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Topic Introduction
Fluorescence Correlation Spectroscopy: Principles and Applications Kirsten Bacia, Elke Haustein, and Petra Schwille
Fluorescence correlation spectroscopy (FCS) is used to study the movements and the interactions of biomolecules at extremely dilute concentrations, yielding results with good spatial and temporal resolutions. Using a number of technical developments, FCS has become a versatile technique that can be used to study a variety of sample types and can be advantageously combined with other methods. Unlike other fluorescence-based techniques, the analysis of FCS data is not based on the average intensity of the fluorescence emission but examines the minute intensity fluctuations caused by spontaneous deviations from the mean at thermal equilibrium. These fluctuations can result from variations in local concentrations owing to molecular mobility or from characteristic intermolecular or intramolecular reactions of fluorescently labeled biomolecules present at low concentrations. Here, we provide a basic introduction to FCS, including its technical development and theoretical basis, experimental setup of an FCS system, adjustment of a setup, data acquisition, and analysis of FCS measurements. Finally, the application of FCS to the study of lipid bilayer membranes and to living cells is discussed.
INTRODUCTION
A steadily increasing demand for better minimally invasive analytical tools to answer specific biological questions has boosted the development of fluorescence-based techniques. Fluorescence correlation spectroscopy (FCS) is a well-established method for analyzing solutions of biomolecules at low concentrations, ranging from nanomolar to low micromolar. One of the advantages of FCS is that it requires only small sample volumes. In a basic type of experiment, the optically delimited, femtoliter-sized FCS detection volume is placed inside a drop of a few microliters of a homogeneous sample solution. In a more sophisticated experiment, the FCS detection volume can be positioned inside or on the membrane of a single eukaryotic cell, on a small biosynthetic container (such as a giant liposome), or inside a microfluidic structure. FCS is a rather flexible method with regard to the sample preparation and the optical setup and can be readily combined with other microscopic techniques such as confocal microscopy or atomic force microscopy. FCS is often referred to as a single-molecule technique because the fluctuations in fluorescence intensity arise from fluctuations in the number or in the brightness of single fluorescent particles. However, each particle is not followed and analyzed individually, which is why FCS may be more accurately described as a single-molecule-sensitive technique. All fluctuating physical parameters influencing the fluorescence signal are, in principle, accessible by FCS. These comprise local concentrations, mobility coefficients (translational diffusion, active transport, rotational diffusion), and rate constants of intramolecular and photophysical reactions (triplet blinking and photon antibunching). Moreover, processes that indirectly influence these parameters can
Adapted from Imaging: A Laboratory Manual (ed. Yuste). CSHL Press, Cold Spring Harbor, NY, USA, 2011. © 2014 Cold Spring Harbor Laboratory Press Cite this introduction as Cold Spring Harb Protoc; doi:10.1101/pdb.top081802
709
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K. Bacia et al.
be analyzed, such as a molecular binding that increases the size and reduces the diffusion coefficient of a molecule. In addition, dual-color fluorescence cross-correlation spectroscopy (dcFCCS) can be implemented to analyze the correlated motion of two distinct fluorescent labels. In this way, cleavage and binding reactions as well as dynamic colocalization during intracellular transport can be studied. TECHNIQUE DEVELOPMENT
The theoretical foundation of FCS is based on the laws of molecular diffusion, formulated from Brown’s observations of random particle motion, and their analysis by Einstein and Smoluchowski at the beginning of the 20th century (Smoluchowski 1906). FCS as a technique was developed in the early 1970s (Magde et al. 1972, 1974; Elson and Magde 1974) as a “miniaturization” of dynamic light scattering. The novelty of FCS lay in the analysis of fluctuations in the fluorescence emission of the sample molecules, induced by spontaneous fluctuations of physical parameters. FCS was first used to measure the diffusion and chemical kinetics of ethidium bromide intercalation into DNA (Magde et al. 1972) and rotational diffusion (Ehrenberg and Rigler 1974). Following these proof-of-principle measurements, a variety of studies were devoted to the investigation of particle concentration and mobility: For example, three-dimensional, and two-dimensional (2D) diffusion (Aragón and Pecora 1976; Fahey et al. 1977) or laminar flow (Magde et al. 1978). In an attempt to restrict the system under investigation to small molecular numbers and, thus, to enhance both detection sensitivity and background suppression, Rigler and coworkers combined FCS with confocal detection (Rigler and Widengren 1990; Rigler et al. 1993). The analytical potential of FCS for the life sciences has been shown in numerous applications since the original work of Rigler and coworkers (Eigen and Rigler 1994; Schwille et al. 1997b). FCS was successfully used to study association and dissociation of nucleic acids (Kinjo and Rigler 1995) and proteins (Rauer et al. 1996). Moreover, a multitude of environmental effects inducing fluctuations in the fluorescence yield of single dye molecules could be studied, including reversible protonation (Haupts et al. 1998), electron transfer, and oxygen and ion concentrations. By introducing a dual-color cross-correlation scheme for the simultaneous observation of different fluorescent species (Schwille et al. 1997a), the detection specificity in bimolecular association and dissociation processes was significantly enhanced. Combined with extremely short data acquisition times, these features even allowed for real-time investigation of enzyme kinetics (Kettling et al. 1998). Cellular measurements were also reported at a very early stage (Elson et al. 1976). However, in turbid media, especially (although not exclusively) inside the cell, the signal-to-noise ratio is influenced by autofluorescence and scattering. In addition to this, photobleaching may irreversibly deplete the limited supply of labeled molecules within the cell, thus, restricting the ability of FCS to measure within small intracellular compartments or small types of cells. The first commercial FCS instrument was released in 1996 by Carl Zeiss (ConfoCor), permitting the first turnkey FCS applications and triggering the evolution of FCS into a standard technique (Weisshart et al. 2004). Subsequent models added laser-scanning modules and were able to perform dual-color cross correlation. Many cell biologists use intracellular FCS applications to investigate in situ dynamics of fluorescent probes. Moreover, the complexity of many biological systems of interest, including developing tissue and organisms, is fostering innovation and improvements in FCS and related optical methods. Various extensions and variations of FCS have been introduced. Because of its inherent depth discrimination, two-photon excitation (TPE) was proposed to reduce the problem of out-of-focus bleaching (Denk et al. 1990). The first two-photon–FCS experiments performed in cells labeled with fluorescent beads were reported by Gratton and colleagues (Berland et al. 1995). Four years later, FCS was sensitive enough to detect single molecules (Schwille et al. 1999). Two-photon-induced transitions to the excited state show selection rules that are different from those of their corresponding onephoton equivalent. This makes it possible to accomplish simultaneous excitation of spectrally distinct dyes (Heinze et al. 2000, 2002, 2004). 710
Cite this introduction as Cold Spring Harb Protoc; doi:10.1101/pdb.top081802
Downloaded from http://cshprotocols.cshlp.org/ at UNIVERSITE LAVAL on July 1, 2014 - Published by Cold Spring Harbor Laboratory Press
Fluorescence Correlation Spectroscopy
Optical configurations other than the standard confocal setup are being explored with the goal of reducing the size of the detection volume. These include total internal reflection (Starr and Thompson 2001); zero-mode waveguides, which consist of subwavelength apertures within a metal film (Levene et al. 2003); and the stimulated emission/depletion approach for obtaining a detection volume below the conventional diffraction limit (Kastrup et al. 2005; Eggeling et al. 2009). Additional information can be obtained from FCS experiments through the use of pulsed excitation with time-resolved detection, which can distinguish fluorophores based on their lifetimes (Felekyan et al. 2005; Kapusta et al. 2007). The use of alternating (pulsed interleaved) excitation with a matching detection scheme in dcFCCS eliminates spectral cross talk artifacts, which can cause a false-positive or increased cross-correlation amplitude, and can complicate the interpretation of dcFCCS data (Muller et al. 2005; Thews et al. 2005). Conventional FCS rapidly provides dynamical information about molecules at one spot within a sample, but it is very slow at providing spatial information because FCS measurements at different locations have to be acquired sequentially. In contrast, conventional laser-scanning microscopy provides spatial information about the time-averaged fluorescence intensity, but is not as well suited for picking up temporal changes. One variant of FCS that can provide spatial information is parallel multispot FCS, which originally used two parallel detection volumes (Brinkmeier et al. 1999) and has more recently been extended by using charge-coupled device detectors (Burkhardt and Schwille 2006; Kannan et al. 2006). Another modification to FCS that makes it possible to obtain both spatial and temporal information is to scan the beam (or the sample) in a linear, a circular, or a random fashion (scanning fluorescence correlation spectroscopy [sFCS]). sFCS is valuable for measuring very slow diffusion phenomena, which are often encountered with membrane probes (Ruan et al. 2004; Ries and Schwille 2006). Raster image correlation spectroscopy was recently developed as a type of sFCS that can be performed using conventional laser-scanning microscopes (Digman et al. 2005). SELECTING A FLUORESCENT LABEL
The outstanding sensitivity and selectivity of FCS is achieved by using strong fluorescent tags for detection because most naturally occurring fluorophores in biological systems are inherently dim. The most crucial decision is, therefore, the selection of the proper fluorescent tag. A good fluorophore undergoes, on average, about 106 excitation cycles before being irreversibly destroyed by photobleaching. To achieve good results, it is important to choose a photostable fluorophore within the proper wavelength range. Moreover, biologically relevant phenomena must be distinguished from potential dye-induced artifacts through control experiments. During the past decade, along with the growing impact of fluorescence techniques on the life sciences, techniques and tools for protein labeling have greatly improved. Autofluorescent proteins, for example, can be incorporated into proteins by recombinant expression, thus allowing for good labeling efficiency and inherent biocompatibility. Novel photostable synthetic chromophores are available from various manufacturers in a wide range of colors from ultraviolet (UV) to near-infrared and with different functional groups. In addition, new developments in mesoscopic physics have made it possible to exploit completely novel fluorescent systems: Semiconductor nanocrystals, also known as quantum dots. The choice of fluorescent dyes may be limited by the available laser lines and the corresponding filter systems. For one-color applications, a wide range of emission wavelengths may be used, although when venturing toward the UV or infrared, spectral region limitations caused by detector sensitivity and the transmission curves of relevant optical components have to be considered. Multicolor applications require that chromophores be spectrally distinguishable (i.e., have minimal spectral overlap). This requirement depends on the form of the emission spectra and the chosen filters, but generally, the larger the separation between the emission peaks, the better. A typical synthetic dye combination is Alexa 488 and Cy5 (or Alexa 647). The green fluorophore is very hydrophilic and, thus, ideal for Cite this introduction as Cold Spring Harb Protoc; doi:10.1101/pdb.top081802
711
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K. Bacia et al.
solution measurements, whereas Cy5 is rather hydrophobic and also displays a quite complicated photophysical behavior. Synthetic Fluorophores
Synthetic fluorophores offer the flexibility to choose a label having suitable spectral and photophysical characteristics. The label does, however, have to be attached to the biomolecule of interest. Proteins are most often labeled on lysine or cysteine residues of purified proteins. Labeling lysine residues typically yields a mixed population of protein molecules carrying varying numbers of fluorophores. Although the resulting heterogeneity in the fluorescence brightness does not affect measurements of the diffusion coefficient, it does affect the correlation amplitudes and is an issue in the quantitative interpretation of dcFCCS measurements (Kim et al. 2005). Therefore, labeling a single engineered cysteine side chain is often the method of choice for obtaining a homogeneously labeled preparation. Alternatively, approaches such as expressed protein ligation (Becker et al. 2006) can be used to obtain stoichiometric coupling of the desired fluorophore to the protein of interest. The small synthetic fluorophores (
Fluorescence Correlation Spectroscopy: Principles and Applications Kirsten Bacia, Elke Haustein and Petra Schwille Cold Spring Harb Protoc; doi: 10.1101/pdb.top081802 Email Alerting Service Subject Categories
Receive free email alerts when new articles cite this article - click here. Browse articles on similar topics from Cold Spring Harbor Protocols. Fluorescence (410 articles) Fluorescence, general (279 articles) Imaging/Microscopy, general (539 articles)
To subscribe to Cold Spring Harbor Protocols go to:
http://cshprotocols.cshlp.org/subscriptions
© 2014 Cold Spring Harbor Laboratory Press
Downloaded from http://cshprotocols.cshlp.org/ at UNIVERSITE LAVAL on July 1, 2014 - Published by Cold Spring Harbor Laboratory Press
Topic Introduction
Fluorescence Correlation Spectroscopy: Principles and Applications Kirsten Bacia, Elke Haustein, and Petra Schwille
Fluorescence correlation spectroscopy (FCS) is used to study the movements and the interactions of biomolecules at extremely dilute concentrations, yielding results with good spatial and temporal resolutions. Using a number of technical developments, FCS has become a versatile technique that can be used to study a variety of sample types and can be advantageously combined with other methods. Unlike other fluorescence-based techniques, the analysis of FCS data is not based on the average intensity of the fluorescence emission but examines the minute intensity fluctuations caused by spontaneous deviations from the mean at thermal equilibrium. These fluctuations can result from variations in local concentrations owing to molecular mobility or from characteristic intermolecular or intramolecular reactions of fluorescently labeled biomolecules present at low concentrations. Here, we provide a basic introduction to FCS, including its technical development and theoretical basis, experimental setup of an FCS system, adjustment of a setup, data acquisition, and analysis of FCS measurements. Finally, the application of FCS to the study of lipid bilayer membranes and to living cells is discussed.
INTRODUCTION
A steadily increasing demand for better minimally invasive analytical tools to answer specific biological questions has boosted the development of fluorescence-based techniques. Fluorescence correlation spectroscopy (FCS) is a well-established method for analyzing solutions of biomolecules at low concentrations, ranging from nanomolar to low micromolar. One of the advantages of FCS is that it requires only small sample volumes. In a basic type of experiment, the optically delimited, femtoliter-sized FCS detection volume is placed inside a drop of a few microliters of a homogeneous sample solution. In a more sophisticated experiment, the FCS detection volume can be positioned inside or on the membrane of a single eukaryotic cell, on a small biosynthetic container (such as a giant liposome), or inside a microfluidic structure. FCS is a rather flexible method with regard to the sample preparation and the optical setup and can be readily combined with other microscopic techniques such as confocal microscopy or atomic force microscopy. FCS is often referred to as a single-molecule technique because the fluctuations in fluorescence intensity arise from fluctuations in the number or in the brightness of single fluorescent particles. However, each particle is not followed and analyzed individually, which is why FCS may be more accurately described as a single-molecule-sensitive technique. All fluctuating physical parameters influencing the fluorescence signal are, in principle, accessible by FCS. These comprise local concentrations, mobility coefficients (translational diffusion, active transport, rotational diffusion), and rate constants of intramolecular and photophysical reactions (triplet blinking and photon antibunching). Moreover, processes that indirectly influence these parameters can
Adapted from Imaging: A Laboratory Manual (ed. Yuste). CSHL Press, Cold Spring Harbor, NY, USA, 2011. © 2014 Cold Spring Harbor Laboratory Press Cite this introduction as Cold Spring Harb Protoc; doi:10.1101/pdb.top081802
709
Downloaded from http://cshprotocols.cshlp.org/ at UNIVERSITE LAVAL on July 1, 2014 - Published by Cold Spring Harbor Laboratory Press
K. Bacia et al.
be analyzed, such as a molecular binding that increases the size and reduces the diffusion coefficient of a molecule. In addition, dual-color fluorescence cross-correlation spectroscopy (dcFCCS) can be implemented to analyze the correlated motion of two distinct fluorescent labels. In this way, cleavage and binding reactions as well as dynamic colocalization during intracellular transport can be studied. TECHNIQUE DEVELOPMENT
The theoretical foundation of FCS is based on the laws of molecular diffusion, formulated from Brown’s observations of random particle motion, and their analysis by Einstein and Smoluchowski at the beginning of the 20th century (Smoluchowski 1906). FCS as a technique was developed in the early 1970s (Magde et al. 1972, 1974; Elson and Magde 1974) as a “miniaturization” of dynamic light scattering. The novelty of FCS lay in the analysis of fluctuations in the fluorescence emission of the sample molecules, induced by spontaneous fluctuations of physical parameters. FCS was first used to measure the diffusion and chemical kinetics of ethidium bromide intercalation into DNA (Magde et al. 1972) and rotational diffusion (Ehrenberg and Rigler 1974). Following these proof-of-principle measurements, a variety of studies were devoted to the investigation of particle concentration and mobility: For example, three-dimensional, and two-dimensional (2D) diffusion (Aragón and Pecora 1976; Fahey et al. 1977) or laminar flow (Magde et al. 1978). In an attempt to restrict the system under investigation to small molecular numbers and, thus, to enhance both detection sensitivity and background suppression, Rigler and coworkers combined FCS with confocal detection (Rigler and Widengren 1990; Rigler et al. 1993). The analytical potential of FCS for the life sciences has been shown in numerous applications since the original work of Rigler and coworkers (Eigen and Rigler 1994; Schwille et al. 1997b). FCS was successfully used to study association and dissociation of nucleic acids (Kinjo and Rigler 1995) and proteins (Rauer et al. 1996). Moreover, a multitude of environmental effects inducing fluctuations in the fluorescence yield of single dye molecules could be studied, including reversible protonation (Haupts et al. 1998), electron transfer, and oxygen and ion concentrations. By introducing a dual-color cross-correlation scheme for the simultaneous observation of different fluorescent species (Schwille et al. 1997a), the detection specificity in bimolecular association and dissociation processes was significantly enhanced. Combined with extremely short data acquisition times, these features even allowed for real-time investigation of enzyme kinetics (Kettling et al. 1998). Cellular measurements were also reported at a very early stage (Elson et al. 1976). However, in turbid media, especially (although not exclusively) inside the cell, the signal-to-noise ratio is influenced by autofluorescence and scattering. In addition to this, photobleaching may irreversibly deplete the limited supply of labeled molecules within the cell, thus, restricting the ability of FCS to measure within small intracellular compartments or small types of cells. The first commercial FCS instrument was released in 1996 by Carl Zeiss (ConfoCor), permitting the first turnkey FCS applications and triggering the evolution of FCS into a standard technique (Weisshart et al. 2004). Subsequent models added laser-scanning modules and were able to perform dual-color cross correlation. Many cell biologists use intracellular FCS applications to investigate in situ dynamics of fluorescent probes. Moreover, the complexity of many biological systems of interest, including developing tissue and organisms, is fostering innovation and improvements in FCS and related optical methods. Various extensions and variations of FCS have been introduced. Because of its inherent depth discrimination, two-photon excitation (TPE) was proposed to reduce the problem of out-of-focus bleaching (Denk et al. 1990). The first two-photon–FCS experiments performed in cells labeled with fluorescent beads were reported by Gratton and colleagues (Berland et al. 1995). Four years later, FCS was sensitive enough to detect single molecules (Schwille et al. 1999). Two-photon-induced transitions to the excited state show selection rules that are different from those of their corresponding onephoton equivalent. This makes it possible to accomplish simultaneous excitation of spectrally distinct dyes (Heinze et al. 2000, 2002, 2004). 710
Cite this introduction as Cold Spring Harb Protoc; doi:10.1101/pdb.top081802
Downloaded from http://cshprotocols.cshlp.org/ at UNIVERSITE LAVAL on July 1, 2014 - Published by Cold Spring Harbor Laboratory Press
Fluorescence Correlation Spectroscopy
Optical configurations other than the standard confocal setup are being explored with the goal of reducing the size of the detection volume. These include total internal reflection (Starr and Thompson 2001); zero-mode waveguides, which consist of subwavelength apertures within a metal film (Levene et al. 2003); and the stimulated emission/depletion approach for obtaining a detection volume below the conventional diffraction limit (Kastrup et al. 2005; Eggeling et al. 2009). Additional information can be obtained from FCS experiments through the use of pulsed excitation with time-resolved detection, which can distinguish fluorophores based on their lifetimes (Felekyan et al. 2005; Kapusta et al. 2007). The use of alternating (pulsed interleaved) excitation with a matching detection scheme in dcFCCS eliminates spectral cross talk artifacts, which can cause a false-positive or increased cross-correlation amplitude, and can complicate the interpretation of dcFCCS data (Muller et al. 2005; Thews et al. 2005). Conventional FCS rapidly provides dynamical information about molecules at one spot within a sample, but it is very slow at providing spatial information because FCS measurements at different locations have to be acquired sequentially. In contrast, conventional laser-scanning microscopy provides spatial information about the time-averaged fluorescence intensity, but is not as well suited for picking up temporal changes. One variant of FCS that can provide spatial information is parallel multispot FCS, which originally used two parallel detection volumes (Brinkmeier et al. 1999) and has more recently been extended by using charge-coupled device detectors (Burkhardt and Schwille 2006; Kannan et al. 2006). Another modification to FCS that makes it possible to obtain both spatial and temporal information is to scan the beam (or the sample) in a linear, a circular, or a random fashion (scanning fluorescence correlation spectroscopy [sFCS]). sFCS is valuable for measuring very slow diffusion phenomena, which are often encountered with membrane probes (Ruan et al. 2004; Ries and Schwille 2006). Raster image correlation spectroscopy was recently developed as a type of sFCS that can be performed using conventional laser-scanning microscopes (Digman et al. 2005). SELECTING A FLUORESCENT LABEL
The outstanding sensitivity and selectivity of FCS is achieved by using strong fluorescent tags for detection because most naturally occurring fluorophores in biological systems are inherently dim. The most crucial decision is, therefore, the selection of the proper fluorescent tag. A good fluorophore undergoes, on average, about 106 excitation cycles before being irreversibly destroyed by photobleaching. To achieve good results, it is important to choose a photostable fluorophore within the proper wavelength range. Moreover, biologically relevant phenomena must be distinguished from potential dye-induced artifacts through control experiments. During the past decade, along with the growing impact of fluorescence techniques on the life sciences, techniques and tools for protein labeling have greatly improved. Autofluorescent proteins, for example, can be incorporated into proteins by recombinant expression, thus allowing for good labeling efficiency and inherent biocompatibility. Novel photostable synthetic chromophores are available from various manufacturers in a wide range of colors from ultraviolet (UV) to near-infrared and with different functional groups. In addition, new developments in mesoscopic physics have made it possible to exploit completely novel fluorescent systems: Semiconductor nanocrystals, also known as quantum dots. The choice of fluorescent dyes may be limited by the available laser lines and the corresponding filter systems. For one-color applications, a wide range of emission wavelengths may be used, although when venturing toward the UV or infrared, spectral region limitations caused by detector sensitivity and the transmission curves of relevant optical components have to be considered. Multicolor applications require that chromophores be spectrally distinguishable (i.e., have minimal spectral overlap). This requirement depends on the form of the emission spectra and the chosen filters, but generally, the larger the separation between the emission peaks, the better. A typical synthetic dye combination is Alexa 488 and Cy5 (or Alexa 647). The green fluorophore is very hydrophilic and, thus, ideal for Cite this introduction as Cold Spring Harb Protoc; doi:10.1101/pdb.top081802
711
Downloaded from http://cshprotocols.cshlp.org/ at UNIVERSITE LAVAL on July 1, 2014 - Published by Cold Spring Harbor Laboratory Press
K. Bacia et al.
solution measurements, whereas Cy5 is rather hydrophobic and also displays a quite complicated photophysical behavior. Synthetic Fluorophores
Synthetic fluorophores offer the flexibility to choose a label having suitable spectral and photophysical characteristics. The label does, however, have to be attached to the biomolecule of interest. Proteins are most often labeled on lysine or cysteine residues of purified proteins. Labeling lysine residues typically yields a mixed population of protein molecules carrying varying numbers of fluorophores. Although the resulting heterogeneity in the fluorescence brightness does not affect measurements of the diffusion coefficient, it does affect the correlation amplitudes and is an issue in the quantitative interpretation of dcFCCS measurements (Kim et al. 2005). Therefore, labeling a single engineered cysteine side chain is often the method of choice for obtaining a homogeneously labeled preparation. Alternatively, approaches such as expressed protein ligation (Becker et al. 2006) can be used to obtain stoichiometric coupling of the desired fluorophore to the protein of interest. The small synthetic fluorophores (
