RESUMO
Modern fluorescence superresolution microscopes are capable of imaging living cells on the nanometer scale. One of those techniques is stimulated emission depletion (STED) which increases the microscope's resolution many times in the lateral and the axial directions. To achieve these high resolutions not only close to the coverslip but also at greater depths, the choice of objective becomes crucial. Oil immersion objectives have frequently been used for STED imaging since their high numerical aperture (NA) leads to high spatial resolutions. But during live-cell imaging, especially at great penetration depths, these objectives have a distinct disadvantage. The refractive index mismatch between the immersion oil and the usually aqueous embedding media of living specimens results in unwanted spherical aberrations. These aberrations distort the point spread functions (PSFs). Notably, during z- and 3D-STED imaging, the resolution increase along the optical axis is majorly hampered if at all possible. To overcome this limitation, we here use a water immersion objective in combination with a spatial light modulator for z-STED measurements of living samples at great depths. This compact design allows for switching between objectives without having to adapt the STED beam path and enables on the fly alterations of the STED PSF to correct for aberrations. Furthermore, we derive the influence of the NA on the axial STED resolution theoretically and experimentally. We show under live-cell imaging conditions that a water immersion objective leads to far superior results than an oil immersion objective at penetration depths of 5-180 µm.
Assuntos
Imageamento Tridimensional/instrumentação , Imageamento Tridimensional/métodos , Microscopia de Fluorescência/instrumentação , Microscopia de Fluorescência/métodos , Água , Artefatos , Células Cultivadas , Fibroblastos/citologia , Corantes Fluorescentes , Compostos de Ouro , Humanos , Nanopartículas Metálicas , Microscopia Confocal/instrumentação , Microscopia Confocal/métodos , Óleos , Poliestirenos , RefratometriaRESUMO
Fluorescence microscopy is rapidly turning into nanoscopy. Among the various nanoscopy methods, the STED/RESOLFT super-resolution family has recently been expanded to image even large fields of view within a few seconds. This advance relies on using light patterns featuring substantial arrays of intensity minima for discerning features by switching their fluorophores between 'on' and 'off' states of fluorescence. Here we show that splitting the light with a grating and recombining it in the focal plane of the objective lens renders arrays of minima with wavelength-independent periodicity. This colour-independent creation of periodic patterns facilitates coaligned on- and off-switching and readout with combinations chosen from a range of wavelengths. Applying up to three such periodic patterns on the switchable fluorescent proteins Dreiklang and rsCherryRev1.4, we demonstrate highly parallelized, multicolour RESOLFT nanoscopy in living cells for ~100 × 100 µm2 fields of view. Individual keratin filaments were rendered at a FWHM of ~60-80 nm, with effective resolution for the filaments of ~80-100 nm. We discuss the impact of novel image reconstruction algorithms featuring background elimination by spatial bandpass filtering, as well as strategies that incorporate complete image formation models.
RESUMO
A marker's dark triplet state is of great importance in fluorescence microscopy: It serves as a means to switch off fluorescent markers and is thus the enabling element for several super-resolution methods. On the other hand, intersystem-crossing to the electronic dark triplet state strongly reduces the fluorescence yield in conventional fluorescence microscopy. The ability to determine the kinetic parameters of transitions into the triplet state is thus of great importance and because fluorescence correlation spectroscopy (FCS) can be applied without disturbing the system under study, it is one of the preferred methods to do so. However, conventional FCS observations of triplet dynamics suffer from bias due to the spatially inhomogeneous irradiance profile of the excitation laser. Herein, we present a novel method to correct this bias and verify it by analyzing both Monte Carlo simulated and experimental data of the organic dye Rhodamine 110 in aqueous solution for both continuous-wave and pulsed excitation. Importantly, our approach can be readily generalized to most other FCS experiments that determine intensity dependent kinetic parameters.
Assuntos
Microscopia de Fluorescência/métodos , Espectrometria de Fluorescência/métodos , Corantes Fluorescentes , Microscopia Confocal/métodos , Modelos Teóricos , Método de Monte CarloRESUMO
In a stimulated emission depletion (STED) microscope the region in which fluorescence markers can emit spontaneously shrinks with continued STED beam action after a singular excitation event. This fact has been recently used to substantially improve the effective spatial resolution in STED nanoscopy using time-gated detection, pulsed excitation and continuous wave (CW) STED beams. We present a theoretical framework and experimental data that characterize the time evolution of the effective point-spread-function of a STED microscope and illustrate the physical basis, the benefits, and the limitations of time-gated detection both for CW and pulsed STED lasers. While gating hardly improves the effective resolution in the all-pulsed modality, in the CW-STED modality gating strongly suppresses low spatial frequencies in the image. Gated CW-STED nanoscopy is in essence limited (only) by the reduction of the signal that is associated with gating. Time-gated detection also reduces/suppresses the influence of local variations of the fluorescence lifetime on STED microscopy resolution.
Assuntos
Aumento da Imagem , Lasers , Microscopia de Fluorescência/métodos , Desenho de Equipamento , Análise de Falha de Equipamento , Microscopia de Fluorescência/instrumentaçãoRESUMO
Exploring the maximum spatial resolution achievable in far-field optical imaging, we show that applying solid immersion lenses (SIL) in stimulated emission depletion (STED) microscopy addresses single spins with a resolution down to 2.4 ± 0.3 nm and with a localization precision of 0.09 nm.
Assuntos
Aumento da Imagem/instrumentação , Lentes , Microscopia de Fluorescência/instrumentação , Nanotecnologia/instrumentação , Desenho de Equipamento , Análise de Falha de Equipamento , SoluçõesRESUMO
In recent years, the diffraction barrier in fluorescence imaging has been broken and optical nanoscopes now routinely image with resolutions of down to 20 nm, an improvement of more than 10 fold. Because this allows imaging much smaller features and because all super-resolution approaches trade off speed for spatial resolution, mechanical instabilities of the microscopes become a limiting factor. Here, we propose a fully data-driven statistical registration method for drift detection and drift correction for single marker switching (SMS) imaging schemes, including a guideline for parameter choice and quality checks of the drift analysis. The necessary assumptions about the drift are minimal, allowing a model-free approach, but more specific models can easily be integrated. We determine the resulting performance on standard SMS measurements and show that the drift determination can be routinely brought to the range of precision achievable by fiducial marker-tracking methods.
Assuntos
Artefatos , Marcadores Fiduciais , Aumento da Imagem/instrumentação , Interpretação de Imagem Assistida por Computador/métodos , Microscopia/instrumentação , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
We demonstrate three-dimensional (3D) super-resolution imaging of stochastically switched fluorophores distributed across whole cells. By evaluating the higher moments of the diffraction spot provided by a 4Pi detection scheme, single markers can be simultaneously localized with <10 nm precision in three dimensions in a layer of 650 nm thickness at an arbitrarily selected depth in the sample. By splitting the fluorescence light into orthogonal polarization states, our 4Pi setup also facilitates the 3D nanoscopy of multiple fluorophores. Offering a combination of multicolor recording, nanoscale resolution and extended axial depth, our method substantially advances the noninvasive 3D imaging of cells and of other transparent materials.
Assuntos
Corantes Fluorescentes , Microscopia de Fluorescência/métodos , Animais , Plaquetas/metabolismo , Células COS , Chlorocebus aethiops , Cor , Humanos , Imageamento Tridimensional , Microtúbulos/ultraestrutura , Nanotecnologia/métodos , Receptores de Fibrinogênio/análise , Processos Estocásticos , Tubulina (Proteína)/análise , Células VeroRESUMO
Current far-field fluorescence nanoscopes provide subdiffraction resolution by exploiting a mechanism of fluorescence inhibition. This mechanism is implemented such that features closer than the diffraction limit emit separately when simultaneously exposed to excitation light. A basic mechanism for such transient fluorescence inhibition is the depletion of the fluorophore ground state by transferring it (via a triplet) in a dark state, a mechanism which is workable in most standard dyes. Here we show that microscopy based on ground state depletion followed by individual molecule return (GSDIM) can effectively provide multicolor diffraction-unlimited resolution imaging of immunolabeled fixed and SNAP-tag labeled living cells. Implemented with standard labeling techniques, GSDIM is demonstrated to separate up to four different conventional fluorophores using just two detection channels and a single laser line. The method can be expanded to even more colors by choosing optimized dichroic mirrors and selecting marker molecules with negligible inhomogeneous emission broadening.
Assuntos
Corantes Fluorescentes/química , Microscopia de Fluorescência/métodos , Nanotecnologia/métodos , Animais , Linhagem Celular , Sobrevivência Celular , Cor , Microscopia de Força Atômica , Potoroidae , Espectrometria de FluorescênciaRESUMO
Synaptic vesicles need to be mobile to reach their release sites during synaptic activity. We investigated vesicle mobility throughout the synaptic vesicle cycle using both conventional and subdiffraction-resolution stimulated emission depletion fluorescence microscopy. Vesicle tracking revealed that recently endocytosed synaptic vesicles are highly mobile for a substantial time period after endocytosis. They later undergo a maturation process and integrate into vesicle clusters where they exhibit little mobility. Despite the differences in mobility, both recently endocytosed and mature vesicles are exchanged between synapses. Electrical stimulation does not seem to affect the mobility of the two types of vesicles. After exocytosis, the vesicle material is mobile in the plasma membrane, although the movement appears to be somewhat limited. Increasing the proportion of fused vesicles (by stimulating exocytosis while simultaneously blocking endocytosis) leads to substantially higher mobility. We conclude that both high- and low-mobility states are characteristic of synaptic vesicle movement.
Assuntos
Vesículas Sinápticas/metabolismo , Animais , Transporte Biológico , Membrana Celular/metabolismo , Eletricidade , Endocitose , Fusão de Membrana , Microscopia , RatosRESUMO
4Pi-microscopy doubles the aperture of the imaging system by coherent addition of the wavefronts for illumination and/or detection through opposing objective lenses. This improves the axial resolution 3-7 fold, but the raw data usually features ghost images which have to be removed by image reconstruction. This straightforward procedure is sometimes precluded by imperfect alignment of the instrument or a specimen with strong variations of its refractive index, because the image formation process now depends on the space-variant phase difference between the counter-propagating wavefronts. Here we present a computationally fast method of parametric blind deconvolution that allows for automatic and robust simultaneous estimation of both the object and the phase function in such cases. We verify the performance of our approach on both synthetic and real data. Because the method does not require a-priori knowledge of the phase function it is major step towards reliable 4Pi-imaging and automatic image restoration by non-expert users.
Assuntos
Algoritmos , Aumento da Imagem/métodos , Interpretação de Imagem Assistida por Computador/métodos , Imageamento Tridimensional/métodos , Microscopia/métodos , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
We compare the performance of video-rate Stimulated Emission Depletion (STED) and confocal microscopy in imaging the interior of living neurons. A lateral resolution of 65 nm is observed in STED movies of 28 frames per second, which is 4-fold higher in spatial resolution than in their confocal counterparts. STED microscopy, but not confocal microscopy, allows discrimination of single features at high spatial densities. Specific patterns of movement within the confined space of the axon are revealed in STED microscopy, while confocal imaging is limited to reporting gross motion. Further progress is to be expected, as we demonstrate that the use of continuous wave (CW) beams for excitation and STED is viable for video-rate STED recording of living neurons. Tentatively providing a larger photon flux, CW beams should facilitate extending fast STED imaging towards imaging fainter living samples.
Assuntos
Microscopia Confocal , Microscopia de Fluorescência/métodos , Nanotecnologia/métodos , Neurônios/fisiologia , Gravação em Vídeo/métodos , Animais , Axônios/fisiologia , Células Cultivadas , Fluorescência , Hipocampo/citologia , Hipocampo/fisiologia , Processamento de Imagem Assistida por Computador , Movimento (Física) , Neurônios/citologia , Fótons , Ratos , Vesículas Sinápticas/fisiologia , Fatores de TempoRESUMO
We propose a maximum a posteriori-based method that solves an important practical problem in the deconvolution of 4Pi images by simultaneously delivering an estimate of both the object and the unknown phase. The method was tested in simulations and on data from both test samples and biological specimen. It generates object estimates that are free from interference artifacts and reliably recovers arbitrary relative phases. Based on vectorial focusing theory, our theoretical analysis allowed for a simple and efficient implementation of the algorithm. Taking several 4Pi images at different relative phases of the interfering beams is shown to improve the robustness of the approach.
Assuntos
Processamento de Imagem Assistida por Computador/métodos , Microscopia/métodos , Algoritmos , Animais , Automação , Chlorocebus aethiops , Imagens de Fantasmas , Células VeroRESUMO
Cholesterol-mediated lipid interactions are thought to have a functional role in many membrane-associated processes such as signalling events. Although several experiments indicate their existence, lipid nanodomains ('rafts') remain controversial owing to the lack of suitable detection techniques in living cells. The controversy is reflected in their putative size of 5-200 nm, spanning the range between the extent of a protein complex and the resolution limit of optical microscopy. Here we demonstrate the ability of stimulated emission depletion (STED) far-field fluorescence nanoscopy to detect single diffusing (lipid) molecules in nanosized areas in the plasma membrane of living cells. Tuning of the probed area to spot sizes approximately 70-fold below the diffraction barrier reveals that unlike phosphoglycerolipids, sphingolipids and glycosylphosphatidylinositol-anchored proteins are transiently ( approximately 10-20 ms) trapped in cholesterol-mediated molecular complexes dwelling within <20-nm diameter areas. The non-invasive optical recording of molecular time traces and fluctuation data in tunable nanoscale domains is a powerful new approach to study the dynamics of biomolecules in living cells.
Assuntos
Membrana Celular/metabolismo , Lipídeos de Membrana/análise , Lipídeos de Membrana/metabolismo , Microscopia de Fluorescência/métodos , Nanotecnologia/métodos , Linhagem Celular , Membrana Celular/química , Sobrevivência Celular , Colesterol/análise , Colesterol/metabolismo , Difusão , Células Epiteliais/citologia , Etanolaminas/análise , Etanolaminas/metabolismo , Glicosilfosfatidilinositóis/metabolismo , Esfingomielinas/análise , Esfingomielinas/metabolismo , Fatores de TempoRESUMO
We propose and analyze a method for isotropic resolution in far-field fluorescence nanoscopy based on switching and mathematically localizing individual emitters. Under typical imaging conditions, the coherent detection of fluorescence light through two opposing high angle lenses strongly improves the 3D-resolution down to 5-10nm in all directions. Furthermore, we give a detailed analysis of the resolution of this and other single molecule switching based approaches using the Fisher information matrix.We verify the results by Monte-Carlo simulations of the imaging process and by applying a simple maximum-likelihood estimator for position determination.
Assuntos
Aumento da Imagem/métodos , Interpretação de Imagem Assistida por Computador/métodos , Imageamento Tridimensional/métodos , Microscopia de Fluorescência/métodos , Nanoestruturas/química , Nanoestruturas/ultraestrutura , Nanotecnologia/métodos , Anisotropia , Método de Monte CarloRESUMO
We combine far-field fluorescence nanoscopy through serialized recording of switchable emitters with polarization-sensitive fluorescence detection. In addition to imaging with nanoscale spatial resolution, this technique allows determination of the fluorescence anisotropy of each detected dipole emitter and thus an estimate of its rotational mobility. Sub-populations of fluorescent markers can thus be separated based on their interaction with the sample. We applied this new functional nanoscopy to imaging of living mammalian cells.
Assuntos
Biopolímeros/química , Biopolímeros/metabolismo , Microscopia de Fluorescência/instrumentação , Microscopia de Polarização/instrumentação , Técnicas de Sonda Molecular/instrumentação , Nanotecnologia/instrumentação , Desenho de Equipamento , Análise de Falha de Equipamento , Microscopia de Fluorescência/métodos , Microscopia de Polarização/métodos , Nanotecnologia/métodos , RotaçãoRESUMO
Fluorescent proteins that can be reversibly photoswitched between a fluorescent and a nonfluorescent state are important for innovative microscopy schemes, such as protein tracking, fluorescence resonance energy transfer imaging, sub-diffraction resolution microscopy and others. However, all available monomeric reversibly switchable fluorescent proteins (RSFPs) have similar properties and switching characteristics, thereby limiting their use. Here, we introduce two bright green fluorescent RSFPs, bsDronpa and Padron, generated by extensive mutagenesis of the RSFP Dronpa, with unique absorption and switching characteristics. Whereas bsDronpa features a broad absorption spectrum extending into the UV, Padron displays a switching behavior that is reversed to that of all green fluorescent RSFPs known to date. These two RSFPs enable live-cell fluorescence microscopy with multiple labels using a single detection color, because they can be distinguished by photoswitching. Furthermore, we demonstrate dual-color fluorescence microscopy with sub-diffraction resolution using bsDronpa and Dronpa whose emission maxima are separated by <20 nm.
Assuntos
Microscopia de Fluorescência/métodos , Nanotecnologia/métodos , Biotecnologia/métodos , Escherichia coli/metabolismo , Fluorescência , Imageamento Tridimensional , Luz , Proteínas Luminescentes/metabolismo , Microscopia Confocal , Mutagênese Sítio-Dirigida , Nanopartículas/química , Fotoquímica/métodos , Plasmídeos/metabolismo , Saccharomyces cerevisiae/metabolismo , Raios UltravioletaRESUMO
By combining the photoswitching and localization of individual fluorophores with spectroscopy on the single molecule level, we demonstrate simultaneous multicolor imaging with low crosstalk and down to 15 nm spatial resolution using only two detection color channels. The applicability of the method to biological specimens is demonstrated on mammalian cells. The combination of far-field fluorescence nanoscopy with the recording of a single switchable molecular species at a time opens up a new class of functional imaging techniques.
Assuntos
Corantes Fluorescentes/química , Microscopia/métodos , Animais , Linhagem Celular , Cor , Estrutura MolecularRESUMO
Reversibly switchable fluorescent proteins (RSFPs) are GFP-like proteins that may be repeatedly switched by irradiation with light from a fluorescent to a nonfluorescent state, and vice versa. They can be utilized as genetically encodable probes and bear large potential for a wide array of applications, in particular for new protein tracking schemes and subdiffraction resolution microscopy. However, the currently described monomeric RSFPs emit only blue-green or green fluorescence; the spectral window for their use is thus rather limited. Using a semirational engineering approach based on the crystal structure of the monomeric nonswitchable red fluorescent protein mCherry, we generated rsCherry and rsCherryRev. These two novel red fluorescent RSFPs exhibit fluorescence emission maxima at approximately 610 nm. They display antagonistic switching modes, i.e., in rsCherry irradiation with yellow light induces the off-to-on transition and blue light the on-to-off transition, whereas in rsCherryRev the effects of the switching wavelengths are reversed. We demonstrate time-lapse live-cell subdiffraction microscopy by imaging rsCherryRev targeted to the endoplasmic reticulum utilizing the switching and localization of single molecules.
Assuntos
Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Nanotecnologia , Engenharia de Proteínas/métodos , Animais , Biomarcadores/metabolismo , Bovinos , Cor , Escherichia coli/citologia , Escherichia coli/genética , Luz , Microscopia , Espectrometria de Fluorescência , Fatores de Tempo , Proteína Vermelha FluorescenteRESUMO
We undertake a comprehensive study of the inverse square root dependence of spatial resolution on the saturation factor in stimulated emission depletion (STED) microscopy and generalize it to account for various focal depletion patterns. We used an experimental platform featuring a high quality depletion pattern which results in operation close to the optimal optical performance. Its superior image brightness and uniform effective resolution <25 nm are evidenced by imaging both isolated and self-organized convectively assembled fluorescent beads. For relevant saturation values, the generalized square-root law is shown to predict the practical resolution with high accuracy.
Assuntos
Algoritmos , Aumento da Imagem/métodos , Interpretação de Imagem Assistida por Computador/métodos , Microscopia de Fluorescência/métodos , Modelos Teóricos , Simulação por ComputadorRESUMO
Tackling biological problems often involves the imaging and localization of cellular structures on the nanometer scale. Although optical super-resolution below 100 nm can be readily attained with stimulated emission depletion (STED) and photoswitching microscopy methods, attaining an axial resolution <100 nm with focused light generally required the use of two lenses in a 4Pi configuration or exceptionally bright photochromic fluorophores. Here, we describe a simple technical solution for 3D nanoscopy of fixed samples: biological specimens are fluorescently labeled, embedded in a polymer resin, cut into thin sections, and then imaged via STED microscopy with nanoscale resolution. This approach allows a 3D image reconstruction with a resolution <80 nm in all directions using available state-of-the art STED microscopes.