| 1. |
- Albertsson, Ingrid, et al.
(author)
-
Functional interactions between nitrite reductase and nitric oxide reductase from Paracoccus denitrificans
- 2019
-
In: Scientific Reports. - : Nature Publishing Group. - 2045-2322. ; 9
-
Journal article (peer-reviewed)abstract
- Denitrification is a microbial pathway that constitutes an important part of the nitrogen cycle on earth. Denitrifying organisms use nitrate as a terminal electron acceptor and reduce it stepwise to nitrogen gas, a process that produces the toxic nitric oxide (NO) molecule as an intermediate. In this work, we have investigated the possible functional interaction between the enzyme that produces NO; the cd(1) nitrite reductase (cd(1)NiR) and the enzyme that reduces NO; the c-type nitric oxide reductase (cNOR), from the model soil bacterium P. denitrificans. Such an interaction was observed previously between purified components from P. aeruginosa and could help channeling the NO (directly from the site of formation to the side of reduction), in order to protect the cell from this toxic intermediate. We find that electron donation to cNOR is inhibited in the presence of cd(1)NiR, presumably because cd(1)NiR binds cNOR at the same location as the electron donor. We further find that the presence of cNOR influences the dimerization of cd(1)NiR. Overall, although we find no evidence for a high-affinity, constant interaction between the two enzymes, our data supports transient interactions between cd(1)NiR and cNOR that influence enzymatic properties of cNOR and oligomerization properties of cd(1)NiR. We speculate that this could be of particular importance in vivo during metabolic switches between aerobic and denitrifying conditions.
|
|
| 2. |
- Bagawath-Singh, Sunitha, et al.
(author)
-
Cytokines Induce Faster Membrane Diffusion of MHC Class I and the Ly49A Receptor in a Subpopulation of Natural Killer Cells
- 2016
-
In: Frontiers in Immunology. - : Frontiers. - 1664-3224. ; 7
-
Journal article (peer-reviewed)abstract
- Cytokines have the potential to drastically augment immune cell activity. Apart from altering the expression of a multitude of proteins, cytokines also affect immune cell dynamics. However, how cytokines affect the molecular dynamics within the cell membrane of immune cells has not been addressed previously. Molecular movement is a vital component of all biological processes, and the rate of motion is, thus, an inherent determining factor for the pace of such processes. Natural killer (NK) cells are cytotoxic lymphocytes, which belong to the innate immune system. By fluorescence correlation spectroscopy, we investigated the influence of cytokine stimulation on the membrane density and molecular dynamics of the inhibitory receptor Ly49A and its ligand, the major histocompatibility complex class I allele H-2D(d), in freshly isolated murine NK cells. H-2D(d) was densely expressed and diffused slowly in resting NK cells. Ly49A was expressed at a lower density and diffused faster. The diffusion rate in resting cells was not altered by disrupting the actin cytoskeleton. A short-term stimulation with interleukin-2 or interferon- alpha + beta did not change the surface density of moving H-2D(d) or Ly49A, despite a slight upregulation at the cellular level of H-2D(d) by interferon-alpha + beta, and of Ly49A by IL-2. However, the molecular diffusion rates of both H-2D(d) and Ly49A increased significantly. A multivariate analysis revealed that the increased diffusion was especially marked in a subpopulation of NK cells, where the diffusion rate was increased around fourfold compared to resting NK cells. After IL-2 stimulation, this subpopulation of NK cells also displayed lower density of Ly49A and higher brightness per entity, indicating that Ly49A may homo-cluster to a larger extent in these cells. A faster diffusion of inhibitory receptors could enable a faster accumulation of these molecules at the immune synapse with a target cell, eventually leading to a more efficient NK cell response. It has previously been assumed that cytokines regulate immune cells primarily via alterations of protein expression levels or posttranslational modifications. These findings suggest that cytokines may also modulate immune cell efficiency by increasing the molecular dynamics early on in the response.
|
|
| 3. |
- Bagheri, Niusha, et al.
(author)
-
Change in the emission saturation and kinetics of upconversion nanoparticles under different light irradiations
- 2019
-
In: Optical materials (Amsterdam). - : Elsevier. - 0925-3467 .- 1873-1252. ; 97
-
Journal article (peer-reviewed)abstract
- Nd3+-sensitized upconversion nanoparticles (UCNPs) can be excited by both 980 and 808 nm light, which is regarded as a particularly advantageous property of these particles. In this work, we demonstrate that the nanoparticles can exhibit significantly different response when excited at these two excitation wavelengths, showing dependence on the intensity of the excitation light and the way it is distributed in time. Specifically, with 808 nm excitation saturation in the emitted luminescence is more readily reached with increasing excitation intensities than upon 980 nm excitation. This is accompanied by delayed upconversion luminescence (UCL) kinetics and weaker UCL intensities. The different luminescence response at 808 and 980 nm excitation reported in this work is relevant in a manifold of applications using UCNPs as labels and sensors. This could also open new possibilities for multi-wavelength excitable UCNPs for upconversion color display and in laser-scanning microscopy providing selective readouts and sub-sectioning of samples.
|
|
| 4. |
|
|
| 5. |
|
|
| 6. |
- Bergstrand, Jan, et al.
(author)
-
Fast, streamlined fluorescence nanoscopy resolves rearrangements of SNARE and cargo proteins in platelets co-incubated with cancer cells
- 2022
-
In: Journal of Nanobiotechnology. - : Springer Nature. - 1477-3155. ; 20:1
-
Journal article (peer-reviewed)abstract
- Background Increasing evidence suggests that platelets play a central role in cancer progression, with altered storage and selective release from platelets of specific tumor-promoting proteins as a major mechanism. Fluorescence-based super-resolution microscopy (SRM) can resolve nanoscale spatial distribution patterns of such proteins, and how they are altered in platelets upon different activations. Analysing such alterations by SRM thus represents a promising, minimally invasive strategy for platelet-based diagnosis and monitoring of cancer progression. However, broader applicability beyond specialized research labs will require objective, more automated imaging procedures. Moreover, for statistically significant analyses many SRM platelet images are needed, of several different platelet proteins. Such proteins, showing alterations in their distributions upon cancer progression additionally need to be identified. Results A fast, streamlined and objective procedure for SRM platelet image acquisition, analysis and classification was developed to overcome these limitations. By stimulated emission depletion SRM we imaged nanoscale patterns of six different platelet proteins; four different SNAREs (soluble N-ethylmaleimide factor attachment protein receptors) mediating protein secretion by membrane fusion of storage granules, and two angiogenesis regulating proteins, representing cargo proteins within these granules coupled to tumor progression. By a streamlined procedure, we recorded about 100 SRM images of platelets, for each of these six proteins, and for five different categories of platelets; incubated with cancer cells (MCF-7, MDA-MB-231, EFO-21), non-cancer cells (MCF-10A), or no cells at all. From these images, structural similarity and protein cluster parameters were determined, and probability functions of these parameters were generated for the different platelet categories. By comparing these probability functions between the categories, we could identify nanoscale alterations in the protein distributions, allowing us to classify the platelets into their correct categories, if they were co-incubated with cancer cells, non-cancer cells, or no cells at all. Conclusions The fast, streamlined and objective acquisition and analysis procedure established in this work confirms the role of SNAREs and angiogenesis-regulating proteins in platelet-mediated cancer progression, provides additional fundamental knowledge on the interplay between tumor cells and platelets, and represent an important step towards using tumor-platelet interactions and redistribution of nanoscale protein patterns in platelets as a basis for cancer diagnostics.
|
|
| 7. |
- Bergstrand, Jan, et al.
(author)
-
On the decay time of upconversion luminescence
- 2019
-
In: Nanoscale. - : Royal Society of Chemistry. - 2040-3364 .- 2040-3372. ; 11:11, s. 4959-4969
-
Journal article (peer-reviewed)abstract
- In this study, we systematically investigate the decay characteristics of upconversion luminescence (UCL) under anti-Stokes excitation through numerical simulations based on rate-equation models. We find that a UCL decay profile generally involves contributions from the sensitizer's excited-state lifetime, energy transfer and cross-relaxation processes. It should thus be regarded as the overall temporal response of the whole upconversion system to the excitation function rather than the intrinsic lifetime of the luminescence emitting state. Only under certain conditions, such as when the effective lifetime of the sensitizer's excited state is significantly shorter than that of the UCL emitting state and of the absence of cross-relaxation processes involving the emitting energy level, the UCL decay time approaches the intrinsic lifetime of the emitting state. Subsequently, Stokes excitation is generally preferred in order to accurately quantify the intrinsic lifetime of the emitting state. However, possible cross-relaxation between doped ions at high doping levels can complicate the decay characteristics of the luminescence and even make the Stokes-excitation approach fail. A strong cross-relaxation process can also account for the power dependence of the decay characteristics of UCL.
|
|
| 8. |
- Bergstrand, Jan, et al.
(author)
-
Scanning inverse fluorescence correlation spectroscopy
- 2014
-
In: Optics Express. - : Optical Society of America. - 1094-4087. ; 22:11, s. 13073-13090
-
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.
|
|
| 9. |
- Bergstrand, Jan
(author)
-
Super resolution fluorescence imaging : analyses, simulations and applications
- 2019
-
Doctoral thesis (other academic/artistic)abstract
- Fluorescence methods offer extraordinary sensitivity and specificity, and are extensively used in the life sciences. In recent years, super resolution fluorescence imaging techniques have developed strongly, uniquely combining ~10 nm sub diffraction resolution and specific labeling with high efficiency. This thesis explores this potential, with a major focus on Stimulated Emission Depletion, STED, microscopy, applications thereof, image analyses and simulation studies. An additional theme in this thesis is development and use of single molecule fluorescence correlation spectroscopy, FCS, and related techniques, as tools to study dynamic processes at the molecular level. In paper I the proteins cytochrome-bo3 and ATP-synthase are studied with fluorescence cross-correlation spectroscopy, FCCS. These two proteins are a part of the energy conversion process in E. coli, converting ADP into ATP. We found that an increased interaction between these proteins, detected by FCCS, correlates with an increase in the ATP production. In paper II an FCS-based imaging method is developed, capable to determine absolute sizes of objects, smaller than the resolution limit of the microscope used. Combined with STED, this may open for studies of membrane nano-domains, such as those investigated by simulations in paper VII. In paper III and paper IV super resolution STED imaging was applied on Streptococcus Pneumoniae, revealing information about function and distribution of proteins involved in the defense mechanism of the bacteria, as well as their role in bacterial meningitis. In paper V, we used STED imaging to investigate protein distributions in platelets. We then found that the adhesion protein P-selectin changes its distribution pattern in platelets incubated with tumor cells, and with machine learning algorithms and classical image analysis of the STED images it is possible to automatically distinguish such platelets from platelets activated by other means. This could provide a strategy for minimally invasive diagnostics of early cancer development, and deeper understanding of the role of platelets in cancer development. Finally, this thesis presents Monte-Carlo simulations of biological processes and their monitoring by FCS. In paper VI, a combination of FCCS and simulations was applied to resolve the interactions between a transcription factor (p53) and an oncoprotein (MDM2) inside live cells. In paper VII, the feasibility of FCS techniques for studying nano-domains in membranes is investigated purely by simulations, identifying the conditions under which such nano-domains would be possible to detect by FCS. In paper VIII, proton exchange dynamics at biological membranes were simulated in a model, verifying experimental FCS data and identifying fundamental mechanisms by which membranes mediate proton exchange on a local (~10nm) scale.
|
|
| 10. |
- Bergstrand, Jan, et al.
(author)
-
Super-resolution microscopy can identify specific protein distribution patterns in platelets incubated with cancer cells
-
Other publication (other academic/artistic)abstract
- Protein contents in platelets are frequently changed upon tumor development and metastasis. However, how cancer cells can influence protein-selective redistribution and release within platelets, thereby promoting tumor development, remains largely elusive. With fluorescence-based super-resolution stimulated emission depletion (STED) imaging we reveal how specific proteins, implicated in tumor progression and metastasis, re-distribute within platelets, when subject to soluble activators (thrombin, adenosine-diphosphate and thromboxaneA2), and when incubated with cancer (MCF-7, MDA-MB-231, EFO21) or non-cancer cells (184A1, MCF10A). Upon cancer cell incubation, the cell-adhesion protein P-selectin was found to re-distribute into circular nano-structures, consistent with accumulation into the membrane of protein-storing alpha-granules within the platelets. These changes were to a significantly lesser extent, if at all, found in platelets incubated with normal cells, or in platelets subject to soluble platelet activators. From these patterns, we developed a classification procedure, whereby platelets exposed to cancer cells, to non-cancer cells, soluble activators as well as non-activated platelets all could be identified in an automatic, objective manner. We demonstrate that STED imaging, in contrast to electron and confocal microscopy, has the necessary spatial resolution and labelling efficiency to identify protein distribution patterns in platelets and can resolve how they specifically change upon different activations. Combined with image analyses of specific protein distribution patterns within the platelets, STED imaging can thus have a role in future platelet-based cancer diagnostics and therapeutic monitoring. The presented approach can also bring further clarity into fundamental mechanisms for cancer cell-platelet interactions, and into non-contact cell-to-cell interactions in general.
|
|
| 11. |
- Bergstrand, Jan, et al.
(author)
-
Super-resolution microscopy can identify specific protein distribution patterns in platelets incubated with cancer cells
- 2019
-
In: Nanoscale. - : ROYAL SOC CHEMISTRY. - 2040-3364 .- 2040-3372. ; 11:20, s. 10023-10033
-
Journal article (peer-reviewed)abstract
- Protein contents in platelets are frequently changed upon tumor development and metastasis. However, how cancer cells can influence protein-selective redistribution and release within platelets, thereby promoting tumor development, remains largely elusive. With fluorescence-based super-resolution stimulated emission depletion (STED) imaging we reveal how specific proteins, implicated in tumor progression and metastasis, re-distribute within platelets, when subject to soluble activators (thrombin, adenosine diphosphate and thromboxane A2), and when incubated with cancer (MCF-7, MDA-MB-231, EFO21) or non-cancer cells (184A1, MCF10A). Upon cancer cell incubation, the cell-adhesion protein P-selectin was found to re-distribute into circular nano-structures, consistent with accumulation into the membrane of protein-storing alpha-granules within the platelets. These changes were to a significantly lesser extent, if at all, found in platelets incubated with normal cells, or in platelets subject to soluble platelet activators. From these patterns, we developed a classification procedure, whereby platelets exposed to cancer cells, to non-cancer cells, soluble activators, as well as non-activated platelets all could be identified in an automatic, objective manner. We demonstrate that STED imaging, in contrast to electron and confocal microscopy, has the necessary spatial resolution and labelling efficiency to identify protein distribution patterns in platelets and can resolve how they specifically change upon different activations. Combined with image analyses of specific protein distribution patterns within the platelets, STED imaging can thus have a role in future platelet-based cancer diagnostics and therapeutic monitoring. The presented approach can also bring further clarity into fundamental mechanisms for cancer cell-platelet interactions, and into non-contact cell-to-cell interactions in general.
|
|
| 12. |
- Blom, Hans, et al.
(author)
-
Electrostatic Interactions of Fluorescent Molecules with Dielectric Interfaces Studied by Total Internal Reflection Fluorescence Correlation Spectroscopy
- 2010
-
In: International Journal of Molecular Sciences. - : MDPI. - 1661-6596 .- 1422-0067. ; 11:2, s. 368-406
-
Journal article (peer-reviewed)abstract
- Electrostatic interactions between dielectric surfaces and different fluorophoresused in ultrasensitive fluorescence microscopy are investigated using objective-based TotalInternal Reflection Fluorescence Correlation Spectroscopy (TIR-FCS). The interfacialdynamics of cationic rhodamine 123 and rhodamine 6G, anionic/dianionic fluorescein,zwitterionic rhodamine 110 and neutral ATTO 488 are monitored at various ionic strengthsat physiological pH. As analyzed by means of the amplitude and time-evolution of theautocorrelation function, the fluorescent molecules experience electrostatic attraction orrepulsion at the glass surface depending on their charges. Influences of the electrostaticinteractions are also monitored through the triplet-state population and triplet relaxationtime, including the amount of detected fluorescence or the count-rate-per-moleculeparameter. These TIR-FCS results provide an increased understanding of how fluorophoresare influenced by the microenvironment of a glass surface, and show a promising approachfor characterizing electrostatic interactions at interfaces.
|
|
| 13. |
- Blom, Hans, et al.
(author)
-
Nearest neighbor analysis of dopamine D1 receptors and Na plus -K plus -ATPases in dendritic spines dissected by STED microscopy
- 2012
-
In: Microscopy research and technique (Print). - : Wiley. - 1059-910X .- 1097-0029. ; 75:2, s. 220-228
-
Journal article (peer-reviewed)abstract
- Protein localization in dendritic spines is the focus of intense investigations within neuroscience. Applications of super-resolution microscopy to dissect nanoscale protein distributions, as shown in this work with dual-color STED, generate spatial correlation coefficients having quite small values. This means that colocalization analysis to some extent looses part of its correlative impact. In this study we thus introduced nearest neighbor analysis to quantify the spatial relations between two important proteins in neurons, the dopamine D1 receptor and Na+,K+-ATPase. The analysis gave new information on how dense the D1 receptor and Na+,K+-ATPase constituting nanoclusters are located both with respect to the homogenous (self to same) and the heterogeneous (same to other) topology. The STED dissected nanoscale topologies provide evidence for both a joint as well as a separated confinement of the D1 receptor and the Na+,K+-ATPase in the postsynaptic areas of dendritic spines. This confined topology may have implications for generation of local sodium gradients and for structural and functional interactions modulating slow synaptic transmission processes. Microsc. Res. Tech., 2011.
|
|
| 14. |
- Blom, Hans, et al.
(author)
-
Spatial Distribution of DARPP-32 in Dendritic Spines
- 2013
-
In: PLOS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 8:9, s. e75155-
-
Journal article (peer-reviewed)abstract
- The phosphoprotein DARPP-32 (dopamine and cyclic adenosine 3́, 5́-monophosphate-regulated phosphoprotein, 32 kDa) is an important component in the molecular regulation of postsynaptic signaling in neostriatum. Despite the importance of this phosphoprotein, there is as yet little known about the nanoscale distribution of DARPP-32. In this study we applied superresolution stimulated emission depletion microscopy (STED) to assess the expression and distribution of DARPP-32 in striatal neurons. Primary culture of striatal neurons were immunofluorescently labeled for DARPP-32 with Alexa-594 and for the dopamine D1 receptor (D1R) with atto-647N. Dual-color STED microscopy revealed discrete localizations of DARPP-32 and D1R in the spine structure, with clustered distributions in both head and neck. Dissected spine structures reveal that the DARPP-32 signal rarely overlapped with the D1R signal. The D1R receptor is positioned in an "aggregated" manner primarily in the spine head and to some extent in the neck, while DARPP-32 forms several neighboring small nanoclusters spanning the whole spine structure. The DARPP-32 clusters have a mean size of 52 +/- 6 nm, which is close to the resolution limit of the microscope and corresponds to the physical size of a few individual phosphoprotein immunocomplexes. Dissection of synaptic proteins using superresolution microscopy gives possibilities to reveal in better detail biologically relevant information, as compared to diffraction-limited microscopy. In this work, the dissected postsynaptic topology of the DARPP-32 phosphoprotein provides strong evidence for a compartmentalized and confined distribution in dendritic spines. The protein topology and the relatively low copy number of phosphoprotein provides a conception of DARPP-32's possibilities to fine-tune the regulation of synaptic signaling, which should have an impact on the performance of the neuronal circuits in which it is expressed.
|
|
| 15. |
- Blom, Hans, et al.
(author)
-
Spatial distribution of Na+-K+-ATPase in dendritic spines dissected by nanoscale superresolution STED microscopy
- 2011
-
In: BMC Neuroscience. - : Springer Science and Business Media LLC. - 1471-2202. ; 12, s. 16-
-
Journal article (peer-reviewed)abstract
- Background: The Na+,K+-ATPase plays an important role for ion homeostasis in virtually all mammalian cells, including neurons. Despite this, there is as yet little known about the isoform specific distribution in neurons. Results: With help of superresolving stimulated emission depletion microscopy the spatial distribution of Na+,K+-ATPase in dendritic spines of cultured striatum neurons have been dissected. The found compartmentalized distribution provides a strong evidence for the confinement of neuronal Na+,K+-ATPase (alpha 3 isoform) in the postsynaptic region of the spine. Conclusions: A compartmentalized distribution may have implications for the generation of local sodium gradients within the spine and for the structural and functional interaction between the sodium pump and other synaptic proteins. Superresolution microscopy has thus opened up a new perspective to elucidate the nature of the physiological function, regulation and signaling role of Na+,K+-ATPase from its topological distribution in dendritic spines.
|
|
| 16. |
- Blom, Hans, et al.
(author)
-
STED microscopy : towards broadened use and scope of applications
- 2014
-
In: Current opinion in chemical biology. - : Elsevier. - 1367-5931 .- 1879-0402. ; 20:1, s. 127-133
-
Research review (peer-reviewed)abstract
- High resolution Stimulated Emission Depletion (STED) microscopy has been demonstrated for fundamental studies in cells, living tissue and organisms. Today, a major trend in the STED technique development is to make the instruments simpler and more user-friendly, without compromising performance. This has become possible by new low-cost, turn-key laser technology and by implementing specifically designed phase plates and polarization elements, extending and simplifying the shaping of the laser beam profiles. These simpler and cheaper realizations of STED are now becoming more broadly available. In parallel with the continuous development of sample preparation and fluorophore reporter molecules ultimately setting the limit of the image quality, contrast and resolution, we can thus expect a significant increase in the use of STED, in science as well as for clinical and drug development purposes.
|
|
| 17. |
- Blom, Hans, et al.
(author)
-
Stimulated Emission Depletion Microscopy
- 2017
-
In: Chemical Reviews. - : American Chemical Society (ACS). - 0009-2665 .- 1520-6890. ; 117:11, s. 7377-7427
-
Research review (peer-reviewed)abstract
- Despite its short history, diffraction-unlimited fluorescence microscopy techniques have already made a substantial imprint in the biological sciences. In this review, we describe how stimulated emission depletion (STED) imaging originally evolved, how it compares to other optical super-resolution imaging techniques, and what advantages it provides compared to previous golden-standards for biological microscopy, such as diffraction-limited optical microscopy and electron microscopy. We outline the prerequisites for successful STED imaging experiments, emphasizing the equally critical roles of instrumentation, sample preparation, and photophysics, and describe major evolving strategies for how to push the borders of STED imaging even further in life science. Finally, we provide examples of how STED nanoscopy can be applied, within three different fields with particular potential for STED imaging experiments: neuroscience, plasma membrane biophysics, and subcellular clinical diagnostics. In these areas, and in many more, STED imaging can be expected to play an increasingly important role in the future.
|
|
| 18. |
- Blom, Hans, et al.
(author)
-
Triplet-State Investigations of Fluorescent Dyes at Dielectric Interfaces Using Total Internal Reflection Fluorescence Correlation Spectroscopy
- 2009
-
In: Journal of Physical Chemistry A. - Washington, DC : American Chemical Society. - 1089-5639 .- 1520-5215. ; 113:19, s. 5554-5566
-
Journal article (peer-reviewed)abstract
- The triplet-state kinetics of several fluorescent dyes used in ultrasensitive fluorescence microscopy are investigated using total internal reflection fluorescence correlation spectroscopy (TIR-FCS). A theoretical outline of the correlation analysis and the physical aspects of evanescent excitation and fluorescence emission at dielectric interfaces are given. From this analysis, the rates of intersystem crossing and triplet decay are deduced for fluorescein, ATTO 488, rhodamine 110, rhodamine 123, and rhodamine 6G in aqueous buffer solutions. All investigated dyes show slightly higher triplet rates at the dielectric interface compared to bulk solution measurements. We attribute this enhancement to possible modifications of the dyes’ photophysical properties near a dielectric interface. In the case of rhodamine 6G, the impact of changes in the dye concentration, ionic strength of the solvent, and potassium iodide concentration are also investigated. This leads to a better understanding of the influences of dye−dye, dye−solvent, and dye−surface interactions on the increased triplet intersystem crossing and triplet decay rates. The study shows that analysis of triplet-state kinetics by TIR-FCS not only results in a better understanding of how the photophysical properties of the dyes are affected by the presence of an interface, but also provides a means for probing the microenvironment near dielectric interfaces.
|
|
| 19. |
|
|
| 20. |
- Cebula, Marcus, et al.
(author)
-
Catalytic Conversion of Lipophilic Substrates by Phase constrained Enzymes in the Aqueous or in the Membrane Phase
- 2016
-
In: Scientific Reports. - : Nature Publishing Group. - 2045-2322. ; 6
-
Journal article (peer-reviewed)abstract
- Both soluble and membrane-bound enzymes can catalyze the conversion of lipophilic substrates. The precise substrate access path, with regard to phase, has however, until now relied on conjecture from enzyme structural data only (certainly giving credible and valuable hypotheses). Alternative methods have been missing. To obtain the first experimental evidence directly determining the access paths (of lipophilic substrates) to phase constrained enzymes we here describe the application of a BODIPY-derived substrate (PS1). Using this tool, which is not accessible to cytosolic enzymes in the presence of detergent and, by contrast, not accessible to membrane embedded enzymes in the absence of detergent, we demonstrate that cytosolic and microsomal glutathione transferases (GSTs), both catalyzing the activation of PS1, do so only within their respective phases. This approach can serve as a guideline to experimentally validate substrate access paths, a fundamental property of phase restricted enzymes. Examples of other enzyme classes with members in both phases are xenobiotic-metabolizing sulphotransferases/UDP-glucuronosyl transferases or epoxide hydrolases. Since specific GSTs have been suggested to contribute to tumor drug resistance, PS1 can also be utilized as a tool to discriminate between phase constrained members of these enzymes by analyzing samples in the absence and presence of Triton X-100.
|
|
| 21. |
- Chen, Xingqi, et al.
(author)
-
Chromatin in situ proximity (ChrISP) : Single-cell analysis of chromatin proximities at a high resolution
- 2014
-
In: BioTechniques. - : Future Science Ltd. - 0736-6205 .- 1940-9818. ; 56:3, s. 117-124
-
Journal article (peer-reviewed)abstract
- Current techniques for analyzing chromatin structures are hampered by either poor resolution at the individual cell level or the need for a large number of cells to obtain higher resolution. This is a major problem as it hampers our understanding of chromatin conformation in single cells and how these respond to environmental cues. Here we describe a new method, chromatin in situ proximity (ChrISP), which reproducibly scores for proximities between two different chromatin fibers in 3-D with a resolution of similar to 170 angstrom in single cells. The technique is based on the in situ proximity ligation assay (ISPLA), but ChrISP omits the rolling circle amplification step (RCA). Instead, the proximities between chromatin fibers are visualized by a fluorescent connector oligonucleotide DNA, here termed splinter, forming a circular DNA.with another circle-forming oligonucleotide, here termed backbone, upon ligation. In contrast to the regular ISPLA technique, our modification enables detection of chromatin fiber proximities independent of steric hindrances from nuclear structures. We use this method to identify higher order structures of individual chromosomes in relation to structural hallmarks of interphase nuclei and beyond the resolution of the light microscope.
|
|
| 22. |
- Chmyrov, Andriy, 1979-, et al.
(author)
-
Characterization of new fluorescent labels for ultra-high resolution microscopy
- 2008
-
In: Photochemical and Photobiological Sciences. - : Springer Science and Business Media LLC. - 1474-905X .- 1474-9092. ; 7:11, s. 1378-1385
-
Journal article (peer-reviewed)abstract
- Photo-induced switching of dyes into dark, long-lived states, such as a triplet state, has recently gained increasing interest, as a means to achieve ultra-high optical resolution. Additionally, these long lived states are often highly environment-sensitive and their photodynamics can thus offer additional independent fluorescence-based information. However, although providing a useful mechanism for photo-induced switching, the triplet state often appears as a precursor state for photobleaching, which potentially can limit its usefulness. In this work, a set of rhodamine and pyronin dyes, modified by substitution of heavy atoms and nitrogen within or close to the central xanthene unit of the dyes, were investigated with respect to their triplet state dynamics and photostabilities, under conditions relevant for ultra-high resolution microscopy. Out of the dyes investigated, in particular the rhodamine and pyronin dyes with a sulfur atom replacing the central oxygen atom in the xanthene unit were found to meet the requirements for ultra-high resolution microscopy, combining a prominent triplet state yield with reasonable photostability.
|
|
| 23. |
- Chmyrov, Andriy, et al.
(author)
-
Characterization of new fluorescent labels for ultrahigh resolution microscopy
- 2009
-
In: Novel Techniques in Microscopy (NTM) 2009. - : Optical Society of America. - 9781557528711
-
Conference paper (peer-reviewed)abstract
- A set of modified dyes was investigated, of which several candidates combine prominent triplet state yield with reasonable photostability. They can be used to achieve ultrahigh optical resolution by photo-induced switching into dark (triplet) states.
|
|
| 24. |
- Chmyrov, Andriy, et al.
(author)
-
Iodide as a Fluorescence Quencher and Promoter-Mechanisms and Possible Implications
- 2010
-
In: Journal of Physical Chemistry B. - : American Chemical Society (ACS). - 1520-6106 .- 1520-5207. ; 114:34, s. 11282-11291
-
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.
|
|
| 25. |
- Chmyrov, Andriy, 1979-, et al.
(author)
-
Iodide as a Triplet State Promoter and Quencher –Mechanisms and Possible Implications
-
Other publication (other academic/artistic)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 arange of organic dyes in the visible wavelength range. Apartfrom a heavy atom effect promoting intersystem crossing tothe triplet states in all dyes, KI was also found to enhancethe triplet state decay by a charge-coupled deactivation.This deactivation was only found for dyes with excitationmaximum in the blue range, not for those with excitationmaxima at wavelengths in the green range or longer. Consequently,under excitation conditions sufficient for tripletstate formation, KI can promote the triplet state build-up ofone dye and reduce it for another, red-shifted dye. The anticorrelated,spectrally separable responses of two dyes to thepresence of one and the same agent are likely to provide auseful readout for biomolecular interaction and micro-environmentalmonitoring studies. In contrast to the typicalnotion of KI as a fluorescence quencher, the FCS measurementsalso revealed that when added in micromolar concentrationsKI can act as an anti-oxidant, promoting the recoveryof photo-oxidized fluorophores. However, in millimolarconcentrations KI also reduces intact, fluorescently viablefluorophores to a considerable extent. In aqueous solutions,an optimal concentration of KI of approximately 5 mM canbe defined at which the fluorescence signal is maximized.This concentration is not high enough to allow full tripletstate quenching. Therefore, as a fluorescence enhancementagent, it is primarily the anti-oxidative properties of KI thatplay a role.
|
|
| 26. |
- Chmyrov, Andriy, 1979-
(author)
-
Photo-induced dark states influorescence spectroscopy – investigations & applications
- 2010
-
Doctoral thesis (other academic/artistic)abstract
- This thesis focuses on investigations of transient dark states of fluorescentmolecules using spectroscopic techniques. The main purpose is to show andconvince the reader that transient dark states are not always a nuisance, butalso represent an additional source of information. Several studies with fluorescencecorrelation spectroscopy were performed, all related to non-fluorescentstates such as triplet state or isomerized states.Photobleaching is one of the main problems in virtually all of the fluorescencetechniques. In this thesis, mechanisms that retard photobleaching arecharacterized. Several compounds, antioxidants and triplet state quenchers,which decrease photobleaching, are studied, and guidelines for achieving optimalfluorescence brightness using these compounds are presented.Triplet state quenching by several compounds was studied. Detailed investigationsof the fluorescence quencher potassium iodide demonstratedthat for some of fluorophores, except of quenching, there is fluorescence enhancementmechanism present. In agreement with the first publication inthis thesis, antioxidative properties were found to play an important role inthe fluorescence enhancement. Quenching of the triplet state is proposedas a tool for monitoring diffusion mediated reactions over a wide range offrequencies.Specially designed fluorophores combining high triplet yields with reasonablefluorescence brightness and photostability were characterized forpossible applications in novel super-resolution imaging techniques based onfluorescence photoswitching. Except of benefits for imaging techniques, photoinducedswitching to non-fluorescent states could be used for monitoringmolecular diffusion, which was also demonstrated in this thesis.Studies of the triplet state kinetics of fluorophores close to dielectric interfaceswere performed using fluorescence spectroscopy. The analysis of thetriplet state kinetic can provide information about the local microenvironmentand electrostatic interactions near dielectric interfaces.
|
|
| 27. |
- Chmyrov, Andriy, 1979-, et al.
(author)
-
Recovery of Photoinduced Reversible Dark States Utilized for Molecular Diffusion Measurements
- 2010
-
In: Analytical Chemistry. - : American Chemical Society (ACS). - 0003-2700 .- 1520-6882. ; 82:24, s. 9998-10005
-
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.
|
|
| 28. |
- Chmyrov, Volodymyr, 1987-
(author)
-
Fluorescence fluctuation studies of biomolecular interactions in solutions, biomembranes and live cells
- 2016
-
Doctoral thesis (other academic/artistic)abstract
- Fluorescence spectroscopy and imaging have a very broad spectrum of applicationswithin the life sciences, in particular for detection and characterization ofbiomolecular dynamics and interactions in different environments. This thesis comprisesprojects that strive to further expand the information content extracted fromthe detected fluorescence, leading to sensitive readout parameters for studies ofbiomolecular dynamics and interactions. Two major strategies are presented toachieve this aim. The first strategy is based on the expansion of the availablereadout parameters beyond the "traditional" fluorescence parameters: intensity,wavelength, polarization and fluorescence lifetime. The additional parameters arebased on blinking properties of fluorescent labels. In particular on transitions betweensinglet and triplet states, and transitions between the trans- and cis-isomersof fluorophores. Two publications in the thesis are based on this strategy (paperI and IV). The second strategy is based on the utilization of fluorescence intensityfluctuations in order to detect the oligomerization mechanisms of fluorescentlylabeled peptides and proteins. This strategy combines the intensity fluctuationanalysis and the readout of distance dependent energy transfer between fluorescentmolecules together with the correlation analysis of fluorescence from two labeledproteins emitting at different wavelengths. Another two publications presented inthe thesis are based on the second comprehensive strategy (papers II and III).The work presented in this thesis shows that the blinking kinetics of fluorescentlabels contain significant information that can be exploited by a combination of fluctuationsanalysis with distance dependent excitation energy transfer between thefluorescent molecules, or by analysis of fluorescence covariance between moleculesthat emit at different wavelengths. These fluorescence-based methods have a significantpotential for molecular interaction studies in the biomedical field.
|
|
| 29. |
- Chmyrov, Volodymyr, et al.
(author)
-
Trans-Cis isomerization of lipophilic dyes probing membrane microviscosity in biological membranes and in live cells
- 2015
-
In: Analytical Chemistry. - : American Chemical Society (ACS). - 0003-2700 .- 1520-6882. ; 87:11, s. 5690-5697
-
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.
|
|
| 30. |
|
|
| 31. |
- Demirbay, Baris
(author)
-
Concepts and biomedical applications of excitation-modulated transient state monitoring of fluorescence emitters
- 2023
-
Doctoral thesis (other academic/artistic)abstract
- Fluorescence methods have developed very strongly in the last decade, allowing single- molecule detection sensitivity, high specificity, high time- and spatial resolution, as well as high readout speeds. Together, this makes fluorescencea very central readout modality for biomolecular and cellular studies. The photophysics of the fluorescence emitters, fluorophores, used is of central importance or the performance. Here, the photostability and brightness ofthe fluorophores, but also their blinking properties set the limits of the performance. Fluorophore blinking, arising from photo-induced, non-fluorescent transient states of fluorophores, can however also be taken advantage of cellular and biomolecular studies. Blinking is a prerequisite for essentially allso-called fluorescence-based super-resolution techniques. Moreover, blinkingis often sensitive to micro-environmental parameters such as pH, oxygen concentrations,redox conditions and viscosity. This follows from the fact that the underlying, non-luminescent, dark transient states typically have 103 to 106 longer lifetimes than the fluorescent excited states of the fluorophores, thereby giving fluorescent molecules in the dark states more time to interactwith their surrounding in biological environment. It can thus be utilized as an alternative readout parameter to provide useful information on molecules and cells and their environments, beyond what can be monitored by traditional fluorescence methods. This thesis takes as one starting point the transient state (TRAST) spectroscopy technique, designed to monitor such long-lived, dark transient states, including triplet, photo-oxidation, photo-reduction and photo-isomerized states of fluorophores, by measuring how the time-averaged fluorescence signal detected in the sample is changed upon systematically varying the excitation modulation.The major focus of the present thesis work is to further extent the use of long-lived dark transient states of fluorescence emitters in solution, lipid membranes and live cells. For this different TRAST modalities were adapted and developed, and then demonstrated as useful characterization methods. First, we showed how the relaxed brightness requirements of TRAST made it possible to characterize the photo-physical properties of the high triplet yield carboxy-fluorescein dye and its brominated derivatives (paper I). By widefield TRAST measurements, we demonstrated its capability to sense heavy atom effect of bromine and iodide atoms, and how they affected the triplet and long-lived photo-oxidation states and their transitions rates. Next, we developed and demonstrated a concept based on TRAST method and its ability to distinguish fluorophores with different blinking properties as a way to perform fluorescence-based barcoding and multiplexing. This concept, demonstrated by exploiting the by TRAST well distinguishable photophysical transitions of two fluorescent dyes, which emit in the same spectral range, was demonstrated in paper II. In the same work, we also developed a TRAST modality for microfluidic measurements of molecules and lipid vesicles, on which the bar-coding concept could be demonstrated on-the-fly, as the molecules and vesicles passed through the microfluidic channel. Furthermore, with TRAST implemented in camera-based wide-field microscopy, multicolor barcoded images of cells with high spatial resolution could be further investigated for the first time due to the specific blinking dynamics of these labels. The last two papers of this thesis describe further extensions of the TRAST concept, and the monitoring of fluorescence blinking to live cell studies. In paper III, TRAST in a widefield microscopy setting was employed in combination with FCS to study the folding of dye-labelled RNA strands into G-quadruplex structures in solution and live cells using photo-isomerization kinetics of cyanine dye as a readout parameter. Here, we took advantage of the high sensitivity of cyanine dye photoisomerization, to viscosity and steric constraints,and the resulting blinking of the cyanines, to monitor conformation changes of RNAs in live cells. Finally, in paper IV, we demonstrated how it by TRAST imaging, taking advantage of the photo-induced dark states of a mitochondrial localization fluorophore (n-Nonyl Acridine Orange, NAO), is possible to give this localization probe environmental sensing properties as well.To sum up, the experimental findings and papers included in this thesis show that fluorescence blinking represent a rich source of information for biomolecular and cellular studies. By the TRAST technique, and the variants further adapted and developed in this work, it is shown that it possible tocapture this rich source of complementary information in a broad range of samples. The work in this thesis suggest that further combination of classical fluorescence readouts and a continued development of different TRAST modalities will open yet new windows and provide insights into molecular interaction studies in biological research.
|
|
| 32. |
|
|
| 33. |
|
|
| 34. |
|
|
| 35. |
- Demirbay, Baris, et al.
(author)
-
Photo-physical characterization of high triplet yield brominated fluoresceins by transient state (TRAST) spectroscopy
- 2023
-
In: Methods and applications in fluorescence. - : IOP Publishing. - 2050-6120. ; 11:4
-
Journal article (peer-reviewed)abstract
- Photo-induced dark transient states of fluorophores can pose a problem in fluorescence spectroscopy. However, their typically long lifetimes also make them highly environment sensitive, suggesting fluorophores with prominent dark-state formation yields to be used as microenvironmental sensors in bio-molecular spectroscopy and imaging. In this work, we analyzed the singlet-triplet transitions of fluorescein and three synthesized carboxy-fluorescein derivatives, with one, two or four bromines linked to the anthracence backbone. Using transient state (TRAST) spectroscopy, we found a prominent internal heavy atom (IHA) enhancement of the intersystem crossing (ISC) rates upon bromination, inferred by density functional theory calculations to take place via a higher triplet state, followed by relaxation to the lowest triplet state. A corresponding external heavy atom (EHA) enhancement was found upon adding potassium iodide (KI). Notably, increased KI concentrations still resulted in lowered triplet state buildup in the brominated fluorophores, due to relatively lower enhancements in ISC, than in the triplet decay. Together with an antioxidative effect on the fluorophores, adding KI thus generated a fluorescence enhancement of the brominated fluorophores. By TRAST measurements, analyzing the average fluorescence intensity of fluorescent molecules subject to a systematically varied excitation modulation, dark state transitions within very high triplet yield (>90%) fluorophores can be directly analyzed under biologically relevant conditions. These measurements, not possible by other techniques such as fluorescence correlation spectroscopy, opens for bio-sensing applications based on high triplet yield fluorophores, and for characterization of high triplet yield photodynamic therapy agents, and how they are influenced by IHA and EHA effects.
|
|
| 36. |
|
|
| 37. |
- Du, Zhixue, et al.
(author)
-
Imaging Fluorescence Blinking of a Mitochondrial Localization Probe : Cellular Localization Probes Turned into Multifunctional Sensors br
- 2022
-
In: Journal of Physical Chemistry B. - : American Chemical Society (ACS). - 1520-6106 .- 1520-5207. ; 126:16, s. 3048-3058
-
Journal article (peer-reviewed)abstract
- Mitochondrial membranes and their microenviron-ments directly influence and reflect cellular metabolic states but aredifficult to probe on site in live cells. Here, we demonstrate astrategy, showing how the widely used mitochondrial membranelocalizationfluorophore 10-nonyl acridine orange (NAO) can betransformed into a multifunctional probe of membrane micro-environments by monitoring its blinking kinetics. By transient state(TRAST) studies of NAO in small unilamellar vesicles (SUVs),together with computational simulations, we found that NAOexhibits prominent reversible singlet-triplet state transitions andcan act as a light-induced Lewis acid forming a red-emissivedoublet radical. The resulting blinking kinetics are highlyenvironment-sensitive, specifically reflecting local membrane oxy-gen concentrations, redox conditions, membrane charge,fluidity, and lipid compositions. Here, not only cardiolipin concentrationbut also the cardiolipin acyl chain composition was found to strongly influence the NAO blinking kinetics. The blinking kinetics alsoreflect hydroxyl ion-dependent transitions to and from thefluorophore doublet radical, closely coupled to the proton-transfer eventsin the membranes, local pH, and two- and three-dimensional buffering properties on and above the membranes. Following the SUVstudies, we show by TRAST imaging that thefluorescence blinking properties of NAO can be imaged in live cells in a spatiallyresolved manner. Generally, the demonstrated blinking imaging strategy can transform existingfluorophore markers intomultiparametric sensors reflecting conditions of large biological relevance, which are difficult to retrieve by other means. This opensadditional possibilities for fundamental membrane studies in lipid vesicles and live cells
|
|
| 38. |
- Du, Zhixue, et al.
(author)
-
In Situ Monitoring of p53 Protein and MDM2 Protein Interaction in Single Living Cells Using Single-Molecule Fluorescence Spectroscopy
- 2018
-
In: Analytical Chemistry. - : American Chemical Society (ACS). - 0003-2700 .- 1520-6882. ; 90:10, s. 6144-6151
-
Journal article (peer-reviewed)abstract
- Protein-protein interactions play a central role in signal transduction, transcription regulations, enzymatic activity, and protein synthesis. The p53 protein is a key transcription factor, and its activity is precisely regulated by the p53-MDM2 interaction. Although the p53-MDM2 interaction has been studied, it is still not clear how p53 structures and external factors influence the p53-MDM2 interaction in living cells. Here, we developed a direct method for monitoring the p53-MDM2 interaction in single living cells using single-molecule fluorescence cross-correlation spectroscopy with a microfluidic chip. First, we labeled p53 and MDM2 proteins with enhanced green fluorescent protein (EGFP) and mCherry, respectively, using lentivirus infection. We then designed various mutants covering the three main domains of p53 (tetramerization, transactivation, and DNA binding domains) and systematically studied effects of p53 protein primary, secondary, and quaternary structures on p53 MDM2 binding affinity in single living cells. We found that p53 dimers and tetramers can bind to MDM2, that the binding affinity of p53 tetramers is higher than that of p53 dimers, and that the affinity is closely correlated to the helicity of the p53 transactivation domain. The hot-spot mutation R175H in the DNA-binding domain reduced the binding of p53 to MDM2. Finally, we studied effects of inhibitors on p53-MDM2 interactions and dissociation dynamics of pS3-MDM2 complexes in single living cells. We found that inhibitors Nutlin 3 alpha and MI773 efficiently inhibited the pS3-MDM2 interaction, but RITA did not work in living cells. This study provides a direct way for quantifying the relationship between protein structure and protein protein interactions and evaluation of inhibitors in living cells.
|
|
| 39. |
- Dumke, Christoph, et al.
(author)
-
SATB1, genomic instability and Gleason grading constitute a novel risk score for prostate cancer
- 2021
-
In: Scientific Reports. - : Springer Nature. - 2045-2322. ; 11:1
-
Journal article (peer-reviewed)abstract
- Current prostate cancer risk classifications rely on clinicopathological parameters resulting in uncertainties for prognostication. To improve individual risk stratification, we examined the predictive value of selected proteins with respect to tumor heterogeneity and genomic instability. We assessed the degree of genomic instability in 50 radical prostatectomy specimens by DNA-Image-Cytometry and evaluated protein expression in related 199 tissue-microarray (TMA) cores. Immunohistochemical data of SATB1, SPIN1, TPM4, VIME and TBB5 were correlated with the degree of genomic instability, established clinical risk factors and overall survival. Genomic instability was associated with a GS >= 7 (p = 0.001) and worse overall survival (p = 0.008). A positive SATB1 expression was associated with a GS <= 6 (p = 0.040), genomic stability (p = 0.027), and was a predictor for increased overall survival (p = 0.023). High expression of SPIN1 was also associated with longer overall survival (p = 0.048) and lower preoperative PSA-values (p = 0.047). The combination of SATB1 expression, genomic instability, and GS lead to a novel Prostate Cancer Prediction Score (PCP-Score) which outperforms the current D'Amico et al. stratification for predicting overall survival. Low SATB1 expression, genomic instability and GS >= 7 were identified as markers for poor prognosis. Their combination overcomes current clinical risk stratification regimes.
|
|
| 40. |
- Eggeling, C., et al.
(author)
-
Analysis of photobleaching in single-molecule multicolor excitation and forster resonance energy transfer measurement
- 2006
-
In: Journal of Physical Chemistry A. - : American Chemical Society (ACS). - 1089-5639 .- 1520-5215. ; 110:9, s. 2979-2995
-
Journal article (peer-reviewed)abstract
- we investigated the influence of photobleaching in fluorescence experiments applying multicolor laser as well as Forster resonance energy transfer (FRET) mediated excitation using several red-emitting dyes frequently used in multicolor experiments or as FRET acceptors. The chosen dyes (cyanine 5 (Cy5), MR121, Alexa660, Alexa680, Atto647N, Atto655) have chemically distinct chromophore systems and can be excited at 650 nm. Several fluorescence analysis techniques have been applied to detect photobleaching and to disclose the underlying photophysics, all of which are based on single-molecule detection: (1) fluorescence correlation spectroscopy (FCS) of bulk solutions, (2) fluorescence cross-correlation of single-molecule trajectories, and (3) multiparameter fluorescence detection (MFD) of single-molecule events. The maximum achievable fluorescence signals as well as the survival times of the red dyes were markedly reduced under additional laser irradiation in the range of 500 nm. Particularly at excitation levels at or close to saturation, the 500 nm irradiation effectively induced transitions to higher excited electronic states on already excited dye molecules, leading to a pronounced bleaching reactivity. A theoretical model for the observed laser irradiance dependence of the fluorescence brightness of a Cy5 FRET acceptor dye has been developed introducing the full description of the underlying photophysics. The model takes into account acceptor as well as donor photobleaching from higher excited electronic states, population of triplet states, and energy transfer to both the ground and excited states of the acceptor dye. Also, photoinduced reverse intersystem crossing via higher excited triplet states is included, which was found to be very efficient for Cy5 attached to DNA. Comparing continuous wave (cw) and pulsed donor excitation, a strong enhancement of acceptor photobleaching by a factor of 5 was observed for the latter. Thus, in the case of fluorescence experiments utilizing multicolor pulsed laser excitation, the application of the appropriate timing of synchronized green and red laser pulses in an alternating excitation mode can circumvent excessive photobleaching. Moreover, important new single-molecule analysis diagnosis tools are presented: (1) For the case of excessive acceptor photobleaching, cross-correlation analysis of single-molecule trajectories of the fluorescence signal detected in the donor and acceptor detection channels and vice versa shows an anticorrelated exponential decay and growth, respectively. (2) The time difference, T-g - T-r of the mean observation times of all photons detected for the donor and acceptor detection channels within a single-molecule fluorescence burst allows one to identify and exclude molecules with an event of acceptor photobleaching. The presented single-molecule analysis methods can be constrained to, for example, FRET-active subpopulations, reducing bias from FRET-inactive molecules. The observations made are of strong relevance for and demand a careful choice of laser action in multicolor and FRET experiments, in particular when performed at or close to saturation.
|
|
| 41. |
|
|
| 42. |
- Evans, Caroline A., et al.
(author)
-
Metastasising Fibroblasts Show an HDAC6-Dependent Increase in Migration Speed and Loss of Directionality Linked to Major Changes in the Vimentin Interactome
- 2022
-
In: International Journal of Molecular Sciences. - : MDPI AG. - 1661-6596 .- 1422-0067. ; 23:4
-
Journal article (peer-reviewed)abstract
- Metastasising cells express the intermediate filament protein vimentin, which is used to diagnose invasive tumours in the clinic. We aimed to clarify how vimentin regulates the motility of metastasising fibroblasts. STED super-resolution microscopy, live-cell imaging and quantitative proteomics revealed that oncogene-expressing and metastasising fibroblasts show a less-elongated cell shape, reduced cell spreading, increased cell migration speed, reduced directionality, and stronger coupling between these migration parameters compared to normal control cells. In total, we identified and compared 555 proteins in the vimentin interactome. In metastasising cells, the levels of keratin 18 and Rab5C were increased, while those of actin and collagen were decreased. Inhibition of HDAC6 reversed the shape, spreading and migration phenotypes of metastasising cells back to normal. Inhibition of HDAC6 also decreased the levels of talin 1, tropomyosin, Rab GDI beta, collagen and emilin 1 in the vimentin interactome, and partially reversed the nanoscale vimentin organisation in oncogene-expressing cells. These findings describe the changes in the vimentin interactome and nanoscale distribution that accompany the defective cell shape, spreading and migration of metastasising cells. These results support the hypothesis that oncogenes can act through HDAC6 to regulate the vimentin binding of the cytoskeletal and cell-extracellular matrix adhesion components that contribute to the defective motility of metastasising cells.
|
|
| 43. |
- Fridberger, Anders, 1966-, et al.
(author)
-
Measuring hearing organ vibration patterns with confocal microscopy and optical flow
- 2004
-
In: Biophysical Journal. - : Cell Press. - 0006-3495 .- 1542-0086. ; 86:1, s. 535-543
-
Journal article (peer-reviewed)abstract
- A new method for visualizing vibrating structures is described. The system provides a means to capture very fast repeating events by relatively minor modi. cations to a standard confocal microscope. An acousto-optic modulator was inserted in the beam path, generating brief pulses of laser light. Images were formed by summing consecutive frames until every pixel of the resulting image had been exposed to a laser pulse. Images were analyzed using a new method for optical flow computation; it was validated through introducing artificial displacements in confocal images. Displacements in the range of 0.8 to 4 pixels were measured with 5% error or better. The lower limit for reliable motion detection was 20% of the pixel size. These methods were used for investigating the motion pattern of the vibrating hearing organ. In contrast to standard theory, we show that the organ of Corti possesses several degrees of freedom during sound-evoked vibration. Outer hair cells showed motion indicative of deformation. After acoustic overstimulation, supporting cells contracted. This slowly developing structural change was visualized during simultaneous intense sound stimulation and its speed measured with the optical flow technique.
|
|
| 44. |
- Gad, Annica K. B., et al.
(author)
-
Rho GTPases link cellular contractile force to the density and distribution of nanoscale adhesions
- 2012
-
In: The FASEB Journal. - : Wiley. - 0892-6638 .- 1530-6860. ; 26:6, s. 2374-2382
-
Journal article (peer-reviewed)abstract
- The ability of cells to adhere and to exert contractile forces governs their capacity to move within an organism. The cytoskeletal regulators of the Rho GTPase proteins are involved in control of the contractile forces of cells. To elucidate the basis of cell migration, we analyzed contractile forces and nanoscale adhesion-related particles in single cells expressing constitutively active variants of Rho GTPases by using traction-force microscopy and ultra-high-resolution stimulated emission depletion microscopy, respectively. RhoAV14 induced large increases in the contractile forces of single cells, with Rac1L61 and RhoDV26 having more moderate effects. The RhoAV14- and RhoDV26-induced forces showed similar spatial distributions and were accompanied by reduced or unaltered cell spreading. In contrast, the Rac1L61-induced force had different, scattered, force distributions that were linked to increased cell spreading. All three of these Rho GTPase activities caused a loss of thick stress fibers and focal adhesions and a more homogenous distribution of nanoscale adhesion-related particles over the ventral surface of the cells. Interestingly, only RhoAV14 increased the density of these particles. Our data suggest a Rac1-specific mode for cells to generate contractile forces. Importantly, increased density and a more homogenous distribution of these small adhesion-related particles promote cellular contractile forces.-Gad, A. K. B., Ronnlund, D., Spaar, A., Savchenko, A. A., Petranyi, G., Blom, H., Szekely, L., Widengren, J., Aspenstrom, P. Rho GTPases link cellular contractile force to the density and distribution of nanoscale adhesions.
|
|
| 45. |
- Guo, Xin, et al.
(author)
-
Achieving low-power single-wavelength-pair nanoscopy with NIR-II continuous-wave laser for multi-chromatic probes
- 2022
-
In: Nature Communications. - : Springer Nature. - 2041-1723. ; 13:1
-
Journal article (peer-reviewed)abstract
- The authors introduce stimulated-emission induced excitation depletion (STExD) nanoscopy using a single pair of low-power, near-infrared, continue-wave lasers. Emission of multichromatic probes is inhibited by cascade amplified depletion in lanthanide upconversion systems induced by manipulating their common sensitizer. Stimulated emission depletion (STED) microscopy is a powerful diffraction-unlimited technique for fluorescence imaging. Despite its rapid evolution, STED fundamentally suffers from high-intensity light illumination, sophisticated probe-defined laser schemes, and limited photon budget of the probes. Here, we demonstrate a versatile strategy, stimulated-emission induced excitation depletion (STExD), to deplete the emission of multi-chromatic probes using a single pair of low-power, near-infrared (NIR), continuous-wave (CW) lasers with fixed wavelengths. With the effect of cascade amplified depletion in lanthanide upconversion systems, we achieve emission inhibition for a wide range of emitters (e.g., Nd3+, Yb3+, Er3+, Ho3+, Pr3+, Eu3+, Tm3+, Gd3+, and Tb3+) by manipulating their common sensitizer, i.e., Nd3+ ions, using a 1064-nm laser. With NaYF4:Nd nanoparticles, we demonstrate an ultrahigh depletion efficiency of 99.3 +/- 0.3% for the 450 nm emission with a low saturation intensity of 23.8 +/- 0.4 kW cm(-2). We further demonstrate nanoscopic imaging with a series of multi-chromatic nanoprobes with a lateral resolution down to 34 nm, two-color STExD imaging, and subcellular imaging of the immunolabelled actin filaments. The strategy expounded here promotes single wavelength-pair nanoscopy for multi-chromatic probes and for multi-color imaging under low-intensity-level NIR-II CW laser depletion.
|
|
| 46. |
- Hassler, Kai, et al.
(author)
-
Dynamic disorder in horseradish peroxidase observed with total internal reflection fluorescence correlation spectroscopy
- 2007
-
In: Optics Express. - 1094-4087. ; 15:9, s. 5366-5375
-
Journal article (peer-reviewed)abstract
- This paper discusses the application of objective-type total internal reflection fluorescence correlation spectroscopy (TIR-FCS) to the study of the kinetics of immobilized horseradish peroxidase on a single molecule level. Objective-type TIR-FCS combines the advantages of FCS with TIRF microscopy in a way that allows for simultaneous ultra-sensitive spectroscopic measurements using a single-point detector and convenient localization of single molecules on a surface by means of parallel imaging.
|
|
| 47. |
|
|
| 48. |
- Hevekerl, Heike, 1982-, et al.
(author)
-
Dark states in ionic oligothiophene bioprobes-evidence from fluorescence correlation spectroscopy and dynamic light scattering
- 2014
-
In: Journal of Physical Chemistry B. - : American Chemical Society (ACS). - 1520-6106 .- 1520-5207. ; 118:22, s. 5924--5933
-
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.
|
|
| 49. |
- Hevekerl, Heike, 1982-, et al.
(author)
-
Determination of molecular stoichiometry without reference samples by analysing fluorescence blinking with and without excitation synchronization
-
Other publication (other academic/artistic)abstract
- Stoichiometry of molecular complexes plays a crucial role in biology. Moreover, for quantitative fluorescence studies, it is often useful to know the number of fluorophores labeled onto the molecules studied. In this work, we propose an approach to determine the number of independent fluorescence emitters on fluorescent molecules based on fluorescence blinking caused by photo-induced triplet state formation, photo-isomerization or charge transfer. The fluorescence blinking is measured under two different excitation regimes, on the same setup, and in one and the same sample. By comparing the fluorescence fluctuations under continuous excitation using Fluorescence Correlation Spectroscopy (FCS), when all the fluorophores are blinking independently of each other, with those occurring under square-pulsed excitation using Transient State (TRAST) spectroscopy, when all fluorophores are blinking in a synchronized manner, the number of fluorophores per molecule can be determined. No calibration sample is needed and the approach is independent of experimental conditions and of the specific environment of the molecules under study.The approach was experimentally validated by labeling double stranded DNA (dsDNA) with different concentrations of the intercalating dye YOYO-1 Iodide. The sample was then measured consecutively by TRAST and FCS and the number of fluorophores per molecule was calculated. The determined numbers were found to agree well with the number of fluorophores per dsDNA, as determined from FCS measurements using additional calibration samples.
|
|
| 50. |
- Hevekerl, Heike, et al.
(author)
-
Determination of molecular stoichiometry without reference samples by analyzing fluorescence blinking with and without excitation synchronization
- 2015
-
In: METHODS AND APPLICATIONS IN FLUORESCENCE. - : IOP Publishing. - 2050-6120. ; 3:2
-
Journal article (peer-reviewed)abstract
- Stoichiometry of molecular complexes plays a crucial role in biology. Moreover, for quantitative fluorescence studies, it is often useful to know the number of fluorophores labeled onto the molecules studied. In this work, we propose an approach to determine the number of independent fluorescence emitters on fluorescent molecules based on fluorescence blinking caused by photo-induced triplet state formation, photo-isomerization or charge transfer. The fluorescence blinking is measured under two different excitation regimes, on the same setup, and in one and the same sample. By comparing the fluorescence fluctuations under continuous excitation using Fluorescence Correlation Spectroscopy (FCS), when all the fluorophores are blinking independently of each other, with those occurring under square-pulsed excitation using Transient State (TRAST) spectroscopy, when all fluorophores are blinking in a synchronized manner, the number of fluorophores per molecule can be determined. No calibration sample is needed and the approach is independent of experimental conditions and of the specific environment of the molecules under study. The approach was experimentally validated by labeling double stranded DNA (dsDNA) with different concentrations of the intercalating dye YOYO-1 Iodide. The sample was then measured consecutively by TRAST and FCS and the number of fluorophores per molecule was calculated. The determined numbers were found to agree well with the number of fluorophores per dsDNA, as determined from FCS measurements using additional calibration samples.
|
|
