Becker W. Advanced Time-Correlated Single Photon Counting Techniques

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81 Advanced Time-Correlated Single

Photon Counting Techniques

By W. Becker

W. B e cker

Advanced Time-Correlated

Single Photon

Counting Techniques

With 349 Figures

123

Dr. Wolfgang Becker

Becker & Hickl GmbH

Nahmitzer Damm 30

12277 Berlin, Germany

E-Mail: becker@becker-hickl.com

Series Editors:

Professor A. W. Castleman, Jr.

Department of Chemistry, The P ennsylvania State University

152 Davey Laboratory, University Park, PA 16802, USA

Professor J.P. Toennies

Max-Planck-Institut für Str

omungsforsch ung, Bunsenstraße 10

37073 G

ottingen, Germany

Professor W. Zinth

Universit

at M

unchen, Institut f

ur Medizinische Optik

Ottingerstr. 67, 80538 M

unchen, Germany

ISSN 0172-6218

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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 Wolfgang Becker

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