| 1. |
- Bergstrand, Jan, et al.
(författare)
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Fast, streamlined fluorescence nanoscopy resolves rearrangements of SNARE and cargo proteins in platelets co-incubated with cancer cells
- 2022
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Ingår i: Journal of Nanobiotechnology. - : Springer Nature. - 1477-3155. ; 20:1
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Tidskriftsartikel (refereegranskat)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.
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| 2. |
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| 3. |
- Jeong, Sejoo, et al.
(författare)
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Pushing the Resolution Limit of Stimulated Emission Depletion Optical Nanoscopy
- 2024
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Ingår i: International Journal of Molecular Sciences. - : MDPI AG. - 1661-6596 .- 1422-0067. ; 25:1
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Forskningsöversikt (refereegranskat)abstract
- Optical nanoscopy, also known as super-resolution optical microscopy, has provided scientists with the means to surpass the diffraction limit of light microscopy and attain new insights into nanoscopic structures and processes that were previously inaccessible. In recent decades, numerous studies have endeavored to enhance super-resolution microscopy in terms of its spatial (lateral) resolution, axial resolution, and temporal resolution. In this review, we discuss recent efforts to push the resolution limit of stimulated emission depletion (STED) optical nanoscopy across multiple dimensions, including lateral resolution, axial resolution, temporal resolution, and labeling precision. We introduce promising techniques and methodologies building on the STED concept that have emerged in the field, such as MINSTED, isotropic STED, and event-triggered STED, and evaluate their respective strengths and limitations. Moreover, we discuss trade-off relationships that exist in far-field optical microscopy and how they come about in STED optical nanoscopy. By examining the latest developments addressing these aspects, we aim to provide an updated overview of the current state of STED nanoscopy and its potential for future research.
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| 4. |
- Sandberg, Elin, et al.
(författare)
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Local monitoring of photosensitizer transient states provides feedback for enhanced efficiency and targeting selectivity in photodynamic therapy
- 2023
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Ingår i: Scientific Reports. - : Springer Nature. - 2045-2322. ; 13:1, s. 16829-
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Tidskriftsartikel (refereegranskat)abstract
- Photodynamic therapy (PDT) fundamentally relies on local generation of PDT precursor states in added photosensitizers (PS), particularly triplet and photo-radical states. Monitoring these states in situ can provide important feedback but is difficult in practice. The states are strongly influenced by local oxygenation, pH and redox conditions, often varying significantly at PDT treatment sites. To overcome this problem, we followed local PDT precursor state populations of PS compounds, via their fluorescence intensity response to systematically varied excitation light modulation. Thereby, we could demonstrate local monitoring of PDT precursor states of methylene blue (MB) and IRdye700DX (IR700), and determined their transitions rates under different oxygenation, pH and redox conditions. By fiber-optics, using one fiber for both excitation and fluorescence detection, the triplet and photo-radical state kinetics of locally applied MB and IR700 could then be monitored in a tissue sample. Finally, potassium iodide and ascorbate were added as possible PDT adjuvants, enhancing intersystem crossing and photoreduction, respectively, and their effects on the PDT precursor states of MB and IR700 could be locally monitored. Taken together, the presented procedure overcomes current methodological limitations and can offer feedback, guiding both excitation and PDT adjuvant application, and thereby more efficient and targeted PDT treatments.
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| 5. |
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| 6. |
- Tabusi, Mahebali, et al.
(författare)
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Neuronal death in pneumococcal meningitis is triggered by pneumolysin and RrgA interactions with beta-actin
- 2021
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Ingår i: PLoS Pathogens. - : Public Library of Science (PLoS). - 1553-7366 .- 1553-7374. ; 17:3
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Tidskriftsartikel (refereegranskat)abstract
- Neuronal damage is a major consequence of bacterial meningitis, but little is known about mechanisms of bacterial interaction with neurons leading to neuronal cell death. Streptococcus pneumoniae (pneumococcus) is a leading cause of bacterial meningitis and many survivors develop neurological sequelae after the acute infection has resolved, possibly due to neuronal damage. Here, we studied mechanisms for pneumococcal interactions with neurons. Using human primary neurons, pull-down experiments and mass spectrometry, we show that pneumococci interact with the cytoskeleton protein beta-actin through the pilus-1 adhesin RrgA and the cytotoxin pneumolysin (Ply), thereby promoting adhesion and invasion of neurons, and neuronal death. Using our bacteremia-derived meningitis mouse model, we observe that RrgA- and Ply-expressing pneumococci co-localize with neuronal beta-actin. Using purified proteins, we show that Ply, through its cholesterol-binding domain 4, interacts with the neuronal plasma membrane, thereby increasing the exposure on the outer surface of beta-actin filaments, leading to more beta-actin binding sites available for RrgA binding, and thus enhanced pneumococcal interactions with neurons. Pneumococcal infection promotes neuronal death possibly due to increased intracellular Ca2+ levels depending on presence of Ply, as well as on actin cytoskeleton disassembly. STED super-resolution microscopy showed disruption of beta-actin filaments in neurons infected with pneumococci expressing RrgA and Ply. Finally, neuronal death caused by pneumococcal infection could be inhibited using antibodies against beta-actin. The generated data potentially helps explaining mechanisms for why pneumococci frequently cause neurological sequelae. Author summary Neuronal damage is a major consequence of meningitis. Streptococcus pneumoniae (pneumococcus) is the leading etiological cause of bacterial meningitis, yet how pneumococci interact with neurons and cause neuronal death is poorly understood. Using human neurons in vitro and our established bacteremia-derived meningitis mouse model in vivo, we found that pneumococci use the pilus-1 adhesin RrgA and the cytotoxin pneumolysin (Ply) to interact with neuronal beta-actin expressed on the plasma membrane. Also, we demonstrate that Ply interaction with the neuronal plasma membrane increase the exposure of beta-actin on the neuronal plasma membrane, allowing more pneumococci to adhere to neurons through RrgA-beta-actin interaction. Moreover, neurons infected with RrgA- and Ply-expressing pneumococci showed increased intracellular Ca2+ levels and disruption of beta-actin filaments, possibly leading to neuronal death. Importantly, by blocking pneumococcal-beta-actin interaction using antibodies, we could reduce neuronal cell death after pneumococcal infection.
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| 7. |
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| 8. |
- Venugopal Srambickal, Chinmaya, et al.
(författare)
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Cumulative effects of photobleaching in volumetric STED imaging-artefacts and possible benefits
- 2021
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Ingår i: Methods and Applications in Fluorescence. - : Institute of Physics (IOP). - 2050-6120. ; 9:1
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Tidskriftsartikel (refereegranskat)abstract
- In stimulated emission depletion (STED) imaging, the excitation and depletion laser beams extend well beyond the focal plane in the imaged sample. We investigated how photobleaching resulting from this irradiation can affect STED images, by acquiring 3D images of fluorescent polystyrene beads using a 2D STED microscope, and applying different Z pixel sizes, scanning speeds, resulting in different laser light doses. While higher STED beam irradiances can increase the spatial resolution, they can also significantly increase photobleaching and thereby reduce signal-to-background levels. In 2D STED imaging, based on a single scan within the focal plane, scan parameters can often be selected to avoid photobleaching effects. Upon 3D optical sectioning experiments however, using the same scan parameters, additional cumulative effects of photobleaching may appear, due to the extension of the excitation and depletion laser beams beyond the focal planes being scanned. Apart from a reduction in signal-to-background levels, such photobleaching can lead to an apparent shift of the axial localization of the objects in the images, but also to an increased resolution in the axial dimension. These findings, confirmed by simulations based on a simplified model for photobleaching, suggests some caution in volumetric STED imaging experiments, but also a possibility for enhanced axial resolution in such experiments.
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| 9. |
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| 10. |
- Venugopal Srambickal, Chinmaya
(författare)
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Super-resolution microscopy – photophysical implications and applications
- 2024
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Unparalleled specificity and high sensitivity have made fluorescence microscopy an indispensable tool for life sciences. Both these aspects come from the use of light-emitting fluorophores, labeled to molecules and structures of interest. The diffraction-limited, maximum achievable resolution of traditional microscopes roughly corresponds to half the wavelength of the light used for observation. In the last decades, however, super-resolution microscopy (SRM) has been developed, which provide spatial information far beyond this diffraction limit. While different SRM techniques use different principles to achieve super resolution, most of these techniques rely on selectively switching the fluorophore emission on and off, within a diffraction-limited volume. Upon excitation, fluorophores can undergo transitions into transient dark states or become permanently photobleached. While reversible transitions may be used to achieve super resolution, they also reduce the overall emission. Characterization and modeling of photophysical dark state transitions are thus important, since they can both provide a basis for, as well as negatively affect the performance of SRM. Nevertheless, SRM has already proven valuable in biological and biomedical research, where the enhanced resolution allows for improved understanding of basic molecular mechanisms in cells and opens for future diagnostic opportunities.This thesis presents two applications of SRM. In paper I, we used STED (stimulated emission depletion) SRM to image the disruption of beta-actin filaments in neurons infected with Streptococcus pneumoniae, suggesting a possible mechanism for neuronal death in bacterial meningitis. In paper II, the nanoscale distribution patterns of six different platelet proteins were imaged with STED to find activation-specific protein rearrangements upon co-incubation of the platelets with cancer cells. Streamlined image acquisition, analysis and classification methods were also developed, opening prospects for SRM-based minimally invasive cancer diagnosis.Photophysical transitions of fluorophores and their implications on SRM were also studied. Cumulative photobleaching in volumetric STED imaging and how it can affect the recorded STED images was studied experimentally and verified by simulations in paper III. The effects of fluorophore transitions into transient dark states in super-resolution MINFLUX (minimal photon fluxes) were studied in paper IV. In this work, photophysical rate parameters of photo-switchable near-infrared (NIR) cyanine dyes were measured using TRAST (transient state) spectroscopy. Time evolutions of their photophysical transitions during MINFLUX localizations were then simulated, showing that fluorophore blinking can be a source of localization errors. However, from the acquired knowledge of the transient states and how they influence the localization in MINFLUX experiments, it was possible to adapt sample and excitation conditions and demonstrate MINFLUX imaging in the NIR. Thereby, it was shown that more weakly emitting and blinking NIR fluorophores can still be used in MINFLUX.
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