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Fluorescence Fluctuation Spectroscopy at single Molecule resolution
Fluorescence Fluctuation Spectroscopy at single Molecule resolution
Confocal fluorescent analysis technologies play an important role in detection and quantitation of interacting biomolecules, leading to wide application in basic and applied science.
Confocal fluorescent analysis technologies play an important role in detection and quantitation of interacting biomolecules, leading to wide application in basic and applied science.
CTB uses confocal fluorescence analysis in three different formats as shown in the three little animations at the right hand side. Top FFS (see below for description), middle, FFC with slow moving objects, like cells, and lower, confocal scanning, via moving the microscopic stage (see bead screening section of this website.)
CTB uses confocal fluorescence analysis in three different formats as shown in the three little animations at the right hand side. Top FFS (see below for description), middle, FFC with slow moving objects, like cells, and lower, confocal scanning, via moving the microscopic stage (see bead screening section of this website.)
In general, we distinguish between single molecule spectroscopy and imaging, and fluorescence fluctuation spectroscopy (FFS). The latter is an analysis technique which works at single molecule resolution but uses mathematical data fitting to derive physical parameters about individual ensembles of molecules in biochemical solutions or in cells. Based on the optical settings of confocal microscopes, FFS comprises a series of micro-spectroscopic techniques capable of retrieving information of small spontaneous variations of intensities of fluorescent molecules at high spatial and temporal resolution, essentially without perturbation of the system, i.e at equilibrium. Fluctuation spectroscopy has been established as ideal method to study the behaviour of biomolecules in-solution and in living cell, and most importantly, at physiological concentrations. At CTB we run 3 confocal microscopes set-up for FFC (PS02, PS04 and PS05, see instruments section). The two main techniques used are fluorescence correlation spectroscopy (FCS) and fluorescence intensity distribution analysis (FIDA). FIDA is often also referred to as photon counting histogram (PCH). These fluctuation dependent and intensity dependent techniques can be applied on one data set and are used to reveal physical parameters like local particle concentration, molecular brightness, diffusion behaviour and internal dynamics of fluorescent molecules. FCS and 1D-FIDA analyse fluorescence fluctuation data collected via one fluorescence excitation and one emission channel, (e.g. for GFP, excitation at 488 nm, and emission through a dichroic mirror e.g. from 520 – 540 nm). With 1 or 2 excitation lasers (optional at 488 nm, 543 nm, 633 nm) and two emission wavelengths, and possible integration of polarizers into both detection paths, we extend the method repertoire to very useful and successful techniques like fluorescence cross correlation spectroscopy (FCCS), 2-colour 2 D-FIDA (2C-2D-FIDA), 2D-FIDA anisotropy (2D-FIDA-r). FCS and FIDA is combined in fluorescence intensity multiple distributions analysis (FIMDA) (Portal et al below). Using pulsed lasers and time correlated detection, fluorescence lifetime measurements become possible and their integration with FIDA, allowing for higher accuracy fluctuation analysis with fluorescence intensity and lifetime distribution analysis (FILDA).
In general, we distinguish between single molecule spectroscopy and imaging, and fluorescence fluctuation spectroscopy (FFS). The latter is an analysis technique which works at single molecule resolution but uses mathematical data fitting to derive physical parameters about individual ensembles of molecules in biochemical solutions or in cells. Based on the optical settings of confocal microscopes, FFS comprises a series of micro-spectroscopic techniques capable of retrieving information of small spontaneous variations of intensities of fluorescent molecules at high spatial and temporal resolution, essentially without perturbation of the system, i.e at equilibrium. Fluctuation spectroscopy has been established as ideal method to study the behaviour of biomolecules in-solution and in living cell, and most importantly, at physiological concentrations. At CTB we run 3 confocal microscopes set-up for FFC (PS02, PS04 and PS05, see instruments section). The two main techniques used are fluorescence correlation spectroscopy (FCS) and fluorescence intensity distribution analysis (FIDA). FIDA is often also referred to as photon counting histogram (PCH). These fluctuation dependent and intensity dependent techniques can be applied on one data set and are used to reveal physical parameters like local particle concentration, molecular brightness, diffusion behaviour and internal dynamics of fluorescent molecules. FCS and 1D-FIDA analyse fluorescence fluctuation data collected via one fluorescence excitation and one emission channel, (e.g. for GFP, excitation at 488 nm, and emission through a dichroic mirror e.g. from 520 – 540 nm). With 1 or 2 excitation lasers (optional at 488 nm, 543 nm, 633 nm) and two emission wavelengths, and possible integration of polarizers into both detection paths, we extend the method repertoire to very useful and successful techniques like fluorescence cross correlation spectroscopy (FCCS), 2-colour 2 D-FIDA (2C-2D-FIDA), 2D-FIDA anisotropy (2D-FIDA-r). FCS and FIDA is combined in fluorescence intensity multiple distributions analysis (FIMDA) (Portal et al below). Using pulsed lasers and time correlated detection, fluorescence lifetime measurements become possible and their integration with FIDA, allowing for higher accuracy fluctuation analysis with fluorescence intensity and lifetime distribution analysis (FILDA).
FFC methods have not only be used in basic science, they have also been translated into drug screening with particularly, the single molecule version of fluorescence anisotropy assays, 2D-FIDA-r, delivering superior high throughput screens in submicroliter volumes per screening well and 1535 well plate formats (see Meisner et al. below).
FFC methods have not only be used in basic science, they have also been translated into drug screening with particularly, the single molecule version of fluorescence anisotropy assays, 2D-FIDA-r, delivering superior high throughput screens in submicroliter volumes per screening well and 1535 well plate formats (see Meisner et al. below).
Portal CF, Seifert JM, Buehler C, Meisner NC, Auer M (2014). A Novel 1:1 Labeling and Purification Process for C-terminal Thioester and Single Cysteine Recombinant Proteins Using Generic Peptidic Toolbox Reagents. Bioconjugate Chemistry 25/7, 1213-1222, DOI: 10.1021/bc5000059, PMID: 24866260.
Portal CF, Seifert JM, Buehler C, Meisner NC, Auer M (2014). A Novel 1:1 Labeling and Purification Process for C-terminal Thioester and Single Cysteine Recombinant Proteins Using Generic Peptidic Toolbox Reagents. Bioconjugate Chemistry 25/7, 1213-1222, DOI: 10.1021/bc5000059, PMID: 24866260.
Meisner NC, Hintersteiner M, Müller K, Bauer R, Seifert JM, Naegeli HU, Ottl J, Oberer L, Guenat C, Moss S, Harrer N, Woisetschläger M, Bühler C, Uhl V, Auer M (2007) Identification and mechanistic characterization of low molecular weight inhibitors for HuR. Full Article. Nature Chemical Biology; 3 (8):508-15. doi:10.1038/nchembio.2007.14, PMID: 17632515.
Meisner NC, Hintersteiner M, Müller K, Bauer R, Seifert JM, Naegeli HU, Ottl J, Oberer L, Guenat C, Moss S, Harrer N, Woisetschläger M, Bühler C, Uhl V, Auer M (2007) Identification and mechanistic characterization of low molecular weight inhibitors for HuR. Full Article. Nature Chemical Biology; 3 (8):508-15. doi:10.1038/nchembio.2007.14, PMID: 17632515.
Single molecule spectroscopy - PS03
Single molecule spectroscopy - PS03
Ensemble measurements which establish the average state of a system are extremely successful in defining biological interactions. Under some circumstances, however, subtle changes in the states of sub-populations are lost when measuring bulk samples, especially where the distribution of states is complex or time-dependent. Single-molecule techniques overcome these limitations by enabling the measurement of the distribution of states within a system, monitoring binding, and the existence of monomers, dimers, and other larger multimers as a function of time. It is achieved by creating a diffraction limited confocal volume of a microscope objective of the order of femtoliters, and then diluting samples to the stage where the probability of more than one molecule being in that volume are insignificant. All the interactions take place in solution, eliminating artefacts caused by attachment to a solid support. The form of single-molecule spectroscopy that is used on this instrument is known as “time-correlated single-photon counting” (TCSPC). In this technique, a pulsed laser is used to excite the samples. Single-photon sensitive APDs detect the emitted photons, and both the “macrotime”, from the start of the experiment, and the “microtime”, between the photon arrival and the pulse that caused the excitation, are recorded for every photon detected. As a single fluorescent molecule traverses the confocal volume, taking from a few milliseconds to hundreds of milliseconds dependent on its size, a large increase in the intensity is recorded, called a burst. Bursts typically contain between several tens and hundreds of individual photons. The measurement yields microtimes for each photon observed from each dye and hence, after compensating for the optical beam path, the distribution of times the fluorophore spent in the excited state. This parameter is known as the lifetime, and is extremely environmentally sensitive. The intensity of the burst can be used to establish the number of fluorophores on each molecule. Furthermore, using a polarizing beamsplitter and two APDs, the anisotropy of each molecule can be established, revealing the rotational correlation time which is strongly size dependent. By increasing the number of detection channels further, multiple colours can also be distinguished, and energy transfer between closely-spaced dyes observed, a parameter which is inversely proportional to the sixth power of distance between them. Combining the analyses can reveal sub-populations lost when the data is analysed individually. The characteristics of members of these sub-populations alone can be subsequently analysed, providing very high quality data about the subset, an approach known as MPFD. From the data, statistical analyses such as FCS, FCCS, and FIDA can also be performed. In the Auer lab, on the custom built single-molecule microscope, known as PS03 has been constructed, with this we are able to monitor bursts in two polarizations and two colours simultaneously.
Ensemble measurements which establish the average state of a system are extremely successful in defining biological interactions. Under some circumstances, however, subtle changes in the states of sub-populations are lost when measuring bulk samples, especially where the distribution of states is complex or time-dependent. Single-molecule techniques overcome these limitations by enabling the measurement of the distribution of states within a system, monitoring binding, and the existence of monomers, dimers, and other larger multimers as a function of time. It is achieved by creating a diffraction limited confocal volume of a microscope objective of the order of femtoliters, and then diluting samples to the stage where the probability of more than one molecule being in that volume are insignificant. All the interactions take place in solution, eliminating artefacts caused by attachment to a solid support. The form of single-molecule spectroscopy that is used on this instrument is known as “time-correlated single-photon counting” (TCSPC). In this technique, a pulsed laser is used to excite the samples. Single-photon sensitive APDs detect the emitted photons, and both the “macrotime”, from the start of the experiment, and the “microtime”, between the photon arrival and the pulse that caused the excitation, are recorded for every photon detected. As a single fluorescent molecule traverses the confocal volume, taking from a few milliseconds to hundreds of milliseconds dependent on its size, a large increase in the intensity is recorded, called a burst. Bursts typically contain between several tens and hundreds of individual photons. The measurement yields microtimes for each photon observed from each dye and hence, after compensating for the optical beam path, the distribution of times the fluorophore spent in the excited state. This parameter is known as the lifetime, and is extremely environmentally sensitive. The intensity of the burst can be used to establish the number of fluorophores on each molecule. Furthermore, using a polarizing beamsplitter and two APDs, the anisotropy of each molecule can be established, revealing the rotational correlation time which is strongly size dependent. By increasing the number of detection channels further, multiple colours can also be distinguished, and energy transfer between closely-spaced dyes observed, a parameter which is inversely proportional to the sixth power of distance between them. Combining the analyses can reveal sub-populations lost when the data is analysed individually. The characteristics of members of these sub-populations alone can be subsequently analysed, providing very high quality data about the subset, an approach known as MPFD. From the data, statistical analyses such as FCS, FCCS, and FIDA can also be performed. In the Auer lab, on the custom built single-molecule microscope, known as PS03 has been constructed, with this we are able to monitor bursts in two polarizations and two colours simultaneously.
High content screening by Fluorescence Imaging
High content screening by Fluorescence Imaging
The Auer lab has extensive high content screening capabilities through utilisation of our 4 color Opera® High Content Screening system (Perkin Elmer). This instrument is used to indicate functionality of active compounds from diverse chemical libraries in a number of cell-based screening methods. Currently applied high content screens include a novel fluorescence-based cellular assay relating inhibition of Staphylococcus aureus bacteria inside macrophage host cells. Up to 4 imaging parameters are detected in 384 well plate format confocal imaging. Macrophage plasma membrane, macrophage nuclei, stably GFP labelled bacteria and a life/dead stain.
The Auer lab has extensive high content screening capabilities through utilisation of our 4 color Opera® High Content Screening system (Perkin Elmer). This instrument is used to indicate functionality of active compounds from diverse chemical libraries in a number of cell-based screening methods. Currently applied high content screens include a novel fluorescence-based cellular assay relating inhibition of Staphylococcus aureus bacteria inside macrophage host cells. Up to 4 imaging parameters are detected in 384 well plate format confocal imaging. Macrophage plasma membrane, macrophage nuclei, stably GFP labelled bacteria and a life/dead stain.
This work is supported by a Wellcome Trust Collaborative Awards in Science:
This work is supported by a Wellcome Trust Collaborative Awards in Science:
Prof Ross Fitzgerald, Prof David Hume, Prof Jose Penades, Prof Manfred Auer
Prof Ross Fitzgerald, Prof David Hume, Prof Jose Penades, Prof Manfred Auer
University of Edinburgh, and University of Glasgow (JP), United Kingdom
University of Edinburgh, and University of Glasgow (JP), United Kingdom
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