A set of monomeric near-infrared fluorescent proteins for multicolor imaging across scales

Bright monomeric near-infrared (NIR) fluorescent proteins (FPs) are in high demand as protein tags for multicolor microscopy and in vivo imaging. Here we apply rational design to engineer a complete set of monomeric NIR FPs, which are the brightest genetically encoded NIR probes. We demonstrate that the enhanced miRFP series of NIR FPs, which combine high effective brightness in mammalian cells and monomeric state, perform well in both nanometer-scale imaging with diffraction unlimited stimulated emission depletion (STED) microscopy and centimeter-scale imaging in mice. In STED we achieve -40nm resolution in live cells. In living mice we detect -10(5) fluorescent cells in deep tissues. Using spectrally distinct monomeric NIR FP variants, we perform two-color live-cell STED microscopy and two-color imaging in vivo. Having emission peaks from 670nm to 720nm, the next generation of miRFPs should become versatile NIR probes for multiplexed imaging across spatial scales in different modalities.Peer reviewe

r-Process Simulation and Heavy-Element Nucleosynthesis.

r-process, short for rapid neutron capture process, is a nucleosynthesis process taking place on short time scales. Rapid neutron captures produce less and less stable neutron-rich nuclei which in turn beta minus decays when the probability for beta decay is higher than the probability for neutron captures, upon which more neutrons are captured and the process repeats itself, creating r-process paths. Very neutron-rich heavy elements are the product of this process taking place at explosive astrophysical sites with high neutron flux. Simulations of r-processes are important for finding out the exact sites, something that is yet not known. To get more accurate simulation results leading to a better understanding of r-processes, the initial parameter dependence of the simulations is important to understand. This report discusses the dependence on three important initial parameters; temperature, density and electron fraction. Furthermore, the dependence on nuclear masses is covered, which is important since no exact model for nuclear masses exists for the neutron-rich nuclei involved. Finally, different stopping criteria are simulated, representing different physical environments in which r-processes may occur. Results from the simulations, carried out using r-Java 2.0, show that r-process simulations are sensitive to all parameters discussed; further research can tell to which extent. A better understanding of the dependence on the parameters will hopefully extend our knowledge of r-processes and where in the universe they occur.r-process, rapid neutron capture process, är en snabb nukleosyntesprocess. Snabba neutroninfångningar producerar allt mer instabila neutronrika atomkärnor som slutligen betaminussönderfaller när sannolikheten för betasönderfall blir högre än sannolikheten för en ny neutroninfångning. Därefeter fångas fler neutroner och processen upprepar sig själv i r-processkedjor. Väldigt neutronrika tunga ämnen bildas under denna process som kräver explosiva astrofysikaliska platser med höga neutronflux. Då det ännu är okänt exakt var dessa platser är så hjälper r-processsimulationer att förstå detta. För att förbättra simuleringsresultaten och därmed förståelsen av r-processer så är det viktigt att förstå hur initiala parametrar påverkar simuleringarna. Temperatur, densitet och förhållandet mellan fria elektroner och nukleoner är tre parametrar som denna rapport behandlar. Påverkan av kärrnmassor diskuteras också, vilket är viktigt då ingen exakt modell för kärnmassor existerar. Slutligen behandlas även olika stoppkriterium vilket representerar olika fysikaliska miljöer där r-processer eventuellt förekommer. Resultat från simuleringar, gjorda i r-Java 2.0, visar på att r-processimuleringar är känsliga för alla parametrar som har behandlats men där vidare forskning får visa till vilken grad. En bättre förståelse för hur simuleringarna påverkas av parametrar kommer förhoppningsvis öka förståelsen för r-processer och var i universum de förekommer

Automating STED microscopy for functional and structural live-cell imaging

Optical microscopy imaging methods are today invaluable tools for studies in life sciences as they allow visualization of biological systems, tissues, cells, and sub-cellular compartments from millimetres down to nanometres. The invention and development of nanoscopy in the past 20 years has pushed fluorescence microscopy down to the nanoscale, reaching beyond the natural diffraction limit of light that does not allow focusing of visible light below sizes of around 200 nm, and into the realm of what was previously only thought possible with electron microscopy. The superior spatial resolution does however come at a price, including complex sample preparation, prolonged recording times, increased illumination doses, and limited fields of view. Stimulated emission depletion (STED) microscopy is one of the techniques that can deliver nanoscale resolution in a range of biological systems, but with all the above-mentioned costs. However, with the right sample the technique can deliver single nanometre spatial resolution, and with the right considerations live-cell imaging is more than possible. In this thesis I present the development of a flexible STED microscope with methodological advancements in a range of directions that aim at facilitating the use of STED microscopy in life sciences and optimising the information extraction from the image data. The developments firstly focused on automation of the data acquisition, to allow the recording of imaging data both with a higher throughput and correlated with fast dynamic processes. I also implemented improved image analysis, both in terms of high throughput and precision as well as in connection with the data acquisition. Furthermore, I worked on control software development, with novel strategies to unify the control software of microscopes and to allow development and implementation of novel acquisition schemes. I also utilized novel fluorophores, to improve live-cell and multicolour possibilities and allow a wider range of applications in STED microscopy. Lastly, I developed a novel concept that takes advantage of STED. Additionally, I present applications of the microscope and image analysis in diverse biological samples such as mammalian cells, tissue sections, and bacteria. Altogether, this work aims at presenting new tools for an imaging technique that is already well-established, to contribute to further development, facilitation of novel experiments, and expansion of the range of applications.Ljusmikroskopi är idag ovärdeligt för forskare inom livsvetenskap för att visualisera och studera biologiska system, vävnader, celler, och beståndsdelar av celler på längdskalor från millimeter ner till nanometer. Uppfinnandet av nanoskopi och dess utveckling de senaste decennenierna har möjliggjort för fluorescensmikroskopi att nå den undre gränsen som tidigare var inom räckhåll endast med elektonmikroskopi. Anledningen till detta är diffraktionsgränsen som dikterar hur väl man kan fokusera elektromagnetisk strålning, och som i praktiken inte tillåter fokusering av synligt ljus till områden mindre än 200 nm i diameter. Nanoskopins överlägsna upplösning kommer däremot inte gratis, utan komplicerad förberedning av prover, förlängda inspelningstider, högre belysningsintensiteter, och begränsade synfält är några av de extra svårigheter som man måste ta hänsyn till. Stimulated emission depletion (STED) mikroskopi är en av dessa metoder som kan avbilda prover från biologiska system med nanometerupplösning, men med alla svårigheter som nämnts ovan. Men med rätt prov så kan metoden leverera en upplösning under 10 nm, och med rätt hänsyn tagen till cellöverlevnad så kan levande celler avbildas.  I denna avhandling presenterar jag utvecklingen av ett STED-mikroskop med en rad tekniska framsteg som fokuserar på att underlätta användningen av STED-mikroskopi i livsvetenskap och optimera utvinningen av information från bilderna. Utvecklingen har fokuserat på automatisering, med möjligheten att samla in bilddata med både högre genomströmning och i samband med snabba processer i de biologiska systemen, men också förbättrad bildanalys både i form av högre genomströmning och precision samt i samband med datainsamlingen. Jag har också utvecklat kontrollmjukvara med nya strategier för att tillåta fortsatt utveckling och implementering av nya datainsamlingssätt för liknande mikroskop. Dessutom har jag utnyttjat nya fluorescenta molekyler för att förbättra möjligheten att använda tekniken i levande celler och med fler inspelningskanaler samt tillåta fler tillämpningssområden. Slutligen har jag utvecklat ett nytt koncept som tar hjälp av STED, och tillämpat mikroskopet och bildanalys på diverse biologiska system såsom däggdjursceller, vävnader och bakterier. Sammantaget siktar mitt arbete på att presentera nya verktyg för en redan etablerad mikroskopiteknik, för att bidra till fortsatt utveckling, underlätta nya typer av experiment och utöka bredden av tillämpningsområden.

Automating STED microscopy for functional and structural live-cell imaging

Optical microscopy imaging methods are today invaluable tools for studies in life sciences as they allow visualization of biological systems, tissues, cells, and sub-cellular compartments from millimetres down to nanometres. The invention and development of nanoscopy in the past 20 years has pushed fluorescence microscopy down to the nanoscale, reaching beyond the natural diffraction limit of light that does not allow focusing of visible light below sizes of around 200 nm, and into the realm of what was previously only thought possible with electron microscopy. The superior spatial resolution does however come at a price, including complex sample preparation, prolonged recording times, increased illumination doses, and limited fields of view. Stimulated emission depletion (STED) microscopy is one of the techniques that can deliver nanoscale resolution in a range of biological systems, but with all the above-mentioned costs. However, with the right sample the technique can deliver single nanometre spatial resolution, and with the right considerations live-cell imaging is more than possible. In this thesis I present the development of a flexible STED microscope with methodological advancements in a range of directions that aim at facilitating the use of STED microscopy in life sciences and optimising the information extraction from the image data. The developments firstly focused on automation of the data acquisition, to allow the recording of imaging data both with a higher throughput and correlated with fast dynamic processes. I also implemented improved image analysis, both in terms of high throughput and precision as well as in connection with the data acquisition. Furthermore, I worked on control software development, with novel strategies to unify the control software of microscopes and to allow development and implementation of novel acquisition schemes. I also utilized novel fluorophores, to improve live-cell and multicolour possibilities and allow a wider range of applications in STED microscopy. Lastly, I developed a novel concept that takes advantage of STED. Additionally, I present applications of the microscope and image analysis in diverse biological samples such as mammalian cells, tissue sections, and bacteria. Altogether, this work aims at presenting new tools for an imaging technique that is already well-established, to contribute to further development, facilitation of novel experiments, and expansion of the range of applications.Ljusmikroskopi är idag ovärdeligt för forskare inom livsvetenskap för att visualisera och studera biologiska system, vävnader, celler, och beståndsdelar av celler på längdskalor från millimeter ner till nanometer. Uppfinnandet av nanoskopi och dess utveckling de senaste decennenierna har möjliggjort för fluorescensmikroskopi att nå den undre gränsen som tidigare var inom räckhåll endast med elektonmikroskopi. Anledningen till detta är diffraktionsgränsen som dikterar hur väl man kan fokusera elektromagnetisk strålning, och som i praktiken inte tillåter fokusering av synligt ljus till områden mindre än 200 nm i diameter. Nanoskopins överlägsna upplösning kommer däremot inte gratis, utan komplicerad förberedning av prover, förlängda inspelningstider, högre belysningsintensiteter, och begränsade synfält är några av de extra svårigheter som man måste ta hänsyn till. Stimulated emission depletion (STED) mikroskopi är en av dessa metoder som kan avbilda prover från biologiska system med nanometerupplösning, men med alla svårigheter som nämnts ovan. Men med rätt prov så kan metoden leverera en upplösning under 10 nm, och med rätt hänsyn tagen till cellöverlevnad så kan levande celler avbildas.  I denna avhandling presenterar jag utvecklingen av ett STED-mikroskop med en rad tekniska framsteg som fokuserar på att underlätta användningen av STED-mikroskopi i livsvetenskap och optimera utvinningen av information från bilderna. Utvecklingen har fokuserat på automatisering, med möjligheten att samla in bilddata med både högre genomströmning och i samband med snabba processer i de biologiska systemen, men också förbättrad bildanalys både i form av högre genomströmning och precision samt i samband med datainsamlingen. Jag har också utvecklat kontrollmjukvara med nya strategier för att tillåta fortsatt utveckling och implementering av nya datainsamlingssätt för liknande mikroskop. Dessutom har jag utnyttjat nya fluorescenta molekyler för att förbättra möjligheten att använda tekniken i levande celler och med fler inspelningskanaler samt tillåta fler tillämpningssområden. Slutligen har jag utvecklat ett nytt koncept som tar hjälp av STED, och tillämpat mikroskopet och bildanalys på diverse biologiska system såsom däggdjursceller, vävnader och bakterier. Sammantaget siktar mitt arbete på att presentera nya verktyg för en redan etablerad mikroskopiteknik, för att bidra till fortsatt utveckling, underlätta nya typer av experiment och utöka bredden av tillämpningsområden.

Far Red‐Shifted CdTe Quantum Dots for Multicolour Stimulated Emission Depletion Nanoscopy

Stimulated emission depletion (STED) nanoscopy is a widely used nanoscopy technique. Two-colour STED imaging in fixed and living cells is standardised today utilising both fluorescent dyes and fluorescent proteins. Solutions to image additional colours have been demonstrated using spectral unmixing, photobleaching steps, or long-Stokes-shift dyes. However, these approaches often compromise speed, spatial resolution, and image quality, and increase complexity. Here, we present multicolour STED nanoscopy with far red-shifted semiconductor CdTe quantum dots (QDs). STED imaging of the QDs is optimized to minimize blinking effects and maximize the number of detected photons. The far-red and compact emission spectra of the investigated QDs free spectral space for the simultaneous use of fluorescent dyes, enabling straightforward three-colour STED imaging with a single depletion beam. We use our method to study the internalization of QDs in cells, opening up the way for future super-resolution studies of particle uptake and internalization.QC 20230607</p

Extending fluorescence anisotropy to large complexes using reversibly switchable proteins

The formation of macromolecular complexes can be measured by detection of changes in rotational mobility using time-resolved fluorescence anisotropy. However, this method is limited to relatively small molecules (~0.1–30 kDa), excluding the majority of the human proteome and its complexes. We describe selective time-resolved anisotropy with reversibly switchable states (STARSS), which overcomes this limitation and extends the observable mass range by more than three orders of magnitude. STARSS is based on long-lived reversible molecular transitions of switchable fluorescent proteins to resolve the relatively slow rotational diffusivity of large complexes. We used STARSS to probe the rotational mobility of several molecular complexes in cells, including chromatin, the retroviral Gag lattice and activity-regulated cytoskeleton-associated protein oligomers. Because STARSS can probe arbitrarily large structures, it is generally applicable to the entire human proteome.QC 20230602</p

Blue-shift photoconversion of near-infrared fluorescent proteins for labeling and tracking in living cells and organisms

Photolabeling of intracellular molecules is an invaluable approach to studying various dynamic processes in living cells with high spatiotemporal precision. Among fluorescent proteins, photoconvertible mechanisms and their products are in the visible spectrum (400–650 nm), limiting their in vivo and multiplexed applications. Here we report the phenomenon of near-infrared to far-red photoconversion in the miRFP family of near infrared fluorescent proteins engineered from bacterial phytochromes. This photoconversion is induced by near-infrared light through a non-linear process, further allowing optical sectioning. Photoconverted miRFP species emit fluorescence at 650 nm enabling photolabeling entirely performed in the near-infrared range. We use miRFPs as photoconvertible fluorescent probes to track organelles in live cells and in vivo, both with conventional and super-resolution microscopy. The spectral properties of miRFPs complement those of GFP-like photoconvertible proteins, allowing strategies for photoconversion and spectral multiplexed applications.Peer reviewe

Blue-shift photoconversion of near-infrared fluorescent proteins for labeling and tracking in living cells and organisms

Abstract Photolabeling of intracellular molecules is an invaluable approach to studying various dynamic processes in living cells with high spatiotemporal precision. Among fluorescent proteins, photoconvertible mechanisms and their products are in the visible spectrum (400–650 nm), limiting their in vivo and multiplexed applications. Here we report the phenomenon of near-infrared to far-red photoconversion in the miRFP family of near infrared fluorescent proteins engineered from bacterial phytochromes. This photoconversion is induced by near-infrared light through a non-linear process, further allowing optical sectioning. Photoconverted miRFP species emit fluorescence at 650 nm enabling photolabeling entirely performed in the near-infrared range. We use miRFPs as photoconvertible fluorescent probes to track organelles in live cells and in vivo, both with conventional and super-resolution microscopy. The spectral properties of miRFPs complement those of GFP-like photoconvertible proteins, allowing strategies for photoconversion and spectral multiplexed applications