Advanced Time-Correlated Single Photon Counting Techniques

415 Pages·2005·5.205 MB·English

by  Wolfgang Becker

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Advanced Time-Correlated
Single Photon
Counting Techniques
With349Figures
123
Dr.WolfgangBecker
Becker&HicklGmbH
NahmitzerDamm30
12277Berlin,Germany
E-Mail:[email protected]
SeriesEditors:
ProfessorA.W.Castleman,Jr.
DepartmentofChemistry,ThePennsylvaniaStateUniversity
152DaveyLaboratory,UniversityPark,PA16802,USA
ProfessorJ.P.Toennies
Max-Planck-InstitutfürStr¨omungsforschung,Bunsenstraße10
37073G¨ottingen,Germany
ProfessorW.Zinth
Universit¨atMu¨nchen,Institutfu¨rMedizinischeOptik
O¨ttingerstr.67,80538Mu¨nchen,Germany
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Preface
In 1984 Desmond O’Connor and David Phillips published their comprehensive
book „Time-correlated Single Photon Counting“. At that time time-correlated sin-
gle photon counting, or TCSPC, was used primarily to record fluorescence decay
functions of dye solutions in cuvettes. From the beginning, TCSPC was an amaz-
ingly sensitive and accurate technique with excellent time-resolution. However,
acquisition times were relatively slow due to the low repetition rate of the light
sources and the limited speed of the electronics of the 70s and early 80s. Moreover,
TCSPC was intrinsically one-dimensional, i.e. limited to the recording of the wave-
form of a periodic light signal. Even with these limitations, it was a wonderful tech-
nique.
More than 20 years have elapsed, and electronics and laser techniques have made
impressive progress. The number of transistors on a single chip has approximately
doubled every 18 months, resulting in a more than 1,000-fold increase in complex-
ity and speed. The repetition rate and power of pulsed light sources have increased
by about the same factor.
One might presume that TCSPC had been rendered obsolete in 20 years. Nothing
like that happened. On the contrary, TCSPC has got a considerable push from the
development of lasers and electronics. It has developed from a sluggish, intrinsi-
cally one-dimensional fluorescence lifetime technique into a fast, multidimensional
optical recording technique. Advanced TCSPC is now used for applications like
single-molecule spectroscopy, fluorescence correlation spectroscopy, time-
resolved laser scanning microscopy, and diffuse optical tomography. Nevertheless,
surprisingly little has been published about the development of TCSPC techniques
in the last 10 years.
Desmond O’Connor and David Phillips wrote in their preface: „It is perhaps ar-
rogance which causes us to believe that new users of the technique do not always
appreciate fully the advantages and pitfalls of the equipment and the curve fitting
routines necessarily used with this hardware.“ The advantages and some of the
pitfalls do still exist, and further advantages and a few pitfalls have emerged. Oper-
ating an instrument as a black box may deliver results at a satisfactory level of accu-
racy. However, if appropriately used, advanced TCSPC not only delivers better
results but also solves highly sophisticated problems. It is the goal of this book to
help existing and potential users understand and make use of the advanced features
of TCSPC.
The book starts with some general remarks about optical signal recording. After
a brief introduction to the most common photon-counting techniques the general
principle of TCSPC is explained. Then the principles of multidimensional TCSPC,
multidetector techniques, sequential recording, imaging techniques, and time-tag
VI Preface
recording are described. Typical applications of advanced TCSPC are introduced
and special technical problems associated with these applications are addressed. An
overview about detectors, detector parameters, their impact on TCSPC measure-
ments, and the performance of selected detectors is presented. A final section gives
practical hints on how to build and use TCSPC systems, including optical compo-
nents and systems, electronics, problems of electromagnetic shielding, optimisation
of TCSPC system parameters, and calibration.
The book does not include data analysis. Analysis of multispectral TCSPC data,
diffuse optical tomography data, or fluorescence correlation data differs considera-
bly from traditional fluorescence decay analysis, and many analysis problems are
not entirely solved yet. The author believes that data analysis should be the subject
of a different book, and leaves this task to someone who is more familiar with it.
I thank Heidrun Wabnitz for encouraging me to write this book, and for countless
helpful hints and discussions. I am also indebted to my collaborators for their work
in the development of TCSPC. My thanks go especially to Helmut Hickl for devel-
oping the digital signal processing techniques, Stefan Smietana for his tremendous
amount of work in designing instrument software, and Axel Bergmann for his data-
analysis developments and his untiring strategic endeavour to push TCSPC into
promising applications.
I am indebted also to those workgroups who have ventured into using advanced
TCSPC features in new applications. I thank in particular Karsten König who was
the first to use fast TCSPC scanning in two-photon microscopy; Dietrich
Schweitzer who built the first time-resolved ophthalmic imager; Herbert Rinneberg,
Britton Chance, and Rinaldo Cubeddu and their collaborators for introducing
TCSPC into diffuse optical tomography; Michael Prummer, Markus Sauer, and
Claus Seidel for their single-molecule applications; and Hans-Erich Wagner, Ronny
Brandenburg, and Kirill Kozlov for their multidimensional TCSPC experiments on
gas discharges. I must also give thanks to Christoph Biskup for his work on FRET
in living cells, and Ammasi Periasamy, Rory Duncan, Brian Bacskai, Enrico Grat-
ton, John White, Dennis Fan, Damain Bird, and Peter So for related microscopy
applications. I am indebted to Advanced Research Technologies (ART), Biorad,
Leica Microsystems, and Zeiss for their cooperation.
Many thanks also to my former professors, Siegfried Dähne and Edgar Klose for
their support and the productive working atmosphere they fostered in the early days
of TCSPC development.
Finally, I thank Sarah Smith for improving the English of this book.
Berlin, January 2005 WolfgangBecker
Contents
List of Technical Terms and Symbols........................................ XIII
1 Optical Signal Recording................................................................. 1
2 Overview of Photon Counting Techniques................................... 11
2.1 Steady-State Photon Counting............................................... 11
2.2 Gated Photon Counting.......................................................... 12
2.3 Multichannel Scalers.............................................................. 16
2.4 Time-Correlated Single Photon Counting (TCSPC).............. 20
2.4.1 General Principle...................................................... 20
2.4.2 The Classic TCSPC Setup........................................ 23
2.4.3 Reversed Start-Stop.................................................. 24
3 Multidimensional TCSPC Techniques......................................... 27
3.1 Multidetector TCSPC............................................................. 29
3.2 Multiplexed TCSPC............................................................... 33
3.3 Sequential Recording Techniques.......................................... 35
3.4 Scanning Techniques.............................................................. 37
3.5 Imaging by Position-Sensitive Detection............................... 39
3.6 Time-Tag Recording.............................................................. 43
3.7 Multimodule Systems............................................................. 45
4 Building Blocks of Advanced TCSPC Devices............................. 47
4.1 Constant-Fraction Discriminators.......................................... 47
4.2 Time Measurement Block...................................................... 50
4.2.1 Time Measurement by Fast TAC / ADC Principle... 50
4.2.2 Digital TDCs............................................................. 55
4.2.3 Sine-Wave Conversion............................................. 59
5 Application of Modern TCSPC Techniques................................. 61
5.1 Classic Fluorescence Lifetime Experiments.......................... 61
5.1.1 Time-Resolved Fluorescence.................................... 61
5.1.2 Fluorescence Lifetime Spectrometers....................... 65
5.1.3 Fluorescence Depolarisation Effects......................... 69
VIII Contents
5.1.4 Reabsorption and Reemission................................... 72
5.1.5 High-Efficiency Detection Systems.......................... 72
5.1.6 Measurement of the Instrument Response
Function.................................................................... 75
5.1.7 What is the Shortest Lifetime that can
be Measured?............................................................ 77
5.1.8 Fluorescence Anisotropy.......................................... 79
5.1.9 Time Resolved Spectra............................................. 82
5.2 Multispectral Fluorescence Lifetime Experiments................. 84
5.3 Excitation-Wavelength Multiplexing..................................... 87
5.4 Transient Fluorescence Lifetime Phenomena........................ 90
5.4.1 Chlorophyll Transients.............................................. 90
5.4.2 Continuous-Flow Mixing Techniques...................... 94
5.4.3 Stopped-Flow Techniques........................................ 95
5.5 Diffuse Optical Tomography (DOT) and Photon Migration.. 97
5.5.1 Principle of Diffuse Optical Tomography................ 97
5.5.2 Scanning Mammography........................................ 102
5.5.3 Brain Imaging......................................................... 106
5.5.4 Muscle and Bone Studies........................................ 112
5.5.5 Exogenous Absorbers............................................. 112
5.5.6 Fluorescence........................................................... 113
5.5.7 Small-Animal Imaging............................................ 115
5.5.8 Technical Aspects of TCSPC-Based DOT............. 117
5.6 Autofluorescence of Biological Tissue................................ 121
5.6.1 Autofluorescence Detection
by Multispectral TCSPC......................................... 122
5.6.2 Two-Photon Autofluorescence............................... 124
5.6.3 Ophthalmic Imaging............................................... 126
5.7 TCSPC Laser Scanning Microscopy.................................... 129
5.7.1 The Laser Scanning Microscope............................. 131
5.7.2 Lifetime Imaging Techniques for Laser
Scanning Microscopy.............................................. 134
5.7.3 Implementation of Multidimensional TCSPC........ 137
5.7.4 Multispectral FLIM................................................. 143
5.7.5 High Count-Rate Systems....................................... 146
5.7.6 FRET Measurements by TCSPC FLIM.................. 149
5.7.7 Technical Details of TCSPC Laser Scanning
Microscopy............................................................. 154
5.8 Other TCSPC Microscopy Techniques................................ 163
5.8.1 TCSPC Lifetime Imaging by Scan Stages.............. 163
5.8.2 Microfluorometry.................................................... 166
Contents IX
5.8.3 Time-Resolved Scanning Near-Field Optical
Microscopy............................................................. 167
5.8.4 TCSPC Wide-Field Microscopy............................. 168
5.9 Picosecond Photon Correlation............................................ 169
5.9.1 AntiBunching Experiments..................................... 170
5.9.2 Practical Details...................................................... 173
5.10 Fluorescence Correlation Spectroscopy............................... 176
5.10.1 Combined FCS/Lifetime Experiments
by TCSPC............................................................... 177
5.10.2 FCS in Laser Scanning Microscopes...................... 182
5.10.3 Practical Tips.......................................................... 184
5.11 Combinations of Correlation Techniques............................. 187
5.11.1 Combining Picosecond Correlation and FCS......... 188
5.11.2 Correlation of Delayed Detector Signals................ 188
5.11.3 Synchronisation of TCSPC Modules...................... 189
5.12 The Photon Counting Histogram.......................................... 191
5.13 Time-Resolved Single Molecule Spectroscopy.................... 193
5.13.1 Burst-Integrated Fluorescence Lifetime (BIFL)
Experiments............................................................ 194
5.13.2 Identification of Single Molecules.......................... 196
5.13.3 Multiparameter Spectroscopy of
Single Molecules..................................................... 198
5.14 Miscellaneous TCSPC Applications.................................... 201
5.14.1 Two-photon Fluorescence with Diode Laser
Excitation................................................................ 201
5.14.2 Remote Sensing...................................................... 204
5.14.3 Laser Ranging......................................................... 206
5.14.4 Positron Lifetime Experiments............................... 206
5.14.5 Diagnostics of Barrier Discharges.......................... 208
5.14.6 Sonoluminescence................................................... 210
5.14.7 The TCSPC Oscilloscope....................................... 211
6 Detectors for Photon Counting.................................................... 213
6.1 Detector Principles............................................................... 213
6.1.1 Conventional Photomultiplier Tubes...................... 213
6.1.2 Channel and Microchannel PMTs........................... 214
6.1.3 Position-Sensitive PMTs......................................... 215
6.1.4 Single-Photon Avalanche Photodiodes................... 217
6.1.5 Hybrid PMTs.......................................................... 220
6.1.6 Other Detector Principles........................................ 221