1. |
- Bergstrand, Jan, et al.
(author)
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Scanning inverse fluorescence correlation spectroscopy
- 2014
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In: Optics Express. - : Optical Society of America. - 1094-4087. ; 22:11, s. 13073-13090
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Journal article (peer-reviewed)abstract
- Scanning Inverse Fluorescence Correlation Spectroscopy (siFCS) is introduced to determine the absolute size of nanodomains on surfaces. We describe here equations for obtaining the domain size from cross-and auto-correlation functions, measurement simulations which enabled testing of these equations, and measurements on model surfaces mimicking membranes containing nanodomains. Using a confocal microscope of 270 nm resolution the size of 250 nm domains were estimated by siFCS to 257 +/- 12 nm diameter, and 40 nm domains were estimated to 65 +/- 26 nm diameter. Applications of siFCS for sizing of nanodomains and protein clusters in cell membranes are discussed.
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2. |
- Chmyrov, Andriy, et al.
(author)
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Iodide as a Fluorescence Quencher and Promoter-Mechanisms and Possible Implications
- 2010
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In: Journal of Physical Chemistry B. - : American Chemical Society (ACS). - 1520-6106 .- 1520-5207. ; 114:34, s. 11282-11291
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Journal article (peer-reviewed)abstract
- In this work, fluorescence correlation spectroscopy (FCS) was used to investigate the effects of potassium iodide (KI) on the electronic-state population kinetics of a range of organic dyes in the visible wavelength range. Apart from a heavy atom effect promoting intersystem crossing to the triplet states in all dyes, KI was also found to enhance the triplet-state decay rate by a charge-coupled deactivation. This deactivation was only found for dyes with excitation maximum in the blue range, not for those with excitation maxima at wavelengths in the green range or longer. Consequently, under excitation conditions sufficient for triplet state formation, KI can promote the triplet state buildup of one dye and reduce it for another, red-shifted dye. This anticorrelated, spectrally separable response of two different dyes to the presence of one and the same agent may provide a useful readout for biomolecular interaction and microenvironmental monitoring studies. In contrast to the typical notion of KI as a fluorescence quencher, the FCS measurements also revealed that when added in micromolar concentrations KI can act as an antioxidant, promoting the recovery of photo-oxidized fluorophores. However, in millimolar concentrations KI also reduces intact, fluorescently viable fluorophores to a considerable extent. In aqueous solutions, for the dye Rhodamine Green, an optimal concentration of KI of approximately 5 mM can be defined at which the fluorescence signal is maximized. This concentration is not high enough to allow full triplet state quenching. Therefore, as a fluorescence enhancement agent, it is primarily the antioxidative properties of KI that play a role.
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3. |
- Chmyrov, Andriy, 1979-, et al.
(author)
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Recovery of Photoinduced Reversible Dark States Utilized for Molecular Diffusion Measurements
- 2010
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In: Analytical Chemistry. - : American Chemical Society (ACS). - 0003-2700 .- 1520-6882. ; 82:24, s. 9998-10005
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Journal article (peer-reviewed)abstract
- For a spatially restricted excitation volume, the effective modulation of the excitation in time is influenced by the passage times of the molecules through the excitation volume. By applying an additional time-modulated excitation, the buildup of photoinduced reversible dark states in fluorescent molecules can be made to vary significantly with their passage times through the excitation volume. The variations in the dark state populations are reflected by the time-averaged fluorescence intensity, which thus can be used to characterize the mobilities of the molecules. The concept was experimentally verified by measuring the fluorescence response of freely diffusing cyanine fluorophores (Cy5), undergoingtrans-cis isomerization when subject to time-modulated excitation in a focused laser beam. From the fluorescence response, and by applying a simple photodynamic model, the transition times of the Cy5 molecules could be well reproduced when applying different laminar flow speeds through the detection volume. The presented approach puts no constraints on sample concentration, no requirements for high time resolution or sensitivity in the detection, nor requires a high fluorescence brightness of the characterized molecules. This can make the concept useful for a broad range of biomolecular mobility studies.
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4. |
- Chmyrov, Volodymyr, et al.
(author)
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Trans-Cis isomerization of lipophilic dyes probing membrane microviscosity in biological membranes and in live cells
- 2015
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In: Analytical Chemistry. - : American Chemical Society (ACS). - 0003-2700 .- 1520-6882. ; 87:11, s. 5690-5697
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Journal article (peer-reviewed)abstract
- Membrane environment and fluidity can modulate the dynamics and interactions of membrane proteins and can thereby strongly influence the function of cells and organisms in general. In this work, we demonstrate that trans-cis isomerization of lipophilic dyes is a useful parameter to monitor packaging and fluidity of biomembranes. Fluorescence fluctuations, generated by trans-cis isomerization of the thiocarbocyanine dye Merocyanine 540 (MC540), were first analyzed by fluorescence correlation spectroscopy (FCS) in different alcohol solutions. Similar isomerization kinetics of MC540 in lipid vesicles could then also be monitored, and the influence of lipid polarity, membrane curvature, and cholesterol content was investigated. While no influence of membrane curvature and lipid polarity could be observed, a clear decrease in the isomerization rates could be observed with increasing cholesterol contents in the vesicle membranes. Finally, procedures to spatially map photoinduced and thermal isomerization rates on live cells by transient state (TRAST) imaging were established. On the basis of these procedures, MC540 isomerization was studied on live MCF7 cells, and TRAST images of the cells at different temperatures were found to reliably detect differences in the isomerization parameters. Our studies indicate that trans-cis isomerization is a useful parameter for probing membrane dynamics and that the TRAST imaging technique can provide spatial maps of photoinduced isomerization as well as both photoinduced and thermal back-isomerization, resolving differences in local membrane microviscosity in live cells.
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5. |
- Hevekerl, Heike, 1982-, et al.
(author)
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Dark states in ionic oligothiophene bioprobes-evidence from fluorescence correlation spectroscopy and dynamic light scattering
- 2014
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In: Journal of Physical Chemistry B. - : American Chemical Society (ACS). - 1520-6106 .- 1520-5207. ; 118:22, s. 5924--5933
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Journal article (peer-reviewed)abstract
- Luminescent conjugated polyelectrolytes (LCPs) can upon interaction with biological macromolecules change their luminescent properties, and thereby serve as conformation- and interaction-sensitive biomolecular probes. However, to exploit this in a more quantitative manner, there is a need to better understand the photophysical processes involved. We report studies of the conjugated pentameric oligothiophene, derivative p-FTAA, which changes optical properties with different p-FTAA concentrations in aqueous buffers, and in a pH and oxygen saturation dependent manner. Using dynamic light scattering, luminescence spectroscopy and fluorescence correlation spectroscopy, we find evidence for a monomer dimer equilibrium, for the formation of large clusters of p-FTAA in aqueous environment, and can couple aggregation to changed emission properties of oligothiophenes. In addition, we observe the presence of at least two dark transient states, one presumably being a triplet state. Oxygen was found to statically quench the p-FTAA fluorescence but also to promote molecular fluorescence by quenching dark transient states of the p-FTAA molecules. Taken together, this study provides knowledge of fluorescence and photophysical features essential for applying p-FTAA and other oligothiophene derivatives for diagnostic purposes, including detection and staining of amyloid aggregates.
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6. |
- Hevekerl, Heike, et al.
(author)
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Forster Resonance Energy Transfer beyond 10 nm : Exploiting the Triplet State Kinetics of Organic Fluorophores
- 2011
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In: Journal of Physical Chemistry B. - : American Chemical Society (ACS). - 1520-6106 .- 1520-5207. ; 115:45, s. 13360-13370
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Journal article (peer-reviewed)abstract
- Inter- or intramolecular distances of biomolecules can be studied by Forster resonance energy transfer (FRET). For most FRET methods, the observable range of distances is limited to 1-10 nm, and the labeling efficiency has to be controlled carefully to obtain accurate distance determinations, especially for intensity-based methods. In this study, we exploit the triplet state of the acceptor fluorophore as a FRET readout using fluorescence correlation spectroscopy and transient state monitoring. The influence of donor fluorescence leaking into the acceptor channel is minimized by a novel suppression algorithm for spectral bleed-through, thereby tolerating a high excess (up to 100-fold) of donor-only labeled samples. The suppression algorithm and the high sensitivity of the triplet state to small changes in the fluorophore excitation rate make it possible to extend the observable range of FRET efficiencies by up to 50% in the presence of large donor-only populations. Given this increased range of FRET efficiencies, its compatibility with organic fluorophores, and the low requirements on the labeling efficiency and instrumentation, we foresee that this approach will be attractive for in vitro and in vivo FRET-based spectroscopy and imaging.
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7. |
- Rönnlund, Daniel, et al.
(author)
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Fluorescence Nanoscopy of Platelets Resolves Platelet-State Specific Storage, Release and Uptake of Proteins, Opening up Future Diagnostic Applications
- 2012
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In: Advanced Healthcare Materials. - : Wiley-Blackwell. - 2192-2640 .- 2192-2659. ; 1:6, s. 707-713
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Journal article (peer-reviewed)abstract
- Dysregulation of how platelets store, sequester and release specific proteins seems to be implicated in many disease states, including cancer. Dual-color immunofluorescence stimulated emission depletion (STED) microscopy with 40 nm resolution is used to map pro-angiogenic VEGF, anti-angiogenic PF-4 and fibrinogen in >300 individual platelets. This reveals that these proteins are stored in a segmented, zonal manner within regional clusters, significantly smaller than the size of an alpha-granule. No colocalization between the different proteins is observed. Upon platelet activation by thrombin or ADP, the proteins undergo significant spatial rearrangements, including alterations in the size and number of the protein clusters, and specific for a certain protein and the type of activation induced. Following these observations, a simple assignment procedure is used to show that the three distinct states of platelets (non-, ADP- and thrombin-activated) can be identified based on the average size, number and peripheral localization profiles of the regional protein clusters within the platelets. Thus, high-resolution spatial mapping of specific proteins is a useful procedure to detect and characterize deviations in the selective storage, release and uptake of these proteins in the platelets. Since these deviations seem to be specific for, and may even underlie, certain patophysiological states, these findings may have interesting diagnostic and therapeutic implications.
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8. |
- Rönnlund, Daniel, et al.
(author)
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Spatial organization of proteins in metastasizing cells
- 2013
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In: Cytometry Part A. - : John Wiley & Sons. - 1552-4922 .- 1552-4930. ; 83:9, s. 855-865
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Journal article (peer-reviewed)abstract
- The ability of tumor cells to invade into the surrounding tissue is linked to defective adhesive and mechanical properties of the cells, which are regulated by cell surface adhesions and the intracellular filamentous cytoskeleton, respectively. With the aim to further reveal the underlying mechanisms and provide new strategies for early cancer diagnostics, we have used ultrahigh resolution stimulated emission depletion (STED) microscopy as a means to identify metastasizing cells, based on their subcellular protein distribution patterns reflecting their specific adhesive and mechanical properties. We have compared the spatial distribution of cell-matrix adhesion sites and the vimentin filamentous systems in a matched pair of primary, normal, and metastatic human fibroblast cells. We found that the metastatic cells showed significantly increased densities and more homogenous distributions of nanoscale adhesion-related particles. Moreover, they showed an increase in the number but reduced sizes of the areas of cell-matrix adhesion complexes. The organization of the vimentin intermediate filaments was also found to be significantly different in the metastasizing cells, showing an increased entanglement and loss of directionality. Image analysis procedures were established, allowing an objective detection and characterization of these features and distinction of metastatic cells from their normal counterparts. In conclusion, our results suggest that STED microscopy provides a novel tool to identify metastasizing cells from a very sparse number of cells, based on the altered spatial distribution of the cell-matrix adhesions and intermediate filaments.
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9. |
- Sandén, Tor, et al.
(author)
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Surface-coupled proton exchange of a membrane-bound proton acceptor
- 2010
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In: Proceedings of the National Academy of Sciences of the United States of America. - Washington : National Academy of Sciences. - 0027-8424 .- 1091-6490. ; 107:9, s. 4129-4134
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Journal article (peer-reviewed)abstract
- Proton-transfer reactions across and at the surface of biological membranes are central for maintaining the transmembrane proton electrochemical gradients involved in cellular energy conversion. In this study, fluorescence correlation spectroscopy was used to measure the local protonation and deprotonation rates of single pH-sensitive fluorophores conjugated to liposome membranes, and the dependence of these rates on lipid composition and ion concentration. Measurements of proton exchange rates over a wide proton concentration range, using two different pH-sensitive fluorophores with different pKas, revealed two distinct proton exchange regimes. At high pH (> 8), proton association increases rapidly with increasing proton concentrations, presumably because the whole membrane acts as a proton-collecting antenna for the fluorophore. In contrast, at low pH (< 7), the increase in the proton association rate is slower and comparable to that of direct protonation of the fluorophore from the bulk solution. In the latter case, the proton exchange rates of the two fluorophores are indistinguishable, indicating that their protonation rates are determined by the local membrane environment. Measurements on membranes of different surface charge and at different ion concentrations made it possible to determine surface potentials, as well as the distance between the surface and the fluorophore. The results from this study define the conditions under which biological membranes can act as proton-collecting antennae and provide fundamental information on the relation between the membrane surface charge density and the local proton exchange kinetics.
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10. |
- Spielmann, Thiemo, 1984-, et al.
(author)
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Transient state imaging probes patterns of altered oxygen consumption in cancer cells
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Other publication (other academic/artistic)abstract
- Altered cellular metabolism plays an important role in many diseases, not least in many forms of cancer, where cellular metabolic pathways requiring lower oxygen consumption are often favored (the so-called Warburg effect). In this work, we have applied fluorescence-based transient state imaging and have exploited the environment sensitivity of long-lived dark states of fluorophores, in particular triplet state decay rates, to image the oxygen consumption of living cells. Our measurements can resolve differences in oxygen concentrations between different regions of individual cells, between different cell types, and also based on what metabolic pathways the cells use. In MCF-7 breast cancer cells, higher oxygen consumption can be detected when they rely on glutamine instead of glucose as their main metabolite, predominantly undergoing oxidative phosphorylation rather than glycolysis. By use of the high triplet yield dye Eosin Y the irradiance requirements during the measurements can be kept low. This reduces the instrumentation requirements, and harmful biological effects from high excitation doses can be avoided. Taken together, our imaging approach is widely applicable and capable of detecting subtle changes in oxygen consumption in live cells, stemming from the Warburg effect or reflecting other differences in the cellular metabolism. This may lead to new diagnostic means as well as advance our understanding of the interplay between cellular metabolism and major disease categories, such as cancer.
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11. |
- Spielmann, Thiemo, et al.
(author)
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Transient State Monitoring by Total Internal Reflection Fluorescence Microscopy
- 2010
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In: Journal of Physical Chemistry B. - : American Chemical Society (ACS). - 1520-6106 .- 1520-5207. ; 114:11, s. 4035-4046
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Journal article (peer-reviewed)abstract
- Triplet, photo-oxidized and other photoinduced, long-lived states Of fluorophores are sensitive to the local environment and thus attractive for microenvironmental imaging purposes. In this work, we introduce an approach where these states are monitored in a total internal reflection (TIR) fluorescence microscope, via the characteristic variations of the time-averaged fluorescence occuring ill response to different excitation modulation schemes. The surface-confined TIR excitation field generates a signal from the fluorescent molecules Close to the glass surface. Thereby, a high selectivity and low background noise is obtained, and in combination with IOW duty Cycles Of excitation, the overall photodegradation of the fluorescent molecules of the sample call be kept low, To verify the approach. the kinetics of the triplet and radical states of the dye Rhodamine 110 were imaged and analyzed in aqueous solutions at different concentrations of dissolved oxygen and of the reducing agent ascorbic acid. The experimental results Were compared to data from corresponding fluorescence correlation spectroscopy (FCS) measurements and simulations based oil finite element analysis. The approach was found to accurately determine relative populations and dynamics of triplet and photooxidized states, Overcoming passage time limitations seen ill FCS measurements. The method circumvents the need for time resolution ill the fluorescence detection, allowing simultaneous readout over the whole SLII-face area subject to excitation. It call be applied over a broad range of concentrations and does not require I strong fluorescence brightness of the sample molecules. Given the sensitivity of the triplet and photooxidized states to oxygen concentrations and not the least to local redox environments, we expect the approach to become an attractive tool for imaging cell metabolism.
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12. |
- Strömqvist, Johan, et al.
(author)
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A modified FCCS procedure applied to Ly49A-MHC class Icis-interaction studies in cell membranes
- 2011
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In: Biophysical Journal. - : Elsevier. - 0006-3495 .- 1542-0086. ; 101:5, s. 1257-1269
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Journal article (peer-reviewed)abstract
- The activity of natural killer (NK) cells is regulated by a fine-tuned balance between activating and inhibitory receptors. Dual-color fluorescence cross-correlation spectroscopy (FCCS) was used to directly demonstrate a so-called cis-interaction between a member of the inhibitory NK cell receptor family Ly49 (Ly49A), and its ligand, the major histocompatibility complex (MHC) class I, within the plasma membrane of the same cell. By a refined FCCS model, calibrated by positive and negative control experiments on cells from the same lymphoid cell line, concentrations and diffusion coefficients of free and interacting proteins could be determined on a collection of cells. Using the intrinsic intercellular variation of their expression levels for titration, it was found that the fraction of Ly49A receptors bound in cis increase with increasing amounts of MHC class I ligand. This increase shows a tendency to be more abrupt than for a diffusion limited three dimensional bimolecular reaction, which most likely reflects the two-dimensional confinement of the reaction. For the Ly49A- MHC class I interaction it indicates that within a critical concentration range the local concentration level of MHC class I can provide a distinct regulation mechanism of the NK cell activity.
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13. |
- Strömqvist, Johan, et al.
(author)
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Quenching of Triplet State Fluorophores for Studying Diffusion-Mediated Reactions in Lipid Membranes
- 2010
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In: Biophysical Journal. - : Elsevier BV. - 0006-3495 .- 1542-0086. ; 99:11, s. 3821-3830
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Journal article (peer-reviewed)abstract
- An approach to study bimolecular interactions in model lipid bilayers and biological membranes is introduced, exploiting the influence of membrane associated electron spin resonance labels on the triplet state kinetics of membrane bound fluorophores Singlet triplet state transitions within the dye Lissamine Rhodamine B (LRB) were studied when free in aqueous solutions, with LRB bound to a lipid in a liposome and in the presence of different local concentrations of the electron spin resonance label TEMPO By monitoring the triplet state kinetics via variations in the fluorescence signal, in this study using fluorescence correlation spectroscopy a strong fluorescence signal can be combined with the ability to monitor low frequency molecular interactions at timescales much longer than the fluorescence lifetimes Both in solution and in membranes the measured relative changes in the singlet triplet transitions rates were found to well reflect the expected collisional frequencies between the LRB and TEMPO molecules These collisional rates could also be monitored at local TEMPO concentrations where practically no quenching of the excited state of the fluorophores can be detected The proposed strategy is broadly applicable in terms of possible read out means types of molecular interactions that can be followed, and in what environments these interactions can be measured
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14. |
- Wennmalm, Stefan, 1970-, et al.
(author)
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Inverse-fluorescence correlation spectroscopy: more information and less labeling
- 2011
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In: Frontiers in Bioscience. - : Frontiers in Bioscience. - 1093-9946 .- 1093-4715 .- 1945-0516 .- 1945-0524. ; s3, s. 385-392
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Journal article (peer-reviewed)abstract
- Inverse-Fluorescence Correlation Spectroscopy(iFCS) is a recently developed modification of standardFCS that allows analysis of particles and biomoleculeswithout labeling. The particles generate no signal; insteadthe signal is generated by a surrounding medium. Particlesdiffusing through the FCS-detection volume displace afraction of the surrounding medium, causing transient dipsin the detected signal. These give information about themobility and concentration of the analyzed particles. Alsolabeled particles can be analyzed, whereby their signal iscross-correlated with that from the surrounding medium(iFCCS). This can give information about the volume of thelabeled particles, or alternatively about the size of thedetection volume. Also the interaction of unlabeledparticles with small, labeled ligands can be analyzed withiFCCS. This allows using cross-correlation as a sensitiveindication of binding, even though only one binding-partneris labeled. This review describes the principles of iFCS andiFCCS and measurements of microspheres dissolved in asurrounding medium containing alexa 488. We also discusspractical considerations, and future possibilities foranalyses of biomolecules.
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15. |
- Widengren, Jerker, 1965-
(author)
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Fluorescence-based transient state monitoring for biomolecular spectroscopy and imaging
- 2010
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In: Journal of the Royal Society Interface. - London : Royal Society Publishing. - 1742-5689 .- 1742-5662. ; 7:49, s. 1135-1144
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Research review (peer-reviewed)abstract
- To increase read-out speed, sensitivity or specificity, an often applied strategy in fluorescence-based biomolecular spectroscopy and imaging is to simultaneously record two or more of the fluorescence parameters: intensity, lifetime, polarization or wavelength. This review highlights how additional, to-date largely unexploited, information can be extracted by monitoring long-lived, photo-induced transient states of organic dyes and their dynamics. Two major approaches are presented, where the transient state information is obtained either from fluorescence fluctuation analysis or by recording the time-averaged fluorescence response to a time-modulated excitation. The two approaches combine the detection sensitivity of the fluorescence signal with the environmental sensitivity of the long-lived transient states. For both techniques, proof-of-principle experiments are reviewed, and advantages, limitations and possible applications for biomolecular cellular biology studies are discussed.
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16. |
- Widengren, Jerker, 1965-
(author)
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Studying ion exchange in solution and at biological membranes by FCS
- 2013
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In: Fluorescence Fluctuation Spectroscopy (FFS), Part B. - : Elsevier. - 9780124055391 ; , s. 231-252
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Book chapter (peer-reviewed)abstract
- By FCS, a wide range of processes can be studied, covering time ranges from subnanoseconds to seconds. In principle, any process at equilibrium conditions can be measured, which reflects itself by a change in the detected fluorescence intensity. In this review, it is described how FCS and variants thereof can be used to monitor ion exchange, in solution and along biological membranes. Analyzing fluorescence fluctuations of ion-sensitive fluorophores by FCS offers selective advantages over other techniques for measuring local ion concentrations, and, in particular, for studying exchange kinetics of ions on a very local scale. This opens for several areas of application. The FCS approach was used to investigate fundamental aspects of proton exchange at and along biological membranes. The protonation relaxation rate, as measured by FCS for a pH-sensitive dye, can also provide information about local accessibility/ interaction of a particular labeling site and conformational states of biomolecules, in a similar fashion as in a fluorescence quenching experiment. The same FCS concept can also be applied to ion exchange studies using other ion-sensitive fluorophores, and by use of dyes sensitive to other ambient conditions the concept can be extended also beyond ion exchange studies.
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