all the camera hardware parameters In the Exposure Parameters subsection it is possible to set the image integration parameters exposure time number of frames for Snap mode and hardware binning Either normal or advanced modes are available There are four numeric fields see Table 3 for an example of settings which can be either editable or automatically adjusted by the software depending of the selected mode namely v Frame exposure time Normal only integration time for each frame required by the user The possible values range between 10 40 us and 2 40 ms This value does not correspond to the Hardware Integration Time of the sensor Longer exposure times are obtained by binning a variable number of frames vV Hardware Integration time physical integration time of the SPC sensor This value is fixed to the Hardware Readout Time in normal mode In advanced mode it can range from 10 ns to 655 25 us v Hardware binning number of frames of duration equal to Hardware Integration Time that are summed up before
8. 8 It is also possible to move along the data using the scrollbar The current frame number and FLIM step if in FLIM mode are also shown v 1 0 1 Micro Photon Devices S r l Italy All Rights Reserved Page 28 SPC3 Single Photon Counting Camera 64x32 x IM PHOTON DEVICES Table 7 Plot settings and control buttons Automatically sets the black and white levels of the image according to the present frame the black level is assigned to the pixel with the minimum number of detected photons in the frame while the white level is assigned to the ones with the maximum count This setting will remain applied also for the following frames Since it is possible to have in the following frames pixels with values lower than black or higher than white they will be respectively indicated in blue or red Resets black and white levels to default values 0 255 for 8 bit images and 0 65535 for 16 bit images Flip the image along the X axis Flip the image along the Y axis Rotate the image by 90 clockwise Keep Windows Docked If selected moving the main window will also move together all the other windows Go to the previous frame Go to the next frame Go to the last acquired frame Play the image sequence backward Continuous rewind When the last first acquired frame is displayed it restarts from the first last frame Play the image sequence forward v 1 0 1 Micro Photon Devices S r l Italy All Rights Reserv
9. dead time 100ns and T 1ms Number of measured Photons 10 10 10 10 10 10 Number of incoming Photons Figure 2 Number of measured photons in an image of 1 ms exposure and dead time of 100 ns as a function of the number of incoming photons PDE 100 for simplicity Let s suppose for example an exposure time T 1 ms a PDE 50 for simplicity and a dead time Tgeag 100 ns In this case the maximum number of photons that can be counted or more in general the maximum T Tde number of SPAD triggerings is 10 000 In our example also Nimp PDE corresponds to 10 000 this ad means that when a photon flux of 20 000 000 photons s hit the detector during the integration time T only 20 000 Nimp photons cross the active area but only 10 000 can be detected due the PDE of 50 and finally only 5 000 events are on average counted Ncounted because of saturation See for example Figure 2 that shows the number of counted photons as a function of the number of the impinging photons for an exposure time T 1 ms a dead time of 100 ns and a PDE of 100 for simplicity Further readings M Ghioni A Gulinatti Rech F Zappa S Cova Progress in Silicon Single Photon Avalanche Diodes IEEE Journal of Selected Topics in Quantum Electronics 2007 13 852 862 D Bronzi F Villa S Tisa A Tosi F Zappa D Durini S Weyers W Brockherde 100 000 Frames s 64 x 32 Single Photon Detector Ar
10. duration of a GATE IN pulse is 5 ns The actual internal gate is delayed by about 15ns Input to select the counting direction of counters 2 and 3 The input has a 50 Ohm DC input DIRECTION v 1 0 1 impedance 3 3V LVCMOS signals are accepted but the input is 5V tolerant A low value means UP counting DIRECTION timings TBD Micro Photon Devices S r l Italy All Rights Reserved Page 7 SPC3 Single Photon Counting Camera 64x32 x IN PHOTON DEVICES MULTI COAXIAL CONNECTOR Figure 3 Camera side views hardware connections 1 TRIGIN 2 Direction 3 SYNC OUT 4 GATE 1 5 GATE 2 N 6 GATE 3 Figure 4 Camera with the cable adapter and the SMA output description TRIG IN Acquisition Start SYNC OUT or Figure 5 Timing of TRIG IN and SYNC OUT signals with respect to the actual start of the acquisition v 1 0 1 Micro Photon Devices S r l Italy All Rights Reserved Page 8 SPC3 Single Photon Counting Camera 64x32 MPD MICRO PHOTON DEVICES Table 2 Electrical Characteristics of the SPC measured at room temperature 25 C Parameter Signal TRIG IN GATE Input logic high voltage 5V tolerant DIRECTION i 2 4 Input logic low voltage IRG oe Soli 0 pee 8 DIRECTION tout SYNC OUT pulse duration frame sync mode SYNC OUT tir Input rise time 10 90 TRIG IN GATE Input fall time 10 90 TRIG IN GATE Output rise time SYNC OUT tof Output fall time SYNC OUT 1 ns tate GATE pul
11. labelled as donors and acceptors Such measurements are used as a research tool in fields such as biology and chemistry Different ways of performing FRET measurement exist The obvious problem with the intensity based steady state FRET measurements is that the emission band of the donor extends into the emission band of the acceptor and the absorption band of the acceptor extends into the absorption band of the donor Furthermore the concentrations of the donor and acceptor and the fraction of donor molecules linked to an acceptor molecule are variable and unknown All this makes the intensity based FRET measurements requiring calibration with the samples containing only donors and acceptors or measurements in which the second step is the destruction of acceptor by photo bleaching obtaining the FRET measurement as the relative increase of donor fluorescence intensity The FLIM based FRET measurement has the advantage that the results are obtained from a single lifetime measurement of the donor and are not affected by the changes of the sample concentration excitation intensity and other factors that limit the intensity based FRET measurements FLIM images are most often obtained by employing the Time Correlated Single Photon Counting TCSPC technique and a point detector such as a PMT which is then scanned in order to reconstruct the decay histogram for each image pixel The exponential decay model is fitted to each pixel histogram in order to deter
12. paragraph Force sbt mode Available only in Advanced mode Data are truncated to 8bit Useful to reduce required bandwidth In the Software Gating subsection it is possible to configure the internal gate generation which applies only to Counter 1 Two gated operation mode are possible Periodic Gate mode and FLIM mode In Periodic Gate mode a periodic signal is continuously applied to Counter 1 as explained in Table 5 Table 5 Periodic Gating section configuration explained Enable Gate Enable Disable the hardware gating 6a Change the delay of the gate signal respect to the internal reference clock The delay value is elay expressed as a percentage of the reference clock period 20 ns E g Delay 10 2 ns delay Duration of the gate on signal This is expressed as a percentage of the internal reference clock Width l period 20 ns E g Width 20 4 ns width In FLIM mode the internal gate is automatically controlled in order to perform a gated FLIM acquisition with parameters explained in Table 6 See paragraph on FLIM operation for further details v 1 0 1 Micro Photon Devices S r l Italy All Rights Reserved Page 27 SPC3 Single Photon Counting Camera 64x32 x IM PHOTON DEVICES Table 6 FLIM mode section configuration explained Enable Disable FLIM mode When FLIM mode is enabled an additional FLIM window pop Enable FLIM mode ups see paragraph below Duration of the gate on signal Thi
13. site http www openmicroscopy org site support ome model ome tiff OME TIFF metadata for FLIM mode acquisition include the ModuloAlongT tag which allows the processing of FLIM data with dedicated FLIM software such as FLIMfit see http www openmicroscopy org site products partner flimfit Exits VisualSPC Micro Photon Devices S r l Italy All Rights Reserved Page 30 SPC3 Single Photon Counting Camera 64x32 x IM PHOTON DEVICES Main Window Acquisition Parameters Summary In this section the information on the acquisition based on the actual parameters is reported The explanation of all the shown parameters is reported in Table 10 Table 10 Explanation of the parameters shown in the Acquisition Parameters Summary panel This is the actual frame rate of a Snap or Continuous Acquisition This is the actual frame exposure time i e the real Hardware Integration Total Frame Time i Time multiplied by the number of internal sums Total acquisition time This is the total time required to conclude the Snap acquisition of Nframes This is the actual inter frame dead time Normally it is lt 10 ns long but may Interframe dead time increase in Advanced mode if very short Hardware Integration Times are selected Bit per pixel Number of bit per pixel 8 or 16 Snap file size File dimension for a Snap acquisition with the current parameters Total saved data only visible during Continuous Acquisition This is t
14. 3 3V LVCMOS are generated at this output for synchronizing the camera with any external device The SPC can be configured by the user to output the internal 50 MHz reference clock synchronised with the internal 100MHz master clock for synchronizing any external instrument such a laser with the internal generated gate The same output can be configured also to generate a signal pulse synchronous with the start of each hardware integration frame The delay between the actual start of the integration frame and the rising edge of this pulse is shown in Figure 5 For more details on gated operation see the corresponding paragraph in the SPC Operation section This coaxial input requires a 3 3V pulse 5V tolerated of minimum duration of 40 ns to start the camera acquisition using a signal from an external device e g a shutter a laser etc The first frame starts between 20ns and 40ns after the TRIG IN rising edge Since the TRIG IN signal is asynchronous respect to the internal clock such delay is of course not deterministic unless it is synched with the SYNC OUT signal The input impedance is 50 Ohm See Figure 5 for reference Active low inputs with 50 Ohm DC input impedance Each input controls one of the three GATE IN 1 GATE IN 2 GATE IN 3 in pixel counters When GATE IN is 1 the incoming photons are not counted by the measuring electronics 3 3V LVCMOS signals are accepted but the inputs are 5V tolerant The minimum
15. 650 5669 2012 Acquisition parameters Integration time Each pixel of the SPC3 camera integrates three 9 bit binary counters two of them with up down capability which measure the number of photons detected during a specified Hardware Integration Time HIT The HIT is thus the time during which the impinging photons are counted without resetting the integrated counters i e the time from when these counters are cleared and started to the time when they are latched and read It can range from the Hardware Readout Time HRT of the array to about 0 65 ms The Hardware Readout Time of the array is proportional to the number of counters Ncounters in use and it always corresponds to the inverse of the frame rate In other words the HRT is the minimum time needed to read the counters in use for all the pixels HRT value is given by HardwareReadoutTime Neounters 5 ns Npixet 160 ns In case of use of a single counter per pixel this time is equal to the time needed to read the 2048 pixels and is 10 40 us for 2 counters it is equal to 20 80 us and for 3 counters it is equal to 31 20 us It must be also noted that even when saving only half the array in order to reduce the mass storage bandwidth requirement the readout will be carried out for the entire imager and so the Hardware Readout Time will not be reduced It is also possible to increase the total integration time by internally inside the camera s internal FPGA accumulating sev
16. Camera 64x32 x JI PHOTON DEVICES 100ns dead time non linearity CORRECTED 100ns dead time non linearity Number of measured Photons 10 10 10 10 10 10 Number of incoming Photons Figure 11 Effect of the dead time corrector in an image of 1 ms exposure and dead time of 500 ns as a function of the number of incoming photons Background subtraction SPC can perform in camera background subtraction For this purpose the user must have previously acquired a dark image i e with the shutter close with the same parameters of the actual acquisition i e the same integration time hold off and gate and then has to enable the background subtraction Additional information Calculation of the Snap size and the Continuous Acquisition Data rate Integration Time and Half Array Saving Mode directly influences the data size of a single Snap mode acquisition and the data rate in Continuous Acquisition mode Snap mode data size in bytes is given by BPP Snap_Size Nyixel_saved Nframes g where Nfames is the number of frames in the Snap and bit per pixel BPP is 8 if there is no internal accumulation of several Hardware Integration Times and both dead time correction and background v 1 0 1 Micro Photon Devices S r l Italy All Rights Reserved Page 23 SPC3 Single Photon Counting Camera 64x32 x IM PHOTON DEVICES subtraction are off 16 otherwise Npixe saved is the number of saved pixel an
17. ICES FLIM Operation Fluorescence Lifetime IMaging FLIM is an imaging technique based on the measurement of the exponential decay rate of the fluorescence from a fluorescent sample rather than of its emission intensity The fluorescence lifetime of a molecule is a measurement of the rate of decay of the emission which is a property of the individual single molecule and thus it is unaffected by changes in probe concentration or excitation intensity FLIM can be used for instance as an imaging technique in confocal microscopy and two photon excitation microscopy A typical application of FLIM is the study of Forster Resonance Energy Transfer FRET the mechanism of energy transfer between two chromophore molecules with the emission band of one overlapping the absorption band of the other When this two components are in close proximity the first one called donor initially in its electronic excited state may non radiatively without emission of the light transfer the energy to the second one called the acceptor The result is the quenching of the donor fluorescence thus the decrease of the donor emission lifetime The efficiency of the energy transfer is inversely proportional to the sixth power of distance between donor and acceptor making the effect noticeable only at distances shorter than 10 nm FRET is used to determine the proximity in the nano meter range between proteins or other elements of interest associated with suitable fluorophores
18. LIM measurement 2 gate shift of 1 FLIM measurement n gate shift of 1 FLIM measurement 1 gate shift of 2 FLIM measurement 2 gate shift of 2 FLIM measurement n gate shift of 2 FLIM measurement etc v 1 0 1 Micro Photon Devices S r l Italy All Rights Reserved Page 31 SPC3 Single Photon Counting Camera 64x32 x IM PHOTON DEVICES Figure 13 Screenshot of the Counter window FLIM Window When FLIM mode is enabled an additional FLIM window pop ups as shown in This graph represents the counted photons in each step Gate Shift of the current FLIM acquisition for a selected pixel and it is thus the TCSPC histogram that reconstructs the waveform under investigation convoluted with the IRF of the system The IRF is roughly a rectangular shape with a width equal to the Gate width To change which pixel is visualized simply click on the desired pixel in Counter 1 window The graph is updated in real time in Live mode In Snap mode it shows the entire FLIM waveform corresponding to the currently visualized FLIM acquisition i e it will not change if in Counter 1 a different FLIM step from the same FLIM acquisition is visualized It is possible to choose between linear and logarithmic scale for the Y axis The Accumulate checkbox if selected will cause the histogram to accumulate counts among frames Y axis Linear Logarithmic _ Accumulate FLIM mode counts per gate 400
19. ach pixel The measurement of light signals by a SPAD has several advantages concerning the signal to noise ratio No analogue measurement of voltage or current is needed since the detector acts like a digital Geiger like counter It follows that no electronic noise is added by analogue to digital converters or amplifiers while measuring the signal Additionally the detector is less sensitive to the electromagnetic interference or the electrical noise generated by external equipment differently from charge coupled devices The primary noise sources for SPADs are 1 Dark counts This noise source comprises all processes which can start an avalanche across the SPAD junction but which are not caused by the detection of a photon The typical source of dark counts is the thermal carrier generation process Dark counts are the dominant noise source for the SPADs of the SPC 2 Afterpulsing The detection of a photon can trigger with a certain probability an additional detection event within few microseconds This spurious event is caused by charge carriers that flew through the junction during the avalanche remained trapped and are then subsequently released These carriers are accelerated by the high electric field across the junction and might trigger another avalanche like the photo generated electron hole pairs when released after the dead time The afterpulsing probability depends on the detector dead time and it is usually reduced t
20. being stored in the computer memory In Advanced mode this field can be changed by the user in Normal mode it is adjusted by the software depending on the chosen Frame Exposure Time v Number of frames number of frames that are acquired during a Snap event This number ranges from 1 to 65534 for 16 bit images and to 131070 for 8 bit images Table 3 Possible configurations of the Exposure Parameters subsection Normal and Advanced mode Normal Acquisition Control Box The duration of a frame is obtained by binning a sequence of frames of Normal mode Advanced mode shorter exposure times In this example a 52 00 us Frame exposure is Frame exposure time Hardware integration obtained by summing 5 frames of 10 40 us This acquisition is then i repeated 1000 times and the data are transferred to the host computer 52 00 ps 10 40 us Practically 5000 frames of 10 40 us are acquired and binned by a factor of Hardware binning Number of frames snap 5 This acquisition mode does not degrade the signal to noise ratio of the K 5 5 1000 images because no read out noise is present Advanced Acquisition Control Box Exposure parameters The user has a full control on the frame acquisition Normal mode Ad ed mod i lone In the example each frame has an effective duration of 103 7 us and no WARNING Possible counter overflows Frame exposure time Hardware integration i r is then repeated 1000 times and the data are transferred to t
21. ciprocal of the dead time Tgead AS a matter of fact since the diode is off for a period equal to Tgead every time it detects a photon the maximum count rate happens when the detector oscillates with a period Tgead Supposing Nimp the number of impinging photons on a SPAD active area during an integration time T Neounted the photons counted by the detector and taking into account the PDE Noounted is equal to Nimp reduced by the PDE and multiplied by the ratio of the actual integration time divided by the set integration time T T N T T Ncountea Nimp PDE n Nimp PDE 1 Ncounted a PDE x Nimp Neounted a ee a a eq 1 1 PDE Nimp x fead By expressing Nimp as a function of Neounteq one obtains also 1 Ncountea N _ ____ 2 tmp PDE i Tl ieaa eq i Ncounted T The total number of detected events is thus smaller than the total number of photons which cross the p n junction because of the saturation effect and the PDE When Nimp PDE o the number of counted J ead events is already reduced of 50 Since a SPAD is blind for Tgead after each triggering it is also clear that ina time period 7 the maximum number of photons that can be counted is T Neounted max T eq 3 dead v 1 0 1 Micro Photon Devices S r l Italy All Rights Reserved Page 5 SPC3 Single Photon Counting Camera 64x32 x IM PHOTON DEVICES No dead time 100ns dead time Max Counts for
22. ction of the background caused by dead time induced non linearity The effect is even more visible in the intensity histogram on the right of Figure 10 The histogram of the reference image deviates from an expected Gauss like profile and shows a remarked shoulder for lower number of photons green curve Conversely the corrected image is closer to the ideal Gauss distribution blue curve It must be observed that the dead time correction is only effective under a moderate illumination of the sensor For this reason we have decided that the SW implemented dead time correction does compensate the non linearity shown in Figure 2 but it does not in any case expand the counting range beyond the natural limit imposed by the hold off time as shown in Figure 11 As a consequence when a strong illumination is applied the corrected values will in any case saturate to the expected saturation level given by the current hold off time Dead time corrected image Dead time corrected image 400 Reference image N UI oO Reference image N O oO Number of pixels vI O Deviations A oO oO uN oO jo 0 0 2 0 4 0 6 0 8 1 Normalized number of photons Figure 10 Improvement of the image quality by using the dead time correction algorithm The intensity histograms of the two images are plot on the right v 1 0 1 Micro Photon Devices S r l Italy All Rights Reserved Page 22 SPC3 Single Photon Counting
23. d currently can be 2048 full array or 1024 half array Continuous Acquisition mode data rate in bytes per second is given by PP CA_DataRate Npyixel saved 8 FrameRate where 1 FrameRate ________ _ ______ Hardware_Readout_Time Noyms being Nsums the number of internal accumulations Camera auto protection The SPC has a protection mechanism that shuts down the SPAD high voltage when too much current is flowing through the array for example when really high photon fluxes are impinging on the camera Solution Reduce the amount of light that falls on the sensor disconnect and reconnect the camera to the host PC and restart the software v 1 0 1 Micro Photon Devices S r l Italy All Rights Reserved Page 24 SPC3 Single Photon Counting Camera 64x32 x IM PHOTON DEVICES SPC3 Software interface VisualSPC Windows only The camera is provided with an acquisition and control software which is running on Microsoft Windows operating systems VisualSPC This software provides a direct access to all the camera functions and it is a basic tool to display images It is recommended to use the Software Development Kit for a more flexible camera control Due to the start up time needed by the internal firmware to be fully working the VisualSPC should be started at least 15 seconds after having powered the SPC Failing to do so will generate an error by the software Simply close and restart the soft
24. depend on the actual throughput of the USB link or PC save speed Snap size is limited by the Capacity of the internal memory 128 MiB For more details on how to calculate snap size see the paragraph on Camera Configuration If you need to acquire more data you should use Continuous Acquisition mode as described below Continuous Acquisition a continuous acquisition of data between a START command and a STOP command from the PC is performed Once the acquisition is started the internal memory is used as Micro Photon Devices S r l Italy All Rights Reserved Page 11 SPC3 Single Photon Counting Camera 64x32 x IM PHOTON DEVICES a buffer and data should be continuously read by a PC In case the internal memory gets full because the PC fails to download the data swiftly enough data loss occurs and an error is issued The maximum achievable frame rate strongly depends on PC performance specially on the HDD write speed Thanks to USB 3 0 it is possible to acquire the full array at the maximum speed about 100kfps However in order to save all generated data at this high throughput it is also crucial that the employed mass storage unit is very fast see the paragraph on Camera Configuration for more details on how to calculate actual data rates SSD storage units are thus recommended If your PC can not sustain such a high throughput you will have to reduce the frame rate or to save only half the array If this is not possible you must r
25. e of clarity let s consider as before 5 000 photons per integration window T both for the background and for the signal Let s also assume T 1 ms a dead time of 100 ns and a PDE 100 for simplicity In this situation if follows that only 3 333 photons are counted when acquiring the 5 000 background BG photons and only 5 000 are counted v 1 0 1 Micro Photon Devices S r l Italy All Rights Reserved Page 21 SPC3 Single Photon Counting Camera 64x32 MPD when acquiring the cumulated 10 000 photons of signal and background SG BG see Eq 1 for reference It is thus clear that subtracting the measured 3 333 BG photons from the 5 000 SG BG photons is totally incorrect and results in the over subtraction of the background which should have been 2 500 and not 3 333 In any case the correct way of proceeding is to correct by means of Eq 2 for the dead time effect the actual measured number of photons and obtain the real number of impinging photons 10 000 and 5 000 for both images and only AFTER perform the subtraction By enabling the embedded dead time correction the effect of the non linearity is mostly removed and a better and faster processing of the data becomes possible Figure 10 shows the improvement of the image quality of two white frames with automatic background subtraction enabled The reference image shows a noisier pattern compared to the dead time corrected one This is due to an over subtra
26. eadout scene Gea T Interframe rai lt ion ns Frame Exposure Time Hardware Integration Time gt Advanced Mode f f f f f f with Hardware Integration Time longer than Readout Time 2 N I SS gt ite Hardware Readout Ta Readout Time Interframe deadtime lt 10 ns Equivalent Hardware Integration Time lt gt Advanced Mode with Hardware Integration Time prm shorter than Readout Time ra Hardware Readout Tin Readout Time Figure 9 Integration times and working modes The dead time strongly affects the image quality It limits the after pulses generated in each single pixel when set at high values e g 150 ns but it introduces a limitation on the total number of photons per second that each pixel can detect This induces a non linearity when bright spots are observed see the introductory section for further details It is possible to improve the quality of the image by correcting for the non linearity introduced by the dead time see section Single Photon Avalanche Diode In fact a simple operation as background subtraction might perform incorrectly when large numbers of photons per second are detected by the pixels This occurs because for example a cumulated flux of 10 000 photons 5 000 signal photons 5 000 optical background photons might suffer a larger non linearity in the detection than the acquisition of only 5 000 optical background photons as predicted by Eq 1 and shown in Figure 2 For sak
27. ed Page 29 SPC3 Single Photon Counting Camera 64x32 x IM PHOTON DEVICES Main Window Control Buttons Table 9 v 1 0 1 N LIVE JN Camera control buttons for starting recording and saving frame acquisitions Start the live mode acquisition Once the acquisition has been started the button becomes a red box Press it again to stop the acquisition In FLIM mode the average counts over all FLIM steps is visualized Starts the acquisition of a sequence of frames Snap mode If no external trigger is requested the acquisition starts immediately and the button becomes a red box If an external trigger is required the button turns first in an orange box then in a red one only when the trigger is detected and the acquisition really starts When the box is orange a click on the button stops the waiting for a trigger When the box is red the user must wait for the end of the acquisition Starts the continuous acquisition of frames If no external trigger is requested the acquisition starts immediately and the button becomes a red box If an external trigger is required the button turns first in an orange box then in a red one only when the trigger is detected and the acquisition really starts For interrupting the waiting for the external trigger signal or the acquisition once it has been started press the button WARNING Continuous mode acquisition may generate huge amount of data in short time depending on acquisition
28. eral Hardware Integration Time in order to obtain the so called Frame Exposure Time This is a viable solution since the SPC3 has no readout noise thus there is no penalty in repeating the v 1 0 1 Micro Photon Devices S r l Italy All Rights Reserved Page 18 SPC3 Single Photon Counting Camera 64x32 MPD readout apart for a negligible dead time or inter frame dead time of less than 10 ns between adjacent Hardware Integration Time The number of Hardware Integration Times that are accumulated to obtain a Frame Exposure Time can range from 1 to 65534 A beneficial side effect of accumulating several Hardware Integration Times instead of using a longer Hardware Integration Time is that the equivalent dynamics of the internal counter is increased In fact since each Hardware Integration Time can accommodate up to 511 photon detections 2 Hardware Integration Time can hold 1022 detections 10 Hardware Integration Time can hold 5110 and so on Finally it is possible to employ the internal detector gating capability of Counter 1 in order to achieve an Equivalent Hardware Integration Time smaller than the Hardware Readout Time This is possible by gating off the detector during a portion of Hardware Integration Time Equivalent Hardware Integration Time can be as short as 10 ns Please note however that using an Equivalent Hardware Integration Time shorter than the Hardware Readout Time results in a corresponding increase in the inter frame dead t
29. esort to the Snap mode at the expense of the experiment duration In both Snap mode and Continuous Acquisition mode it is possible to trigger the actual start of the acquisition with an external signal In all three modes it is possible to output a pulse synchronous with the start of each integration time It is also possible to internally subtract a background image previously stored into the camera Gated operation Normally the counters of each pixel of the SPC will just continuously count all the pulses generated by the SPADs This operating mode is called free running and it is the default operation mode Anyway each of the three integrated binary counters which register the detected number of photons in each pixel can be enabled or disabled by a gate signal This operating mode is called time gating The gate signal for counters 2 and 3 is provided only by the two coaxial inputs GATE IN 2 and GATE IN 3 The gate signal for counter 1 instead is the logical OR between two independent digital signals GATE IN 1 i e one of the camera coaxial inputs and Software gate generated internally by the SPC electronics which can be set by the user through the provided software Each of the three external GATE IN signals can be periodic or aperiodic and it will be applied asynchronously and directly through the OR gate to the SPADs The Software Gate is a periodic digital signal which is synchronized with the internal Reference clock 20 ns pe
30. f a standard acquisition FLIM mode acquisitions are possible in Snap mode and Continuous Acquisition mode whereas a real Live mode in not available since each FLIM acquisition is composed by several elementary frames However the VisualSPC software see below offers a seamlessly equivalent Live mode which is internally implemented performing repetitive Snap acquisitions Due to the time required for internal gate shifting which is also dependent on the shift parameters the time required for a full FLIM acquisition is not simply the sum of the integration time of each step and it is instead conveniently calculated by the camera software and reported to the user A reference clock inside the camera guarantees that every FLIM acquisition starts only after this time is elapsed thus ensuring the isochronicity of FLIM acquisitions and allowing the recording of FLIM movies Exactly as for the gated operation the gate signal is generated synchronized with the internal 50MHz reference clock Its width can be varied in the range 0 100 of the reference clock period 20 ns in steps of 1 while the gate shift can be varied in the range 40 40 of the same period in nominal steps of 0 1 corresponding to 20 ps Of course in order to make the Gated FLIM work the external laser has to be synchronised with the gate and thus in case of FLIM the SYNC OUT outputs the internal reference clock Finally as shown in Figure 8 since the reference c
31. files data represent measured Photon Detection Efficiency for each pixel Values range from 0 to 10000 i e a 32 5 PDE will be expressed as 3250 Each frame represents a wavelength whose range and step are reported in the header v 1 0 1 Micro Photon Devices S r l Italy All Rights Reserved Page 33 SPC3 Single Photon Counting Camera 64x32 x IM PHOTON DEVICES The header is composed by a signature of 8 bytes and a metadata section of 32 bytes as reported in Table 11 Note that multibyte fields are little endian Table 11 Header structure of SPC3 proprietary file formats spc3 spcf spce Byte offset Number of bytes Description 0 8 File signature 0x4d5044ff03000000 8 1 Number of rows 9 1 Number of colums 10 1 Bit per pixel 11 1 Counters in use 12 2 Hardware integration time multiples of 10ns 14 2 Summed frames 16 1 Dead time correction enabled 17 1 Internal gate duty cycle 0 100 18 2 Holdoff time ns 20 1 Background subtraction enabled 21 1 Data for counters 1 and 2 are signed 22 1 FLIM enabled 23 1 FLIM shift 24 1 FLIM steps 25 4 FLIM frame length multiples of 10ns 29 2 FLIM bin width fs 31 1 PDE measurement 32 2 Start wavelength nm 34 2 Stop wavelength nm 36 2 Step nm 38 2 Unused In order to easily visualize and elaborate data stored in the proprietary spc3 spcf and spce file formats a plugin for ImageJ software http rsb info nih gov ij is also provided The plugin is compatible bo
32. he actual size of data so far saved to the disk The number is updated in real time after every write operation Counter Window VisualSPC will show up to three windows see Figure 13 showing the map of the accumulated counts in each counter Window for Counter 1 is always visible windows for Counter 2 and 3 will pop up if selected in the Exposure Parameters subsection of the Control Panel In addition to settings shown in Table 7 it is possible to manually adjust the black and white levels of the image and its contrast by using the controls on the color bar at the left of the image After a Live or Snap mode acquisition is stopped it is possible to directly read the number of photons detected by each pixel by simply moving the mouse pointer over the image available only if window size is set to the default value Check Keep windows docked to revert to default size In FLIM mode only Counter 1 window is available In Live mode the average counts over all FLIM steps are visualized whereas in Snap mode counts for each individual FLIM step are shown For the sake of clarity let s consider a Snap of 10 FLIM acquisitions camera in FLIM mode and let s suppose that each acquisition is composed by 50 steps Gate Shifts i e Gate positions a total of 500 frames will be thus shown in Counter 1 window following a FLIM first time second scheme With such scheme the frame sequence would follow this ordering 1 gate shift of 1 F
33. he host binning of the frames is performed Hardware binning 1 The acquisition 103 70 us computer Frame exposures down to 10 ns can be obtained Hardware binning Number of frames snap WARNING the integrated counters can overflow at long exposure times v 1 0 1 Micro Photon Devices S r l Italy All Rights Reserved Page 26 SPC Single Photon Counting Camera 64x32 MPD In this subsection there is also a set of checkboxes to further customizing the acquisition The full list and their explanation can be found in Table 4 Table 4 List of checkboxes in the Exposure Parameter subsection and their explanation Acquire half array Only the central 32x32 portion of the array is acquired Useful to reduce required bandwidth Enable Counter 2 Enable the extra in pixel counters When a counter is enabled a new Counter window pop ups when a counter is disabled the Enable Counter 3 l l corresponding window is closed When at least Counter 2 is enabled this checkbox indicates if the data Signed data from Counter 2 and 3 has to be considered signed or not Useful when these counters are employed in up down mode Wait for external trigger The camera waits for an external trigger on the TRIG IN input before starting the acquisition of an image sequence The acquired dark frame is subtracted by the output image A dark Enable background subtraction frame acquisition must have been previously performed see the Control Buttons
34. images the time between two images can not be precise and strongly depends on how the PC software behaves and on the PC s current work load This mode should not be used for actual scientific acquisition but it is instead useful for acquiring a low frame rate live movie i e real time for instance when aligning the camera in the optical setup or adjusting the acquisition parameters with the current illumination conditions Considering the practical limitations of the USB connection when transferring small data blocks typical achievable frame rates are in the range of 50 100 fps actual values depend on the performance of both the PC hardware and the software employed As a consequence the PC requests of live images should be limited to a maximum of 1 every 10 ms or longer time Snap a sequence of images is acquired and stored into the internal memory of the camera The dead period between subsequent frames inter frame dead time is less than 10 ns and the frames timing is accurately determined by the internal clock Once the required number of frames is measured the electronic interface transfers the data to the computer The images can then be displayed and saved on the hard drive This mode can not be used for live acquisition since all data will be downloaded to the PC only at the end of the entire snap which can last few seconds Instead this approach allows data acquisitions at the maximum frame rate possible since it does not
35. ime by an amount equal to the difference between the Hardware Readout Time and the chosen Equivalent Hardware Integration Time Since in this particular working mode the gate is controlled directly by the camera it is not recommended to apply any external gate signal or to change the internal gate settings either with the PC software or with the standard gate control procedures provided in the SDK Particularly the new gate settings will overwrite the already applied ones thus interfering with the Equivalent Hardware Integration Time generation process In order to configure these three parameters the SPC can be set into two different modes In normal mode the camera settings are constrained so that the Hardware Integration Time is equal to the Hardware Readout Time in order to avoid or at least reduce to a minimum the probability of the occurrence of an overflow of the integrated counters In fact the internal 9 bit binary counters can overflow at long Hardware Integration Times and thus induce artefacts in the detected image As already shown the maximum number of photons Nmax that can be counted per second given a dead time of Tgeaa IS 1 Nmax counts second Taead thus the maximum number of photons that can be counted during a Hardware Integration Time equal to T IS 1 Nmax HIT T T dead For instance when using a single counter T is equal to 10 40 us and with the minimum Tgeag of 50 ns the maximum counted photons is
36. lock period is 20ns the nominal maximum observation time window is also 20ns Actually since the gate shift range is 40 40 the gate can be moved from 2 ns to 18 ns and thus the actual maximum observation window is 16 ns The reason for the reduced range is due to the fact that for shifts longer than 40 of period the shift accuracy is not satisfactory and thus the SPC FPGA does not allow their selection The actual width and phase of the gate signal obtained for a given set of parameters may change among different SPC3 units due to fabrication tolerances of the FPGA That s why the gate width is calibrated by MPD before delivering the camera The initial position of the gate in a FLIM sequence is also internally calibrated and has an accuracy better than 20ps plus a constant offset see also paragraph on Gated Operation This shift offset is not calibrated since in case of a FLIM experimental set up what is really important to measure is the full offset of the gate signal with respect to the laser pulse This offset is not only determined by the internal shift offset due to the camera but also and probably largely by the delays introduced by the used external interconnection cables and the laser itself A calibration of the actual gate shift with respect to the laser excitation is therefore necessary after the camera is mounted into the measurement setup This can be easily done by directly shining the laser onto the array wi
37. mine the lifetime The colour of each image pixel represents the determined lifetime giving the possibility to obtain images with contrast between materials with different decay rates even if they v 1 0 1 Micro Photon Devices S r l Italy All Rights Reserved Page 14 SPC3 Single Photon Counting Camera 64x32 x IM PHOTON DEVICES fluoresce at the same wavelength or on the other hand identify two region of the same material even if they have different intensity The use of an array of SPADs has major advantages for imaging applications since aS opposed to single pixel systems it does not require any scan of the sample A second major advantage of the presented pixel structure concerns the short dead time which has a lower limit of about 50 ns thus allowing the counting of very high photon fluxes The embedded short gate capability enables the use of SPC as a FLIM camera even though the employed SPAD imager chip is only counting the detected photons and it is not time tagging them In fact it is possible to employ the gate in order to implement a time gated FLIM detection system In time gated FLIM the decay of the fluorescence is reconstructed by repetitively counting the number of detected photons in short time windows that are progressively shifted with respect to the excitation laser pulse as shown in Figure 8 In order to perform this type of measurement the SYNC_OUT output form the SPC camera has to be connected to the trigger in
38. n the actual average gate shift bin i e minimum gate shift this value is reported to the user through the provided software and it should be used as a scale factor on the time axis of the reconstructed FLIM waveform FLIM data acquired can be either saved on a multipage TIFF with embedded acquisition metadata according to the OME TIFF format or in the proprietary SPCF format For both formats image data is composed by a set of images following a FLIM first time second scheme i e with the following frame sequence 1 gate shift of 1 FLIM measurement 2 gate shift of 1 FLIM measurement n gate shift of 1 FLIM measurement 1 gate shift of 2 FLIM measurement 2 gate shift of 2 FLIM measurement n gate shift of 2 FLIM measurement etc OME TIFF file could be opened with any image reader compatible with TIFF file since metadata are saved into the Image Description tag in XML format In order to decode OME TIFF metadata it is possible to use free OME TIFF readers such as OMERO or the Bio Formats plugin for ImageJ For more details see the OME TIFF web site http www openmicroscopy org site support ome model ome tiff OME TIFF metadata include the ModuloAlongT tag which allows the processing of FLIM data with dedicated FLIM softwares such as FLIMfit see http www openmicroscopy org site products partner flimfit A tutorial on basic usage of FLIMfit is available at http help openmicroscopy org flimfit html
39. o about 1 2 at the normal operating conditions Longer dead times decrease the afterpulsing probability v 1 0 1 Micro Photon Devices S r l Italy All Rights Reserved Page 4 SPC3 Single Photon Counting Camera 64x32 x IM PHOTON DEVICES 3 Cross talk During the avalanche process photons are emitted by hot carriers These can propagate through the silicon and trigger a detection event in SPADs which are placed in close proximity Practically the Cross talk probability is very low among the pixels of the SPC about 10 due to the large distance between the SPADs Its contribution to the total noise is mostly negligible Since the read out noise of CCD or CMOS cameras is normally slightly higher than one electron per integration time and since the dark counting rates of the SPAD pixels of the SPC3 is about one hundred per second it follows that the optimal operating condition for the camera is at fast or moderate frame rates Particularly the dark counts contribution is low with exposure times of about 0 1s By reducing even further the integration time the dark count noise becomes negligible Due to the dead time the SPAD has a linear working range i e the number of detection events within a defined time period 7 depends linearly on the illumination intensity only at low or moderate photon fluxes At large photon fluxes the number of detected photons deviates from linearity and saturates to a constant value which is the re
40. of photons which are detected by the sensor during the acquisition time Figure 1 SPC Single photon Counting Camera v 1 0 1 Micro Photon Devices S r l Italy All Rights Reserved Page 3 SPC3 Single Photon Counting Camera 64x32 x IM PHOTON DEVICES Single Photon Avalanche Diode The SPAD is a p n junction which is reverse biased well above its breakdown voltage Under this operating condition the absorption of a single photon generates an electron hole pair which is accelerated by the high electric field across the junction The energy of the charge carriers is eventually sufficient to trigger a macroscopic avalanche current of few milliamperes through the device The SPAD biasing electronics namely the Active Quenching Circuit AQC is integrated on the same silicon chip The AQC senses the avalanche current quenches it and resets the diode to its initial state The avalanche is quenched by decreasing the voltage across the SPAD junction below breakdown for an adjustable time hold off that ranges from few tens to few hundreds of nanoseconds The SPAD bias voltage is then restored to the initial state and thus the diode is ready to detect the next photon The total time which is required to restore the initial state of the SPAD after the detection of a photon is named dead time During the dead time no photons can be detected The AQC provides additionally a voltage pulse to an associated counter which is integrated in e
41. oscopy org site support ome model ome tiff Proprietary format files produced by SPC3 camera are based on the same binary structure but different file extensions are used for differentiate among data Photon counting acquisitions are stored in spc3 files whereas FLIM mode acquisitions are saved in spcf files In addition each camera is provided with an spce file containing measured photon detection efficiency All these files contain binary data composed by a header with acquisition metadata followed by raw image data which contain the 8 16 bit pixel values in row major order The byte order is little endian for the 16 bit data Raw data have the following meaning spc3 files data represents photon counts in each pixels during each Exposure Time with acquisition parameters reported in the header In case more counters are used data are interlaced i e the sequence of frames is the following 1 frame of 1 counter 1 frame of 2 counter 1 frame of 3 counter 2 frame of 1 counter etc spcf files data represents photon counts in each pixels during each Exposure Time with acquisition parameters reported in the header Images follow a FLIM first time second scheme 1 gate shift of 1 FLIM measurement 2 gate shift of 1 FLIM measurement n gate shift of 1 FLIM measurement 1 gate shift of 2 FLIM measurement 2 gate shift of 2 FLIM measurement nl gate shift of 2 FLIM measurement etc spce
42. put of a pulsed laser source able to generate optical pulses with width of at most few hundreds of picoseconds Such laser is then used to excite the fluorescence in the sample under test The internally generated Gate signal activates the counters after the generation of the laser pulse for the time defined by gate width Accordingly the fluorescence decay kinetics is measured by changing gate shift position over time Both gate shift and gate width have optimal values depending on the lifetime of the excited state of the fluorescent molecules and on the imaging frame rate In order to speed up FLIM acquisitions and increase the frame rate the SPC includes an automatic FLIM mode in which the internal gate signal is automatically generated with user defined step width and shifted of the user desired number of steps fj Detected Photons PEALLEN E Discarded Photons SYNC_OUT Int_Clk Int_Clk jj cate ia i Gate Shift 2 Int_Gate 3 Gate aed Width JJ Optical Signal JJ Figure 8 Optical waveform reconstruction using the Gated FLIM approach with the SPC3 camera v 1 0 1 Micro Photon Devices S r l Italy All Rights Reserved Page 15 SPC3 Single Photon Counting Camera 64x32 x IM PHOTON DEVICES Each FLIM acquisition is thus composed by a sequence of FLIM elementary step frames one for each desired gate position step each one consisting of an acquisition with the same exposure parameters o
43. ray for 2 D Imaging and 3 D Ranging IEEE Journal of Selected Topics in Quantum Flectronics 2014 vol 20 no 6 pp 354 363 M Vitali D Bronzi A J Krmpot S N Nikolic F J Schmitt C Junghans S Tisa T Friedrich V Vukojevic L Terenius F Zappa R Rigler A Single Photon Avalanche Camera for Fluorescence Lifetime Imaging Microscopy and Correlation Spectroscopy IEEE Journal of Selected Topics in Quantum Electronics 2014 vol 20 no 6 pp 344 353 v 1 0 1 Micro Photon Devices S r l Italy All Rights Reserved Page 6 SPC3 Single Photon Counting Camera 64x32 x IM PHOTON DEVICES SPC3 hardware characteristics Electrical connections The SPC has power and USB connections on one side and a multi coaxial connector on the other side for various control signals as shown in Figure 1 and Figure 3 SPC is provided with a cable adapter from the multi coaxial connector to standard SMA connectors as shown in Figure 4 A description of the input and the outputs can be found in Table 1 The Electrical specifications of the input and outputs are reported in Table 2 Table 1 USB SPC3 Inputs and Outputs description High speed USB 3 0 connector It is recommended to use short USB cables lt 2 m 12Vbc SYNC OUT Jack for connecting the provided 12V power supply Coaxial output which can drive 50 Ohm terminated transmission lines A 50 Ohm DC load TRIG IN termination is required Electric pulses of
44. riod The user can set the Delay i e the time shift between the rising edge of the Reference clock and the high to low transition of Software gate and the parameter Width which defines the duration of the counter enabled Software gate pulse Essentially the Software gate generates a periodic gate signal of a given Width and Delay from the reference clock Note that the Width is the duration of the Software Gate s low state as shown in Figure 7 The Width of the Software Gate which is generated by the control electronics can be varied in the range O 100 of the reference clock in steps of 1 Actually during a factory calibration the Gate width has v 1 0 1 Micro Photon Devices S r l Italy All Rights Reserved Page 12 SPC3 Single Photon Counting Camera 64x32 MPD been internally discretized with 1000 points over the full range In this way whenever one of the 100 nominal selectable values is requested by the user the SPC camera is set to the closed possible one i e the one with the smallest possible error Note that however values below 2ns could be unreliable Particularly the minimum achievable Gate signal is about 1 5ns The actual set value can be reported to the user by the software The Delay can be varied in the range 40 40 of the Software Gate period in nominal steps of 0 1 corresponding to 20 ps The actual gate delay step can actually vary depending on the used camera however an internal calibration en
45. s is expressed as a percentage of the internal reference Gate Width clock period 20 ns E g Width 20 4 ns width FLIM steps Number of steps to be performed for each FLIM acquisition Gate shift for each FLIM steps in thousandths of reference period E g Step 50 o gt 1 ns step FLIM shift per step Start position of the of the first FLIM step with respect to the internal reference clock FLIM Start position The delay value is expressed as a percentage of the reference clock period 20 ns E g Delay 10 2 ns delay In the Deadtime subsection it is possible to adjust the hold off time in the range 50 150 ns and enable the embedded dead time correction In the Synchronization Output subsection it is possible to enable the generation of voltage pulses on the SYNC OUT output Two possible synchronization signals are available v Frame sync a 50 ns pulse at the beginning of each frame v Ref Clock the internal 50 MHz clock is replicated to this output Main Window Plot settings and Playback Control The plot settings apply only to the images displayed on the screen No changes are introduced to the data in the memory buffer Therefore these parameters do not affect the saved images All this settings can be changed in real time during display The plot settings can be adjusted as explained in Table 7 After a Snap has been acquired it is possible to review the images by using the controls shown in Table
46. se duration GATE tgate delay GATE delay GATE DIRECTION delay XXX DIRECTION tair dela Internal reference clock frequency SYNC OUT internal l Internal reference clock duty SYNC OUT internal 50 v 1 0 1 Micro Photon Devices S r l Italy All Rights Reserved Page 9 SPC3 Single Photon Counting Camera 64x32 x IM PHOTON DEVICES Camera mechanical dimensions 34 75 0 2 UNLESS OTHERWISE SPECIFIED DIMENSONS ARE IN MILLIMETERS Figure 6 SPC mechanical dimensions All dimensions in mm v 1 0 1 Micro Photon Devices S r l Italy All Rights Reserved Page 10 SPC3 Single Photon Counting Camera 64x32 x IM PHOTON DEVICES SPC Operation The SPC camera features three different modes of operation that can be optimised through the use of several working parameters all controllable from a computer using either the delivered SDK library or the provided Windows PC software The following paragraphs describe in detail all these features Standard operation The camera is operated in its standard mode when each pixel is simply used to count photons in a user defined integration time and the counters are not gated see the next paragraph for details When the SPC is operated in this mode three different acquisition modes are possible v 1 0 1 Live acquisition single images from the camera are acquired and downloaded to the computer Since in this mode it is the PC that continuously asks for new
47. settings Check Total saved data in the Acquisition Parameters Summary box for the actual size of data saved on the HDD Performs the acquisition of a background frame Once the light is prevented to reach the detector only dark counts are measured A dark frame is generated by averaging the number of frames acquired This frame is then sent to the acquisition electronics to perform hardware background subtraction The acquisition of the background frame must be performed using the same parameters that will be used for the actual measurement Any change in the acquisition parameters including the gate parameters and the dead time correction settings REQUIRES a new acquisition of the background frame The background frame is not stored inside the camera and is lost when the camera is switched off Saves the acquired data by a Snap into a SPC3 format file or special SPC3 FLIM format Saves the data acquired by a Snap into a multipage TIFF file The file is compliant with the OME TIFF specification which allows embedding in the file metadata all the acquisition parameters for the specific measurement as well as FLIM mode parameters OME TIFF file can be opened with any image reader compatible with TIFF file since metadata are saved into the Image Description tag in XML format In order to decode OME TIFF metadata it is possible to use free OME TIFF reader such as OMERO or the Bio Formats plugin for ImageJ For more details see the OME TIFF web
48. sing the internal gate generation is enabled Internal gate is automatically enabled when the user selects a Hardware Integration Time shorter than the Hardware Readout Time It is then up to the user the calculus of the actual inter frame dead time for this purpose since the Hardware Readout Time is equal to the inverse of the frame rate in Snap or Continuous Acquisition modes it could be useful to have a look at the actual frame rate by enabling and checking the Sync Out output Please also note that when this feature is enabled only Counter 1 will be automatically gated whereas Counters 2 and 3 will have an actual Hardware Integration Time equal to the Hardware Readout Time Figure 9 summarizes the relationship between Hardware Integration Time Readout Time Frame Exposure Time in the two working modes Dead time time and dead time correction SPAD dead time can be selected between 50 ns and 150 ns both in normal and in advanced mode There are about 60 discrete values that can be assumed over the full range thus whenever a value is input by the user the SPC3 camera is set to the closest possible value according to factory calibration The actual value can be reported to the user by the software v 1 0 1 Micro Photon Devices S r l Italy All Rights Reserved Page 20 SPC3 Single Photon Counting Camera 64x32 x IM PHOTON DEVICES Frame Exposure Time Hardware Integration Time tc i Normal mode E Zz Hardware R
49. sures that the integral non linearity in delay setting is always less than 20ps An initial offset may also be present This offset is on purpose not calibrated since it will sum up with other setup depended offsets e g from different cable lengths The overall system offset can be evaluated through optical measurements employing a short pulse laser i e less than 100ps pulse width When the SPC is operated in gated mode the same three acquisition modes of the standard operation are possible this time in conjunction with the use of the three gates Reference Clock SYNC OUT E B B i B B gate gt Width Delay 5 ns off lon off 5 ns gt lt GATE IN Gate on Gate off Gate on Gate signal Software gate OR GATE IN Detected photons Valid Valid Notvalid Valid eS O XX O Photon Gateon _ Gate off T Figure 7 Hardware gating example Delay 5 ns and Width 5ns both of 25 of the Reference Clock period The reference clock has been sent to the SYNC OUT output of the camera Additionally a GATE IN signal was provided by the user Only the photons which are detected when the Gate signal is on are counted Example valid for counter 1 for counter 2 and 3 the example remains the same without the software gate In such case only the 4 photon would not bet detected v 1 0 1 Micro Photon Devices S r l Italy All Rights Reserved Page 13 SPC3 Single Photon Counting Camera 64x32 x IM PHOTON DEV
50. th with ImageJ tested on v 1 49q and with Fiji http fiji sc Fiji which is essentially a distribution of ImageJ together with several useful plugins In order to install the plugin follow this procedure v 1 0 1 Micro Photon Devices S r l Italy All Rights Reserved Page 34 SPC3 Single Photon Counting Camera 64x32 x IM PHOTON DEVICES e for ImageJ tested on v 1 49q launch ImageJ and select the menu item Plugins Install then follow the instructions and select the file SPC3_Reader java provided in SPC3 USB key The Save Plugin Macro or Script dialog appears with a list of your currently installed ImageJ plugins Select a folder such as the Input Output folder and save the SPC3_Reader java file there Do not change the name SPC3_Reader java as it follows ImageJ naming conventions and it is not seen by Image if it does not contain an underscore e for Fiji launch Fiji and select the menu item Plugins Install Plugin then follow the instructions and select the file SPC3_ Reader java provided in SPC3 USB key Restart Fiji In order to use the plugin select SPC3 Reader under the Plugins menu It will be in the location you ve previously chosen for ImageJ or at the end of the menu for Fiji An Open SPC3 file dialog appears Select the spc3 spcf or spce file you wish to load ImageJ will now ask you how to deal with signed data If data is 8bit there are three options e Convert data to 32bit float
51. thout interposing any fluorescent sample or filter and performing a full measurement over the entire 20 ns shift range By inspecting the measurement and v 1 0 1 Micro Photon Devices S r l Italy All Rights Reserved Page 16 SPC3 Single Photon Counting Camera 64x32 x IM PHOTON DEVICES finding the laser peak it is possible to evaluate the shift between the actual laser pulse and the internal reference clock Actually it is not even important to measure it What needs to be done is simply the adjustment of the cables length from the SYNC_OUT of the camera to the TRIG_IN of the laser in order to place the laser pulse at the beginning of the FLIM observation time window In this way the fluorescence decay will be centred inside the 16 ns FLIM window This would also prevent any folding of the fluorescence signal and the associated measurement distortion Of course since the first and the last 2 ns of the FLIM window are ignored the laser pulse is not seen when it falls there If this happens making 1 m longer of shorter the cable that connects the camera to the laser will make the laser pulse reappear in the FLIM observation window Such calibration could be useful also to zero any skew among pixels but in many applications like the FLIM it is usually not needed should it be the SPC3 user can contact MPD at the following email address for more details imaging micro photon devices com Finally a factory calibration is performed also o
52. us acquisition Supported operating systems o VisualSPC Microsoft Windows 7 8 32 or 64 bit versions o SDK and ImageJ Plugin Microsoft Windows 7 8 32 or 64 bit versions Linux Ubuntu 12 04 LTS CentOS 6 5 6 6 6 7 or compatible distributions 32 or 64 bit versions Different distributions should work but were not tested Mac OS X 10 8 and above Tested ImageJ versions different versions should work but were not tested o Standalone ImageJ version 1 49q o Fiji distribution version 2 0 0 rc 28 1 50b Copyright and disclaimer No part of this manual including the products and software described in it may be reproduced transmitted transcribed stored in a retrieval system or translated into any language in any form or by any means except for the documentation kept by the purchaser for backup purposes without the express written permission of Micro Photon Devices S r l All trademarks mentioned herein are property of their respective companies Micro Photon Devices S r l reserves the right to modify or change the design and the specifications the products described in this document without notice v 1 0 1 Micro Photon Devices S r l Italy All Rights Reserved Page 36
53. ware Installation Double click the desired installer 32 bit or 64 bit and follow the guided installation procedure Please note that device driver from OpalKelly provided on the SPC3 USB Key must be installed before connecting the camera to the PC Software Interface The VisualSPC interface is composed a main window divided in several sections as showed in Figure 12 and a separate window for each active counter see Figure 13 An additional window will also pop up when FLIM mode is enabled see 2 38983 SPC3 camera firmware version 1 01 Cpese SPC3 SDK version 1 01 rete MICRO PHOTON DEVICES 5 PL VisualSPC3 version 1 01 Plot settings only for visualization Acquisition Parameters Summary Frame rate 19 230 77 frames s Total frame time 52 00 us Total acquisition time 52 00 ms Flip image on X Flip image on Y Rotate 90 CCW Keep windows docked Interframe deadtime 0 01 us go Bit per pixel 16 l Snap file size 3 91 MiB Auto BW Level Reset BW Level Control panel Exposure parameters Software gating Nomal mode Advanced mode Periodic Gate FLIM mode Frame exposure time Hardware integration Enable gate lt gt 10 Gate delay 52 00 us 10 40 ps Hardware binning Number of frames snap 5 1000 gt 50 Gate Width Deadtime lt _ Acquire half aray Enable Counter 2 Enable deadtime correction Synchronization output
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