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2015 | Buch

Advanced Photon Counting

Applications, Methods, Instrumentation

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This volume focuses on Time-Correlated Single Photon Counting (TCSPC), a powerful tool allowing luminescence lifetime measurements to be made with high temporal resolution, even on single molecules. Combining spectrum and lifetime provides a “fingerprint” for identifying such molecules in the presence of a background. Used together with confocal detection, this permits single-molecule spectroscopy and microscopy in addition to ensemble measurements, opening up an enormous range of hot life science applications such as fluorescence lifetime imaging (FLIM) and measurement of Förster Resonant Energy Transfer (FRET) for the investigation of protein folding and interaction. Several technology-related chapters present both the basics and current state-of-the-art, in particular of TCSPC electronics, photon detectors and lasers. The remaining chapters cover a broad range of applications and methodologies for experiments and data analysis, including the life sciences, defect centers in diamonds, super-resolution microscopy, and optical tomography. The chapters detailing new options arising from the combination of classic TCSPC and fluorescence lifetime with methods based on intensity fluctuation represent a particularly unique highlight.

Inhaltsverzeichnis

Frontmatter
Modern TCSPC Electronics: Principles and Acquisition Modes
Abstract
Time-correlated single-photon counting (TCSPC) is an extraordinarily versatile and sensitive technique. While it was initially used almost only to measure excited state lifetimes, it can today be used much more flexibly, embracing and combining experimental methods that in the past required separate instrumentation. This has become possible by time-tagged event recording and modern time measurement circuitry. This chapter shows how such technologies operate with regard to electronics, data processing, and applications. Some implementation details will be exemplified by state-of-the-art TCSPC instruments and a recent software package for TCSPC data acquisition and analysis.
Michael Wahl
Single-Photon Counting Detectors for the Visible Range Between 300 and 1,000 nm
Abstract
Single-photon counting in the visible spectral range has become a standard method for many applications today, ranging from fluorescence spectroscopy to single-molecule detection and quantum optics. One of the key components for every setup is single-photon sensitive detectors. Unfortunately a detector with “ultimate” features, i.e., high detection efficiency at a large wavelength range, high temporal resolution, and low dark counts, does not exist. For most of the applications, it is therefore necessary to choose a detector based on the most crucial parameters for the targeted application.
This chapter provides an overview about the typically used single-pixel detectors for photon counting in the visible range. It provides information about the key parameters such as detection efficiency, dark counts and timing resolution that principally allow to choose the best suited detector for a targeted application.
Andreas Bülter
Single-Photon Detectors for Infrared Wavelengths in the Range 1–1.7 μm
Abstract
The ongoing progress of scientific research in areas such as quantum communications, low-light level laser ranging, and material science (to name but a few) has led to increased interest in the detection of single photons in the wavelength range 1–1.7 μm. Several technologies have been used to detect photons with wavelengths in this range – each with different characteristic parameters that affect their suitability for specific applications. This chapter will provide a review of progress in the development of detectors for use in this spectral region and will highlight some notable results.
Gerald S. Buller, Robert J. Collins
Modern Pulsed Diode Laser Sources for Time-Correlated Photon Counting
Abstract
Time-correlated single-photon counting applications require pulsed excitation sources at various wavelengths from the UV to the IR that feature a short pulse width (usually picoseconds or femtoseconds) as well as repetition rates in the kilohertz to megahertz range. The repetition rate should ideally be tunable in order to adapt the pulse period to the required measurement window. In the blue, red, and IR spectral range, such pulses with energies of up to 100 pJ can be readily provided by single gain-switched laser diodes which can be housed in compact and robust packages. Laser pulses in the UV or green-yellow spectral range are, however, not directly accessible and require more elaborate setups that are based on power amplification and frequency conversion. An alternative excitation source that has also become popular in the recent years is the supercontinuum laser as it gives direct access to a broad wavelength spectrum that spans from the blue to the IR.
This chapter provides an overview about the fundamental aspects and parameters of pulsed diode lasers as well as a short introduction into pulsed LEDs and supercontinuum lasers that are usually used for time-correlated single-photon counting applications.
Thomas Schönau, Sina Riecke, Andreas Bülter, Kristian Lauritsen
Advanced FCS: An Introduction to Fluorescence Lifetime Correlation Spectroscopy and Dual-Focus FCS
Abstract
This chapter focuses on two advanced fluorescence correlation spectroscopy (FCS) methods; fluorescence lifetime correlation spectroscopy (FLCS) and dual-focus FCS (2fFCS). We decided to put our focus on a detailed discussion of these two – and in our eyes well-merited – advanced methods, rather than giving an overview over the broad variety of advanced FCS methods that would consequently lack detail and leave the reader rather uneducated on all these methods. For this reason we had to exclude some candidates that would very well deserve the same amount of attention as the methods that we chose to focus on. Amongst these candidates camera-FCS, Bayes-FCS, and scanning-FCS are to be kept on the radar for sure.
The great benefit of FLCS is that it provides a general tool that allows filtering for sub-populations, afterpulsing-artifacts, background effects, and basically anything that can be distinguished by its fluorescence lifetime. Complementarily, 2fFCS has brought a new level of accuracy to the table that has been previously reached only by complementary methods such as for example pulsed-field gradient NMR.
Thomas Dertinger, Steffen Rüttinger
Lifetime-Weighted FCS and 2D FLCS: Advanced Application of Time-Tagged TCSPC
Abstract
Time-tagged TCSPC (time-correlated single photon counting) is a special acquisition mode of TCSPC with which one determines not only the excitation-emission delay time of detected photons but also their arrival times measured from the start of the experiment. Time-tagged TCSPC enables us to examine slow fluctuation of fluorescence lifetimes, which is particularly important in the study of heterogeneous or fluctuating systems at the single-molecule level. In this chapter, we describe recent development of new methods using time-tagged TCSPC, aiming at showing their high potential in studying dynamics of complex systems. We depict two closely related methods based on fluorescence correlation spectroscopy (FCS), i.e., lifetime-weighted FCS and two-dimensional fluorescence lifetime correlation spectroscopy (2D FLCS). These methods enable us to quantify fluorescence lifetime fluctuations on the microsecond timescale. Showing examples including the study of a biological macromolecule, we demonstrate the usefulness of these two methods in real applications. In addition, we present another application of time-tagged TCSPC, which analyzes photon interval time for characterizing timing instability of photon detectors.
Kunihiko Ishii, Takuhiro Otosu, Tahei Tahara
MFD-PIE and PIE-FI: Ways to Extract More Information with TCSPC
Abstract
Pulsed interleaved excitation (PIE) is the method of fast alternation of pulsed lasers for quasi-simultaneous observation of fluorophores with different spectral properties. PIE was originally introduced to enable artifact-free fluorescence cross-correlation measurements, while first experiments with alternating laser excitation (ALEX) used the dual excitation of donor and acceptor for single-pair Förster resonance energy transfer (spFRET). In this article, we will review the benefit of PIE for spFRET experiments with multiparameter fluorescence detection (MFD). The direct probing of the acceptor fluorophore in PIE increases the robustness of the quantitative MFD analysis and extends it to even more parameters.
Recently, PIE has been combined with commonly used fluorescence fluctuation imaging techniques such as raster image correlation spectroscopy (RICS) and the number and brightness analysis (N&B). We highlight how PIE improves these methods, and how artifacts in the analysis can be avoided. Similar to PIE-FCS, quantitative cross-correlation raster image correlation spectroscopy (ccRICS) is greatly simplified. Additionally, the lifetime information can be used to further increase the contrast and sensitivity of the method with raster lifetime image correlation spectroscopy (RLICS).
Anders Barth, Lena Voith von Voithenberg, Don C. Lamb
Photon Antibunching in Single Molecule Fluorescence Spectroscopy
Abstract
Single molecule fluorescence spectroscopy (SMFS) opened an important pathway for studying properties of individual quantum systems under ambient conditions. One such fundamental characteristic is based on the fact that single emitters can only emit single photons. This leads to the phenomenon of antibunching, that is, the probability for detecting multiple photons approaches zero for decreasing detection time windows shorter than the excited state lifetime. In the last decade photon antibunching has regained interest by many researchers in the field of SMFS for two main reasons. First, the observation of antibunching by measuring photon correlations could easily be transferred to become the only direct proof that a single fluorescing molecule is observed. This is crucial for quantum information processing, quantum cryptography, and metrology. Second, its characteristic photon statistics could be exploited for estimating the number of independently emitting molecules with a confocal fluorescence microscope. Recent applications aim at understanding mechanisms of energy transfer in multichromophoric molecules and photo-systems and at quantifying copy numbers in protein complexes. This chapter highlights different methods to measure photon antibunching in SMFS experiments. Aside from technical aspects we will consider the fundamental theories that are used for data analysis. Each methodological approach is then followed by a section illustrating the respective applications of photon antibunching.
Kristin S. Grußmayer, Dirk-Peter Herten
FLIM Strategies for Intracellular Sensing
Fluorescence Lifetime Imaging as a Tool to Quantify Analytes of Interest
Abstract
Since the very early years of microscopy development, scientists have pursued the ability to observe live cellular activity in order to probe the processes occurring inside cells in real time. Fluorescence microscopy has become an extraordinary tool for unraveling the myriad cellular processes and interactions that are relevant to understanding cell physiology. The intracellular sensing of certain analytes is of crucial importance to understanding some of these processes, such as the relation between cellular pH and metabolic states or the roles of specific ions in signaling pathways. However, the acquisition of quantitative information from the interiors of cells is not a trivial challenge. Ratiometric, intensity-based fluorescence microscopy approaches are commonly used, but they suffer from many systematic difficulties that make them unsuitable for quantitative sensing. Fluorescence lifetime imaging microscopy (FLIM) detects the time duration of fluorescence emission, taking advantage of the multidimensional nature of photon emission. FLIM-based intracellular sensing approaches, especially in the time domain in single-photon timing (SPT) mode, overcome many of the limitations of fluorescence-intensity methods. Herein, we review strategies for the FLIM-based intracellular sensing of local pH, ion concentration, and biomolecular interactions. In the first section, we demonstrate how in-depth knowledge of the photophysics of dyes can be useful in the development of FLIM sensors. Then, we review the growing field of nanoparticle-based FLIM sensors. Finally, the expanding detection capabilities of FRET and the use of FLIM for the larger-scale analysis of tissues are discussed.
Maria J. Ruedas-Rama, Jose M. Alvarez-Pez, Luis Crovetto, Jose M. Paredes, Angel Orte
Multiple-Pulse Pumping with Time-Gated Detection for Enhanced Fluorescence Imaging in Cells and Tissue
Abstract
Fluorescence-based sensing and imaging experiments are constrained by the background signal generated on a sample. A main contribution to the background, besides direct scattering of excitation and Raman scattering of the solvent, comes from sample autofluorescence and additives used for sample preparation. Such unwanted signals from endogenous chromophores and fixatives typically are broad and spectrally overlap with the probe signal; thus becoming a major limitation for sensitive detection and quantitative imaging. Since the fluorescence lifetimes of the majority of naturally occurring chromophores are relatively short, long-lived fluorophores allow for background discrimination by time-gated detection. Unfortunately, the brightness of long-lived, red-emitting fluorescent probes is inherently very low, consequently limiting many applications. Recently we reported a simple new approach with bursts of closely spaced laser excitation pulses for excitation (multi-pulse excitation) that allows for many-fold increase in the intensity of a long-lived probe over the background signal. This technology can be easily implemented for biomedical diagnostics and imaging to significantly enhance the signal of long-lived probes over the background. In this report, we are discussing an example of the Ruthenium-based dye tris(2,2′-bipyridyl)dichlororuthenium(II) hexahydrate (Ru) (Sigma–Aldrich) (~2% quantum yield and ~350 ns fluorescence lifetime) that when used with the multi-pulse approach and time-gated detection allows for high quality imaging that can easily be enhanced two orders of magnitude as compared to the normal approach (imaging with typical fluorescence microscopy).
Rafal Fudala, Ryan M. Rich, Joe Kimball, Ignacy Gryczynski, Sangram Raut, Julian Borejdo, Dorota L. Stankowska, Raghu R. Krishnamoorthy, Karol Gryczynski, Badri P. Maliwal, Zygmunt Gryczynski
Pattern-Based Linear Unmixing for Efficient and Reliable Analysis of Multicomponent TCSPC Data
Abstract
A method for a reliable quantitative analysis of fluorescence lifetime imaging microscopy (FLIM) data is presented. It is based on the linear unmixing of the intensity decay on the basis of selected reference patterns. This approach allows to use decays that are not mono-exponential without increasing the complexity of the analysis. This is a major benefit when working with labeled biomolecules or using autofluorescent cellular chromophores.
The method can be used intuitively and is fast. Furthermore, based on the reference patterns and the amount of recorded photons, one can easily determine confidence levels of the obtained results. We demonstrate that for a decomposition to three patterns of common chromophores, one achieves a standard deviation of better than 10% for as few as 1,000 photons per pixel, where the total amplitude of such a signal will show an error of 3% due to shot noise. Indeed, the accuracy of the results is very close to a maximum-likelihood estimator that defines the absolute limit for this kind of problem.
Ingo Gregor, Matthias Patting
Metal-Induced Energy Transfer
Abstract
This chapter presents an overview of the recently introduced concept of metal-induced energy transfer and two of its applications. We discuss the basic principle of the method and its application to the mapping of the membrane of a living cell and to the single-molecule axial localization with 2–3 nm accuracy.
Narain Karedla, Daja Ruhlandt, Anna M. Chizhik, Jörg Enderlein, Alexey I. Chizhik
The Importance of Photon Arrival Times in STED Microscopy
Abstract
Lens-based or far-field fluorescence microscopy is a very popular technique for investigating the living cell. However, the spatial resolution of its standard versions is limited to about 200 nm due to diffraction, impeding the imaging of molecular assemblies at smaller scales. The turn of the twenty-first century has witnessed the advent of far-field fluorescence super-resolution microscopy or nanoscopy, a fluorescence microscopy featuring a spatial resolution down to molecular scales. STED microscopy was the first of such nanoscopy techniques, but was for a long time considered as a very complex technique, hard to apply in everyday biological research. Based on developments in label and laser technology, recent years have however seen major improvements of the STED nanoscopy approach, one of which is gated continuous-wave STED (gCW-STED) microscopy. gCW-STED microscopy reduces complexity by combining STED laser operating in CW with pulsed excitation and time-gated photon detection. Here, we describe the physical principles of gCW-STED, formulate the theoretical framework which characterizes its main benefits and limitations, as well as show experimental data.
Giuseppe Vicidomini, Ivàn Coto Hernàndez, Alberto Diaspro, Silvia Galiani, Christian Eggeling
Single-Color Centers in Diamond as Single-Photon Sources and Quantum Sensors
Abstract
Single quantum systems in the solid state have a potential application for quantum information processing. Among these, color defects in diamond seem to be the most promising ones, as they can operate at ambient conditions. In this chapter the optical and spin properties of the widely investigated nitrogen-vacancy (NV) and silicon-vacancy (SiV) centers in diamonds will be reviewed. We will present the latest experiments showing their application as single-photon sources, qubits, and sensitive magnetic field sensors with nanometer spatial resolution.
Boris Naydenov, Fedor Jelezko
Photon Counting and Timing in Quantum Optics Experiments
Abstract
In this chapter we briefly review present implementations of single-photon and photon-pair sources. After providing the basic fundamentals of nonclassical light, the role of photon detection to characterize these sources is highlighted. Then, we motivate why ongoing experiments heading at the realization of more complex quantum optical devices make very high demands on detectors and detection electronics. First, results towards quantum repeater architectures and hybrid quantum systems are discussed. Finally, we outline future prospects of all-optical quantum technologies.
Andreas Ahlrichs, Benjamin Sprenger, Oliver Benson
Photon Counting in Diffuse Optical Imaging
Abstract
The high sensitivity and the picosecond time resolution of time-correlated single photon counting have led to the application of this technique for diffuse optical imaging of biological tissue in vivo in the near-infrared spectral range. In this chapter the fundamentals of photon propagation in biological tissue and the concept of the distribution of times of flight of scattered photons are briefly discussed. Then the main features of time-resolved, frequency-domain, and continuous-wave techniques are compared. An overview is given on the application of time-correlated single photon counting for investigations on human breast tissue, on the brain, and on muscle tissue. In the second part, experimental approaches and clinical studies on the detection and characterization of breast tumors based on oxy- and deoxyhemoglobin concentrations are considered in more detail. The application of time-resolved measurements to monitor breast tumor degeneration by neoadjuvant chemotherapy is discussed. Finally, fluorescence mammography with the contrast agent indocyanine green is considered as a tool to improve differentiation between malignant and benign breast lesions.
Dirk Grosenick
Backmatter
Metadaten
Titel
Advanced Photon Counting
herausgegeben von
Peter Kapusta
Michael Wahl
Rainer Erdmann
Copyright-Jahr
2015
Electronic ISBN
978-3-319-15636-1
Print ISBN
978-3-319-15635-4
DOI
https://doi.org/10.1007/978-3-319-15636-1

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