Contents
Overview about
bh TCSPC Instrumentation......................................................................................................... 1
General Features of the bh TCSPC Devices.................................................................................................... 1
TSPC Module Types.......................................................................................................................................... 2
Measurement Modes of the bh TCSPC Modules.......................................................................................... 6
Simple-Tau TCSPC Systems.............................................................................................................................. 14
bh DSC‑120 Confocal Scanning FLIM
Systems............................................................................................. 14
bh FLIM Systems for Zeiss LSM 510 and
710 Laser Scanning Microscopes........................................... 15
bh FLIM Systems for Leica SP2 and SP5 Laser
Scanning Microscopes.................................................... 17
bh FLIM Systems for Olympus FV 300 and
FV 1000..................................................................................... 18
bh FLIM Systems for Nikon C1 scanners....................................................................................................... 19
DPC‑230 16 Channel Correlator / TCSPC
module........................................................................................... 19
PMS and MSA Multichannel Scalers.............................................................................................................. 19
Picosecond Diode Lasers.................................................................................................................................. 20
Detectors.............................................................................................................................................................. 21
Detector Electronics........................................................................................................................................... 24
Connecting Electronics...................................................................................................................................... 25
Instrument Software........................................................................................................................................... 26
Data Analysis Software..................................................................................................................................... 26
Classic Time-Correlated Single Photon
Counting................................................................................................ 29
Single-Photon Detection.................................................................................................................................... 29
Time-Correlated Single Photon Counting....................................................................................................... 29
The Classic TCSPC Architecture...................................................................................................................... 33
Multi-Dimensional TCSPC....................................................................................................................................... 37
Multi-Detector and Multi-Wavelength TCSPC.............................................................................................. 38
Multiplexed TCSPC............................................................................................................................................. 41
Sequential Recording......................................................................................................................................... 42
TCSPC Scanning FLIM Techniques................................................................................................................ 44
Time-Tag Recording........................................................................................................................................... 45
Imaging in the Time-Tag Mode........................................................................................................................ 47
Multi-Module TCSPC Systems......................................................................................................................... 48
Architecture of the bh TCSPC Modules................................................................................................................ 51
General Principle.................................................................................................................................................. 51
CFD and SYNC circuits................................................................................................................................ 51
TAC................................................................................................................................................................ 52
ADC................................................................................................................................................................ 52
Channel Register........................................................................................................................................... 53
Sequencer...................................................................................................................................................... 53
Memory Control............................................................................................................................................ 53
Recording of single curves................................................................................................................... 54
Multi-Detector Operation...................................................................................................................... 54
Multiplexed TCSPC................................................................................................................................ 55
Sequential Recording............................................................................................................................. 55
Continuous Flow Mode........................................................................................................................ 56
Scan Modes............................................................................................................................................ 56
CNT and ADD Control.......................................................................................................................... 57
Time-Tag (FIFO) Mode......................................................................................................................... 57
Detailed Description of Building Blocks......................................................................................................... 58
CFD in the Detector (Start) Channel.......................................................................................................... 58
SPC-x00 Versions.................................................................................................................................... 58
SPC-x30 Versions.................................................................................................................................... 59
CFD in the Synchronisation (Stop) Channel............................................................................................ 60
SPC-x00 Versions.................................................................................................................................... 60
SPC-x30 Versions.................................................................................................................................... 61
Time-to-Amplitude Converter..................................................................................................................... 62
Analog-to-Digital Conversion.................................................................................................................... 64
ADC Error Correction............................................................................................................................ 64
Variable ADC Resolution...................................................................................................................... 65
ADC Zoom.............................................................................................................................................. 66
Detectors for TCSPC................................................................................................................................................. 67
Detector Principles.............................................................................................................................................. 67
Conventional PMTs..................................................................................................................................... 67
Channel and Microchannel PMTs............................................................................................................. 67
Hybrid PMTs................................................................................................................................................. 68
Single-Photon Avalanche Photodiodes.................................................................................................... 69
Detector Parameters............................................................................................................................................ 70
Gain................................................................................................................................................................. 70
Single Electron Response............................................................................................................................ 71
Pulse Height Distribution............................................................................................................................ 72
Signal Transit Time....................................................................................................................................... 73
Transit Time Spread and Timing Jitter....................................................................................................... 73
Cathode Efficiency....................................................................................................................................... 74
Dark Count Rate............................................................................................................................................ 76
Afterpulsing.................................................................................................................................................. 77
Pre-Pulses...................................................................................................................................................... 78
Description of Selected Detectors.................................................................................................................... 80
MCP PMTs.................................................................................................................................................... 80
Hamamatsu R5600 and R7400 Miniature PMTs....................................................................................... 81
H5783 and H5773 Photosensor Modules.................................................................................................. 82
PMH-100 and PMC-100............................................................................................................................... 83
Hamamatsu H7422 and H8632..................................................................................................................... 84
Hamamatsu H7421........................................................................................................................................ 85
Hamamatsu R10467 Hybrid PMT............................................................................................................... 85
PML-16C Multichannel Detector Head..................................................................................................... 87
Conventional PMTs..................................................................................................................................... 88
Single-Photon Avalanche Photodiodes.................................................................................................... 89
Preamplifiers and Detector Control......................................................................................................................... 93
Preamplifiers......................................................................................................................................................... 93
The DCC-100 Detector Controller..................................................................................................................... 94
Electromagnetic Shielding................................................................................................................................. 96
Safety Recommendations for Using Detectors.............................................................................................. 100
Installation of the bh TCSPC Modules.................................................................................................................. 103
General Requirements......................................................................................................................................... 103
Software Installation........................................................................................................................................... 103
First Installation............................................................................................................................................ 104
Installing the DLL and Lab View Libraries................................................................................................ 105
Software Update........................................................................................................................................... 105
Update from the Web................................................................................................................................... 106
Installing New Software Components....................................................................................................... 106
Software Repair............................................................................................................................................. 107
Removing the TCSPC Package................................................................................................................... 107
Hardware Installation - Single SPC Modules.................................................................................................. 107
Driver Installation......................................................................................................................................... 107
Hardware Installation - Several SPC Modules................................................................................................ 108
Software Start...................................................................................................................................................... 108
Module Test Program......................................................................................................................................... 109
Installation Problems.......................................................................................................................................... 110
Starting the SPC Software without an SPC
Module...................................................................................... 110
Operating the SPC Module...................................................................................................................................... 113
Detector and Reference Inputs......................................................................................................................... 113
Generating the Reference Signal....................................................................................................................... 113
Delayed Stop Operation..................................................................................................................................... 115
Synchronisation to Random Signals................................................................................................................ 116
System Connection............................................................................................................................................. 117
Single-Detector Systems............................................................................................................................. 117
Multi-Detector Systems............................................................................................................................... 120
Using the Experiment Trigger............................................................................................................................ 123
Resolving Cable Conflicts................................................................................................................................. 125
FLIM Systems............................................................................................................................................................ 126
Modular FLIM Systems for Confocal and
Multiphoton Laser Scanning Microscopes.......................... 126
Single-Module TCSPC FLIM Systems...................................................................................................... 126
Multi-Module TCSPC FLIM Systems....................................................................................................... 129
Wiring Diagrams of typical FLIM Systems.............................................................................................. 130
Single-Detector NDD FLIM Systems.................................................................................................. 130
Dual-Detector NDD FLIM Systems with a Single
TCSPC Module................................................ 131
Multi-Wavelength FLIM Systems....................................................................................................... 133
Multi-Module FLIM Systems............................................................................................................... 134
Other Scanning Systems....................................................................................................................... 134
DCS-120 Confocal Scanning FLIM System.................................................................................................... 135
General System Architecture...................................................................................................................... 135
DCS-120 System Connections.................................................................................................................... 136
Scanning Systems with Piezo Scan Stages..................................................................................................... 140
First Light: Getting Started................................................................................................................................ 142
Recommended System Parameters............................................................................................................. 142
Setup of Standard Fluorescence Lifetime
Systems................................................................................. 143
Setup of Scanning Systems........................................................................................................................ 145
Multi-Detector Systems............................................................................................................................... 149
Setup Procedure for Other TCSPC Applications..................................................................................... 150
Optimising a TCSPC System.................................................................................................................................... 151
General Recommendations................................................................................................................................ 151
Configuring the CFD and SYNC Inputs.......................................................................................................... 152
Pulse Shaping................................................................................................................................................ 152
Configuration for Positive Input (SPC-x00
only)..................................................................................... 153
Optimising the CFD Parameters........................................................................................................................ 154
Adjusting the CFD Threshold.................................................................................................................... 154
CFD Zero Cross Level.................................................................................................................................. 156
Optimising the SYNC Parameters..................................................................................................................... 157
Adjusting the SYNC and CFD Cable Length.................................................................................................. 158
Adjusting the TAC Parameters......................................................................................................................... 160
TAC Linearity...................................................................................................................................................... 162
Dead Time............................................................................................................................................................ 163
Counting Loss..................................................................................................................................................... 165
Dead-Time Compensation................................................................................................................................. 166
Pile-Up Effects..................................................................................................................................................... 167
Classic Pile-Up.............................................................................................................................................. 167
Inter-Pulse Pile-Up........................................................................................................................................ 168
Fighting Pile-Up Effects..................................................................................................................................... 169
Low Repetition Rate Measurements.......................................................................................................... 169
High Repetition Rate Signals...................................................................................................................... 170
Optimising the Photomultiplier......................................................................................................................... 172
Voltage Divider............................................................................................................................................. 172
Illuminated Area............................................................................................................................................ 172
Signal-Dependent Background.................................................................................................................. 173
Dark Count Rate............................................................................................................................................ 174
Checking the SER of PMTs......................................................................................................................... 174
Quick Test of a PMT.................................................................................................................................... 175
Optical System..................................................................................................................................................... 176
Lenses............................................................................................................................................................ 176
Absorptive Colour Filters............................................................................................................................ 176
Interference Filters........................................................................................................................................ 176
Monochromators and Polychromators..................................................................................................... 177
Optical Fibres................................................................................................................................................ 177
Baffles and Aperture Stops......................................................................................................................... 177
Avoiding Optical Reflections..................................................................................................................... 178
Fluorescence Depolarisation...................................................................................................................... 178
Re-Absorption.............................................................................................................................................. 180
TCSPC Applications................................................................................................................................................. 181
Measurement of Luminescence Decay Curves.............................................................................................. 181
Monochromator-Based Systems................................................................................................................ 181
Time-Resolved Spectra................................................................................................................................ 183
Filter-Based Systems.................................................................................................................................... 185
Multi-Spectral Lifetime Experiments.......................................................................................................... 186
Measurement of Time-Resolved Fluorescence
Anisotropy.................................................................. 188
Lock-in TCSPC.............................................................................................................................................. 191
Transient Fluorescence Phenomena................................................................................................................ 193
Chlorophyll Transients................................................................................................................................ 193
Stopped Flow Techniques.......................................................................................................................... 196
Continuous Flow Mixing Techniques....................................................................................................... 197
Simultaneous Measurement of Fluorescence and
Phosphorescence Decay...................................... 198
Time-Resolved Laser Scanning Microscopy.................................................................................................. 200
The Laser Scanning Microscope............................................................................................................... 201
Implementing TCSPC in Laser Scanning
Microscopes.......................................................................... 203
One-Photon Confocal Microscopes.................................................................................................... 203
Excitation........................................................................................................................................... 203
Detection........................................................................................................................................... 203
Two-Photon Microscopes.................................................................................................................... 205
Excitation........................................................................................................................................... 205
Detection........................................................................................................................................... 205
Multi-Spectral FLIM.............................................................................................................................. 207
Multi-Spectral Confocal FLIM....................................................................................................... 208
Multiphoton Multi-Spectral NDD FLIM...................................................................................... 209
High-Speed FLIM................................................................................................................................... 210
Acquisition Time of TCSPC FLIM....................................................................................................... 212
Multi-Exponential Decay Functions.................................................................................................... 216
IRF Recording......................................................................................................................................... 217
Effect of Fluorescence Depolarisation on
Measured Decay Curves............................................. 218
Special Microscopy Techniques................................................................................................................ 221
Multi-Beam Scanning Techniques...................................................................................................... 221
Stimulated Emission Depletion Microscopy
(STED)........................................................................ 222
Stage Scanning Systems....................................................................................................................... 223
Time-Resolved Optical Near-Field Microscopy
(SNOM)................................................................ 224
Biological FLIM Applications.................................................................................................................... 225
Measurement of Local Environment Parameters............................................................................... 225
Fluorescence Resonance Energy Transfer (FRET)........................................................................... 226
Autofluorescence Microscopy of Tissue.......................................................................................... 233
Internalisation and Conversion of
Photosensitisers for Photodynamic Therapy........................ 236
Other FLIM Applications...................................................................................................................... 236
Diffuse Optical Tomography............................................................................................................................. 237
Scanning Mammography............................................................................................................................ 240
Static Brain Imaging..................................................................................................................................... 242
Dynamic Brain Imaging................................................................................................................................ 243
Perfusion Measurements............................................................................................................................. 245
Other DOT Experiments............................................................................................................................... 247
Muscle and Bone Studies..................................................................................................................... 247
Fluorescence Lifetime in DOT.............................................................................................................. 247
Small-Animal Imaging............................................................................................................................ 248
Technical Aspects of DOT......................................................................................................................... 248
Optical Fibres.......................................................................................................................................... 248
Detectors................................................................................................................................................. 249
Autofluorescence of Tissue.............................................................................................................................. 251
Single-Point Multi-Wavelength Measurement........................................................................................ 251
Autofluorescence Lifetime Imaging........................................................................................................... 252
Ophthalmic Imaging..................................................................................................................................... 253
Picosecond Photon Correlation........................................................................................................................ 256
Antibunching................................................................................................................................................ 257
Technical Aspects........................................................................................................................................ 258
Fluorescence Correlation Spectroscopy......................................................................................................... 259
Principle.......................................................................................................................................................... 259
Calculation of FCS curves from TCSPC data............................................................................................ 260
FCS in Solution............................................................................................................................................. 263
FCS in Live Cells........................................................................................................................................... 263
Gated and Lifetime-Selective FCS.............................................................................................................. 264
Scanning FCS................................................................................................................................................ 265
Raster Image Correlation Spectroscopy (RICS)................................................................................. 265
FCS Imaging by slow scanning............................................................................................................ 266
Correlation of Delayed Detector Signals................................................................................................... 267
Fluorescence Correlation down to Picoseconds..................................................................................... 267
Practical Tips................................................................................................................................................. 269
TCSPC Setup Parameters for FCS........................................................................................................ 269
TCSPC-FCS with CW Excitation.......................................................................................................... 272
Interference of the Laser Period with the
Recording Clock............................................................. 273
Detectors................................................................................................................................................. 273
Afterpulsing of Detectors..................................................................................................................... 274
Background Signals............................................................................................................................... 275
Beam Jitter............................................................................................................................................... 275
Sample Volume........................................................................................................................................ 275
Optical Aberrations................................................................................................................................ 276
FCS with Commercial Laser Scanning
Microscopes......................................................................... 277
Time-Resolved Single-Molecule Spectroscopy............................................................................................. 278
Burst-Integrated Fluorescence Lifetime (BIFL)
Techniques.................................................................. 278
Multi-Parameter Fluorescence Detection and
Analysis......................................................................... 280
Single-Molecule FRET Experiments........................................................................................................... 284
Identification of Single Molecules............................................................................................................. 285
Correlation of Lifetime Fluctuations.......................................................................................................... 286
Photon Counting Histograms..................................................................................................................... 286
Two-Photon Excitation by Picosecond Diode
Lasers................................................................................... 289
Barrier Discharges............................................................................................................................................... 290
Positron Lifetime Measurements...................................................................................................................... 291
Ranging Systems................................................................................................................................................ 292
Remote Sensing................................................................................................................................................... 293
Measurement of Absolute Detector Quantum
Efficiency............................................................................ 294
Optical Oscilloscope........................................................................................................................................... 294
SPCM Software.......................................................................................................................................................... 297
Overview.............................................................................................................................................................. 297
Configuration of the SPC Main Panel........................................................................................................ 297
Changing Between Different Configurations........................................................................................... 299
Display and Trace Parameters.................................................................................................................... 299
Resizing and Positioning the Display Windows..................................................................................... 300
Cursors in the Display Windows of the Main
Panel............................................................................... 301
Trace Statistics.............................................................................................................................................. 302
On-Line Display............................................................................................................................................ 302
System Parameter Settings.......................................................................................................................... 302
Module Select (Multi-SPC Systems)......................................................................................................... 303
Status Information........................................................................................................................................ 303
Main............................................................................................................................................................................ 305
Load Panel............................................................................................................................................................ 305
Save Panel............................................................................................................................................................ 307
Predefined Setups............................................................................................................................................... 309
Multi-File View.................................................................................................................................................... 310
Data Export: Convert Functions....................................................................................................................... 311
Converting .sdt Files.................................................................................................................................... 311
Converting FIFO Files.................................................................................................................................. 312
Link to SPCImage Data Analysis...................................................................................................................... 314
Print....................................................................................................................................................................... 314
Sleep policy.......................................................................................................................................................... 315
System Parameters.................................................................................................................................................... 317
Operation Modes................................................................................................................................................ 317
‘Single’ Mode................................................................................................................................................ 318
Oscilloscope Mode...................................................................................................................................... 319
f(txy) Mode.................................................................................................................................................... 320
f(t,T) Mode.................................................................................................................................................... 322
f(t,EXT) Mode............................................................................................................................................... 324
fi(T) Mode...................................................................................................................................................... 326
fi(EXT) Mode................................................................................................................................................ 328
Continuous Flow Mode............................................................................................................................... 330
Scan Sync Out Mode................................................................................................................................... 333
Sequential Recording in the Scan Sync Out
mode.................................................................................. 335
Scan Sync In Mode...................................................................................................................................... 336
Scan Sync In, Continuous Flow Imaging.................................................................................................. 340
Scan XY Out Mode (SPC-700/730 only).................................................................................................... 341
Scan Mode Display...................................................................................................................................... 342
FIFO Mode.................................................................................................................................................... 344
FIFO Imaging Mode..................................................................................................................................... 348
Control Parameters (Photon Distribution
Modes)......................................................................................... 352
Stop Condition and Overflow Handling.................................................................................................... 352
Steps............................................................................................................................................................... 353
Cycles and Autosave................................................................................................................................... 353
Accumulate.................................................................................................................................................... 353
Repeat............................................................................................................................................................. 353
Trigger............................................................................................................................................................ 353
Display after each step and each cycle..................................................................................................... 354
Add / Sub Signal........................................................................................................................................... 354
Stepping Device............................................................................................................................................ 354
Timing Control Parameters................................................................................................................................ 354
Collection Time............................................................................................................................................. 354
Repeat Time................................................................................................................................................... 355
Display Time.................................................................................................................................................. 355
Dead Time Compensation........................................................................................................................... 356
CFD Parameters................................................................................................................................................... 356
SYNC Parameters................................................................................................................................................ 356
TAC Parameters.................................................................................................................................................. 357
Data Format.......................................................................................................................................................... 358
Page Control........................................................................................................................................................ 362
More Parameters................................................................................................................................................. 363
Parameter Management for Multi-Module SPC
systems.............................................................................. 363
Display Parameters.................................................................................................................................................... 365
General Display Parameters............................................................................................................................... 365
2D Display Parameters....................................................................................................................................... 366
3D Display Parameters....................................................................................................................................... 366
Displaying Subsets of Multidimensional Data............................................................................................... 368
Display Parameters for Different Display
Windows...................................................................................... 370
2D Trace Parameters................................................................................................................................................. 371
Trace Parameters for 2D Curve Mode.............................................................................................................. 371
Trace Parameters for 2D Block Mode.............................................................................................................. 373
Block Info............................................................................................................................................................. 374
Export of Trace Data........................................................................................................................................... 374
3D Trace Parameters................................................................................................................................................. 375
Window Intervals...................................................................................................................................................... 377
Time Windows.................................................................................................................................................... 377
Routing X and Y Windows............................................................................................................................... 379
Scan X and Y Windows..................................................................................................................................... 382
Auto Set Functions............................................................................................................................................ 383
Adjust Parameters..................................................................................................................................................... 385
Production Data.................................................................................................................................................. 385
Adjust Values...................................................................................................................................................... 385
Scanner Control......................................................................................................................................................... 387
General Scan Parameters.................................................................................................................................... 387
Control of Lasers................................................................................................................................................. 388
Preview Function................................................................................................................................................ 388
Scan Area............................................................................................................................................................. 389
Park Beam Function............................................................................................................................................ 389
Scanner Configuration....................................................................................................................................... 390
Assigning Names to the Laser Buttons.......................................................................................................... 391
Z Scanning........................................................................................................................................................... 391
Display Routines....................................................................................................................................................... 393
2D Display............................................................................................................................................................ 393
Cursors........................................................................................................................................................... 393
2D Data Processing...................................................................................................................................... 394
3D Display............................................................................................................................................................ 395
Display of Multi-Dimensional Data........................................................................................................... 395
Cursor and Zoom Functions....................................................................................................................... 396
3D Data Processing...................................................................................................................................... 398
Starting and Stopping a Measurement.................................................................................................................. 399
Start....................................................................................................................................................................... 399
Interrupt................................................................................................................................................................ 399
Stop....................................................................................................................................................................... 399
Exit......................................................................................................................................................................... 400
Data file structure...................................................................................................................................................... 401
Data of the Photon Distribution Modes.......................................................................................................... 401
File Header..................................................................................................................................................... 401
File Information............................................................................................................................................. 402
Setup............................................................................................................................................................... 402
Measurement Description Blocks.............................................................................................................. 403
Data Blocks.................................................................................................................................................... 403
FIFO Files............................................................................................................................................................. 405
Setup Files..................................................................................................................................................... 405
FIFO Data Files (SPC-600/630, 4096 Channel
Mode).............................................................................. 405
FIFO Data Files (SPC-600/630 256 Channel
Mode)................................................................................. 406
FIFO Data Files (SPC-134, SPC-144, SPC‑154,
SPC-830)......................................................................... 407
Trouble Shooting...................................................................................................................................................... 409
How to Avoid Damage....................................................................................................................................... 409
Testing the Module by the SPC Test Program............................................................................................... 410
Test for General Function and for
Differential Nonlinearity......................................................................... 410
Test for Time Resolution................................................................................................................................... 411
Frequently Encountered Problems................................................................................................................... 411
Assistance through bh...................................................................................................................................... 417
Routing and Control Signals................................................................................................................................... 418
SPC-600/630......................................................................................................................................................... 418
SPC-700/730 and SPC-830.................................................................................................................................. 419
SPC‑130, SPC-134................................................................................................................................................ 422
SPC‑140, SPC-144, SPC‑150, SPC‑154............................................................................................................... 423
DCC‑100 Detector Controller................................................................................................................................... 425
Specifications............................................................................................................................................................. 427
SPC-600/630......................................................................................................................................................... 427
SPC-700/730......................................................................................................................................................... 428
SPC-830................................................................................................................................................................. 429
SPC-130/134......................................................................................................................................................... 430
SPC-140/144......................................................................................................................................................... 431
SPC-150/154......................................................................................................................................................... 432
DCC-100 Detector Controller............................................................................................................................. 433
Absolute Maximum Ratings (for all SPC
modules)........................................................................................ 434
References.................................................................................................................................................................. 435
Index............................................................................................................................................................................ 454