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1.
Nat Methods ; 21(5): 882-888, 2024 May.
Artículo en Inglés | MEDLINE | ID: mdl-38395993

RESUMEN

Light-sheet fluorescence microscopy is an invaluable tool for four-dimensional biological imaging of multicellular systems due to the rapid volumetric imaging and minimal illumination dosage. However, it is challenging to retrieve fine subcellular information, especially in living cells, due to the width of the sheet of light (>1 µm). Here, using reversibly switchable fluorescent proteins (RSFPs) and a periodic light pattern for photoswitching, we demonstrate a super-resolution imaging method for rapid volumetric imaging of subcellular structures called multi-sheet RESOLFT. Multiple emission-sheets with a width that is far below the diffraction limit are created in parallel increasing recording speed (1-2 Hz) to provide super-sectioning ability (<100 nm). Our technology is compatible with various RSFPs due to its minimal requirement in the number of switching cycles and can be used to study a plethora of cellular structures. We track cellular processes such as cell division, actin motion and the dynamics of virus-like particles in three dimensions.


Asunto(s)
Microscopía Fluorescente , Microscopía Fluorescente/métodos , Humanos , Proteínas Luminiscentes/química , Proteínas Luminiscentes/metabolismo , Animales , Actinas/metabolismo , Imagenología Tridimensional/métodos , Proteínas Fluorescentes Verdes/metabolismo , Proteínas Fluorescentes Verdes/química , Células HeLa
2.
Nat Biotechnol ; 41(4): 552-559, 2023 04.
Artículo en Inglés | MEDLINE | ID: mdl-36217028

RESUMEN

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.


Asunto(s)
Proteoma , Humanos , Polarización de Fluorescencia/métodos , Espectrometría de Fluorescencia/métodos
3.
Nat Methods ; 19(4): 461-469, 2022 04.
Artículo en Inglés | MEDLINE | ID: mdl-35314838

RESUMEN

The promise of single-objective light-sheet microscopy is to combine the convenience of standard single-objective microscopes with the speed, coverage, resolution and gentleness of light-sheet microscopes. We present DaXi, a single-objective light-sheet microscope design based on oblique plane illumination that achieves: (1) a wider field of view and high-resolution imaging via a custom remote focusing objective; (2) fast volumetric imaging over larger volumes without compromising image quality or necessitating tiled acquisition; (3) fuller image coverage for large samples via multi-view imaging and (4) higher throughput multi-well imaging via remote coverslip placement. Our instrument achieves a resolution of 450 nm laterally and 2 µm axially over an imaging volume of 3,000 × 800 × 300 µm. We demonstrate the speed, field of view, resolution and versatility of our instrument by imaging various systems, including Drosophila egg chamber development, zebrafish whole-brain activity and zebrafish embryonic development - up to nine embryos at a time.


Asunto(s)
Encéfalo , Pez Cebra , Animales , Encéfalo/diagnóstico por imagen , Drosophila , Desarrollo Embrionario , Microscopía Fluorescente/métodos
4.
Biomed Opt Express ; 13(11): 5616-5627, 2022 Nov 01.
Artículo en Inglés | MEDLINE | ID: mdl-36733723

RESUMEN

Fast volumetric imaging of large fluorescent samples with high-resolution is required for many biological applications. Oblique plane microscopy (OPM) provides high spatiotemporal resolution, but the field of view is typically limited by its optical train and the pixel number of the camera. Mechanically scanning the sample or decreasing the overall magnification of the imaging system can partially address this challenge, albeit by reducing the volumetric imaging speed or spatial resolution, respectively. Here, we introduce a novel dual-axis scan unit for OPM that facilitates rapid and high-resolution volumetric imaging throughout a volume of 800 × 500 × 200 microns. This enables us to perform volumetric imaging of cell monolayers, spheroids and zebrafish embryos with subcellular resolution. Furthermore, we combined this microscope with a multi-perspective projection imaging technique that increases the volumetric interrogation rate to more than 10 Hz. This allows us to rapidly probe a large field of view in a dimensionality reduced format, identify features of interest, and volumetrically image these regions with high spatiotemporal resolution.

5.
Nat Methods ; 18(7): 829-834, 2021 07.
Artículo en Inglés | MEDLINE | ID: mdl-34183831

RESUMEN

We introduce a cost-effective and easily implementable scan unit that converts any camera-based microscope with optical sectioning capability into a multi-angle projection imaging system. Projection imaging reduces data overhead and accelerates imaging by a factor of >100, while also allowing users to readily view biological phenomena of interest from multiple perspectives on the fly. By rapidly interrogating the sample from just two perspectives, our method also enables real-time stereoscopic imaging and three-dimensional particle localization. We demonstrate projection imaging with spinning disk confocal, lattice light-sheet, multidirectional illumination light-sheet and oblique plane microscopes on specimens that range from organelles in single cells to the vasculature of a zebrafish embryo. Furthermore, we leverage our projection method to rapidly image cancer cell morphodynamics and calcium signaling in cultured neurons at rates up to 119 Hz as well as to simultaneously image orthogonal views of a beating embryonic zebrafish heart.


Asunto(s)
Procesamiento de Imagen Asistido por Computador/métodos , Microscopía Confocal/instrumentación , Microscopía Confocal/métodos , Animales , Colon/citología , Embrión no Mamífero/citología , Femenino , Corazón/diagnóstico por imagen , Corazón/embriología , Humanos , Imagenología Tridimensional , Masculino , Ratones , Ratones Transgénicos , Neuronas/citología , Ratas Sprague-Dawley , Esferoides Celulares/patología , Pez Cebra/embriología
6.
Genetics ; 217(4)2021 04 15.
Artículo en Inglés | MEDLINE | ID: mdl-33693628

RESUMEN

We create and share a new red fluorophore, along with a set of strains, reagents and protocols, to make it faster and easier to label endogenous Caenorhabditis elegans proteins with fluorescent tags. CRISPR-mediated fluorescent labeling of C. elegans proteins is an invaluable tool, but it is much more difficult to insert fluorophore-size DNA segments than it is to make small gene edits. In principle, high-affinity asymmetrically split fluorescent proteins solve this problem in C. elegans: the small fragment can quickly and easily be fused to almost any protein of interest, and can be detected wherever the large fragment is expressed and complemented. However, there is currently only one available strain stably expressing the large fragment of a split fluorescent protein, restricting this solution to a single tissue (the germline) in the highly autofluorescent green channel. No available C. elegans lines express unbound large fragments of split red fluorescent proteins, and even state-of-the-art split red fluorescent proteins are dim compared to the canonical split-sfGFP protein. In this study, we engineer a bright, high-affinity new split red fluorophore, split-wrmScarlet. We generate transgenic C. elegans lines to allow easy single-color labeling in muscle or germline cells and dual-color labeling in somatic cells. We also describe a novel expression strategy for the germline, where traditional expression strategies struggle. We validate these strains by targeting split-wrmScarlet to several genes whose products label distinct organelles, and we provide a protocol for easy, cloning-free CRISPR/Cas9 editing. As the collection of split-FP strains for labeling in different tissues or organelles expands, we will post updates at doi.org/10.5281/zenodo.3993663.


Asunto(s)
Proteínas de Caenorhabditis elegans/metabolismo , Proteínas Fluorescentes Verdes/metabolismo , Proteínas Luminiscentes/metabolismo , Animales , Caenorhabditis elegans , Proteínas de Caenorhabditis elegans/genética , Ingeniería Genética/métodos , Células Germinativas/citología , Células Germinativas/metabolismo , Proteínas Fluorescentes Verdes/genética , Proteínas Luminiscentes/genética , Transporte de Proteínas , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteína Fluorescente Roja
7.
Elife ; 92020 11 12.
Artículo en Inglés | MEDLINE | ID: mdl-33179596

RESUMEN

We present an oblique plane microscope (OPM) that uses a bespoke glass-tipped tertiary objective to improve the resolution, field of view, and usability over previous variants. Owing to its high numerical aperture optics, this microscope achieves lateral and axial resolutions that are comparable to the square illumination mode of lattice light-sheet microscopy, but in a user friendly and versatile format. Given this performance, we demonstrate high-resolution imaging of clathrin-mediated endocytosis, vimentin, the endoplasmic reticulum, membrane dynamics, and Natural Killer-mediated cytotoxicity. Furthermore, we image biological phenomena that would be otherwise challenging or impossible to perform in a traditional light-sheet microscope geometry, including cell migration through confined spaces within a microfluidic device, subcellular photoactivation of Rac1, diffusion of cytoplasmic rheological tracers at a volumetric rate of 14 Hz, and large field of view imaging of neurons, developing embryos, and centimeter-scale tissue sections.


Asunto(s)
Microscopía Confocal/instrumentación , Microscopía Confocal/métodos , Análisis de la Célula Individual/métodos , Animales , Células Cultivadas , Humanos , Ratones , Técnicas Analíticas Microfluídicas/instrumentación , Plásmidos , Ratas
8.
Biomed Opt Express ; 11(5): 2313-2327, 2020 May 01.
Artículo en Inglés | MEDLINE | ID: mdl-32499925

RESUMEN

The performance of fluorescence microscopy and nanoscopy is often discussed by the effective point spread function and the optical transfer function. However, due to the complexity of the fluorophore properties such as photobleaching or other forms of photoswitching, which introduce a variance in photon emission, it is not trivial to choose optimal imaging parameters and to predict the spatial resolution. In this paper, we analytically derive a theoretical framework for estimating the achievable resolution of a microscope depending on parameters such as photoswitching, labeling densities, exposure time and sampling. We developed a numerical simulation software to analyze the impact of reversibly switchable probes in RESOLFT imaging.

9.
PLoS One ; 12(10): e0185849, 2017.
Artículo en Inglés | MEDLINE | ID: mdl-28973013

RESUMEN

We describe a method to speed up microelectromechanical system (MEMS) mirror scanning by > 20x, while also improving scan accuracy. We use Landweber deconvolution to determine an input voltage which would produce a desired output, based on the measured MEMS impulse response. Since the MEMS is weakly nonlinear, the observed behavior deviates from expectations, and we iteratively improve our input to minimize this deviation. This allows customizable MEMS angle vs. time with <1% deviation from the desired scan pattern. We demonstrate our technique by optimizing a point scanning microscope's raster patterns to image mammal submandibular gland and pollen at ~10 frames/s.


Asunto(s)
Diseño de Equipo , Sistemas Microelectromecánicos/métodos , Microscopía Confocal/métodos , Lentes , Polen , Glándula Submandibular/diagnóstico por imagen
10.
Nat Commun ; 6: 8184, 2015 Sep 03.
Artículo en Inglés | MEDLINE | ID: mdl-26333365

RESUMEN

We describe two-step fluorescence microscopy, a new approach to non-linear imaging based on positive reversible photoswitchable fluorescent probes. The protein Padron approximates ideal two-step fluorescent behaviour: it equilibrates to an inactive state, converts to an active state under blue light, and blue light also excites this active state to fluoresce. Both activation and excitation are linear processes, but the total fluorescent signal is quadratic, proportional to the square of the illumination dose. Here, we use Padron's quadratic non-linearity to demonstrate the principle of two-step microscopy, similar in principle to two-photon microscopy but with orders-of-magnitude better cross-section. As with two-photon, quadratic non-linearity from two-step fluorescence improves resolution and reduces unwanted out-of-focus excitation, and is compatible with structured illumination microscopy. We also show two-step and two-photon imaging can be combined to give quartic non-linearity, further improving imaging in challenging samples. With further improvements, two-step fluorophores could replace conventional fluorophores for many imaging applications.


Asunto(s)
Colorantes Fluorescentes , Microscopía Fluorescente/métodos , Línea Celular Tumoral/ultraestructura , Humanos , Dinámicas no Lineales
11.
Methods Mol Biol ; 1251: 231-61, 2015.
Artículo en Inglés | MEDLINE | ID: mdl-25391803

RESUMEN

Photoactivated localization microscopy (PALM) and related single-molecule imaging techniques enable biological image acquisition at ~20 nm lateral and ~50-100 nm axial resolution. Although such techniques were originally demonstrated on single imaging planes close to the coverslip surface, recent technical developments now enable the 3D imaging of whole fixed cells. We describe methods for converting a 2D PALM into a system capable of acquiring such 3D images, with a particular emphasis on instrumentation that is compatible with choosing relatively dim, genetically expressed photoactivatable fluorescent proteins (PA-FPs) as PALM probes. After reviewing the basics of 2D PALM, we detail astigmatic and multiphoton imaging approaches well suited to working with PA-FPs. We also discuss the use of open-source localization software appropriate for 3D PALM.


Asunto(s)
Técnicas Citológicas/métodos , Colorantes Fluorescentes , Imagenología Tridimensional/métodos , Microscopía Fluorescente/instrumentación , Microscopía Fluorescente/métodos , Imagen Molecular/métodos , Programas Informáticos , Colorantes Fluorescentes/metabolismo
12.
Optica ; 1(3): 181-191, 2014 Sep 20.
Artículo en Inglés | MEDLINE | ID: mdl-25485291

RESUMEN

Fluorescence imaging methods that achieve spatial resolution beyond the diffraction limit (super-resolution) are of great interest in biology. We describe a super-resolution method that combines two-photon excitation with structured illumination microscopy (SIM), enabling three-dimensional interrogation of live organisms with ~150 nm lateral and ~400 nm axial resolution, at frame rates of ~1 Hz. By performing optical rather than digital processing operations to improve resolution, our microscope permits super-resolution imaging with no additional cost in acquisition time or phototoxicity relative to the point-scanning two-photon microscope upon which it is based. Our method provides better depth penetration and inherent optical sectioning than all previously reported super-resolution SIM implementations, enabling super-resolution imaging at depths exceeding 100 µm from the coverslip surface. The capability of our system for interrogating thick live specimens at high resolution is demonstrated by imaging whole nematode embryos and larvae, and tissues and organs inside zebrafish embryos.

13.
PLoS Genet ; 10(8): e1004526, 2014 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-25101664

RESUMEN

Sporulation in the bacterium Bacillus subtilis is a developmental program in which a progenitor cell differentiates into two different cell types, the smaller of which eventually becomes a dormant cell called a spore. The process begins with an asymmetric cell division event, followed by the activation of a transcription factor, σF, specifically in the smaller cell. Here, we show that the structural protein DivIVA localizes to the polar septum during sporulation and is required for asymmetric division and the compartment-specific activation of σF. Both events are known to require a protein called SpoIIE, which also localizes to the polar septum. We show that DivIVA copurifies with SpoIIE and that DivIVA may anchor SpoIIE briefly to the assembling polar septum before SpoIIE is subsequently released into the forespore membrane and recaptured at the polar septum. Finally, using super-resolution microscopy, we demonstrate that DivIVA and SpoIIE ultimately display a biased localization on the side of the polar septum that faces the smaller compartment in which σF is activated.


Asunto(s)
Proteínas Bacterianas/genética , Proteínas de Ciclo Celular/genética , Factor sigma/genética , Esporas Bacterianas/genética , División Celular Asimétrica/genética , Bacillus subtilis/genética , Bacillus subtilis/crecimiento & desarrollo , Polaridad Celular , Regulación Bacteriana de la Expresión Génica , Esporas Bacterianas/crecimiento & desarrollo
14.
Proc Natl Acad Sci U S A ; 111(14): 5254-9, 2014 Apr 08.
Artículo en Inglés | MEDLINE | ID: mdl-24706872

RESUMEN

Multifocal structured illumination microscopy (MSIM) provides a twofold resolution enhancement beyond the diffraction limit at sample depths up to 50 µm, but scattered and out-of-focus light in thick samples degrades MSIM performance. Here we implement MSIM with a microlens array to enable efficient two-photon excitation. Two-photon MSIM gives resolution-doubled images with better sectioning and contrast in thick scattering samples such as Caenorhabditis elegans embryos, Drosophila melanogaster larval salivary glands, and mouse liver tissue.


Asunto(s)
Iluminación , Microscopía/métodos , Fotones , Animales , Caenorhabditis elegans/embriología , Drosophila melanogaster/crecimiento & desarrollo , Larva/química , Hígado/química , Ratones
15.
Chemphyschem ; 15(4): 794-800, 2014 Mar 17.
Artículo en Inglés | MEDLINE | ID: mdl-24436314

RESUMEN

We use Richardson-Lucy (RL) deconvolution to combine multiple images of a simulated object into a single image in the context of modern fluorescence microscopy techniques. RL deconvolution can merge images with very different point-spread functions, such as in multiview light-sheet microscopes,1, 2 while preserving the best resolution information present in each image. We show that RL deconvolution is also easily applied to merge high-resolution, high-noise images with low-resolution, low-noise images, relevant when complementing conventional microscopy with localization microscopy. We also use RL deconvolution to merge images produced by different simulated illumination patterns, relevant to structured illumination microscopy (SIM)3, 4 and image scanning microscopy (ISM). The quality of our ISM reconstructions is at least as good as reconstructions using standard inversion algorithms for ISM data, but our method follows a simpler recipe that requires no mathematical insight. Finally, we apply RL deconvolution to merge a series of ten images with varying signal and resolution levels. This combination is relevant to gated stimulated-emission depletion (STED) microscopy, and shows that merges of high-quality images are possible even in cases for which a non-iterative inversion algorithm is unknown.


Asunto(s)
Procesamiento de Imagen Asistido por Computador , Microscopía/métodos , Algoritmos
16.
Nat Biotechnol ; 31(11): 1032-8, 2013 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-24108093

RESUMEN

Optimal four-dimensional imaging requires high spatial resolution in all dimensions, high speed and minimal photobleaching and damage. We developed a dual-view, plane illumination microscope with improved spatiotemporal resolution by switching illumination and detection between two perpendicular objectives in an alternating duty cycle. Computationally fusing the resulting volumetric views provides an isotropic resolution of 330 nm. As the sample is stationary and only two views are required, we achieve an imaging speed of 200 images/s (i.e., 0.5 s for a 50-plane volume). Unlike spinning-disk confocal or Bessel beam methods, which illuminate the sample outside the focal plane, we maintain high spatiotemporal resolution over hundreds of volumes with negligible photobleaching. To illustrate the ability of our method to study biological systems that require high-speed volumetric visualization and/or low photobleaching, we describe microtubule tracking in live cells, nuclear imaging over 14 h during nematode embryogenesis and imaging of neural wiring during Caenorhabditis elegans brain development over 5 h.


Asunto(s)
Rastreo Celular/métodos , Imagenología Tridimensional/métodos , Microscopía/instrumentación , Microscopía/métodos , Animales , Encéfalo/anatomía & histología , Encéfalo/crecimiento & desarrollo , Encéfalo/ultraestructura , Caenorhabditis elegans/embriología , Células Endoteliales de la Vena Umbilical Humana , Humanos , Iluminación , Fotoblanqueo
17.
Nat Methods ; 10(11): 1122-6, 2013 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-24097271

RESUMEN

Existing super-resolution fluorescence microscopes compromise acquisition speed to provide subdiffractive sample information. We report an analog implementation of structured illumination microscopy that enables three-dimensional (3D) super-resolution imaging with a lateral resolution of 145 nm and an axial resolution of 350 nm at acquisition speeds up to 100 Hz. By using optical instead of digital image-processing operations, we removed the need to capture, store and combine multiple camera exposures, increasing data acquisition rates 10- to 100-fold over other super-resolution microscopes and acquiring and displaying super-resolution images in real time. Low excitation intensities allow imaging over hundreds of 2D sections, and combined physical and computational sectioning allow similar depth penetration to spinning-disk confocal microscopy. We demonstrate the capability of our system by imaging fine, rapidly moving structures including motor-driven organelles in human lung fibroblasts and the cytoskeleton of flowing blood cells within developing zebrafish embryos.


Asunto(s)
Embrión de Mamíferos/citología , Animales , Microscopía Fluorescente
18.
Nat Methods ; 9(7): 749-54, 2012 May 13.
Artículo en Inglés | MEDLINE | ID: mdl-22581372

RESUMEN

We demonstrate three-dimensional (3D) super-resolution in live multicellular organisms using structured illumination microscopy (SIM). Sparse multifocal illumination patterns generated by a digital micromirror device (DMD) allowed us to physically reject out-of-focus light, enabling 3D subdiffractive imaging in samples eightfold thicker than had been previously imaged with SIM. We imaged samples at one 2D image per second, at resolutions as low as 145 nm laterally and 400 nm axially. In addition to dual-labeled, whole fixed cells, we imaged GFP-labeled microtubules in live transgenic zebrafish embryos at depths >45 µm. We captured dynamic changes in the zebrafish lateral line primordium and observed interactions between myosin IIA and F-actin in cells encapsulated in collagen gels, obtaining two-color 4D super-resolution data sets spanning tens of time points and minutes without apparent phototoxicity. Our method uses commercially available parts and open-source software and is simpler than existing SIM implementations, allowing easy integration with wide-field microscopes.


Asunto(s)
Embrión no Mamífero , Aumento de la Imagen/métodos , Imagenología Tridimensional/métodos , Microscopía Confocal/métodos , Animales , Animales Modificados Genéticamente/embriología , Animales Modificados Genéticamente/genética , Embrión no Mamífero/metabolismo , Embrión no Mamífero/ultraestructura , Proteínas Fluorescentes Verdes/genética , Aumento de la Imagen/instrumentación , Imagenología Tridimensional/instrumentación , Iluminación , Microscopía Confocal/instrumentación , Transgenes , Pez Cebra/embriología , Pez Cebra/genética
19.
Nat Methods ; 8(4): 327-33, 2011 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-21317909

RESUMEN

We demonstrate three-dimensional (3D) super-resolution microscopy in whole fixed cells using photoactivated localization microscopy (PALM). The use of the bright, genetically expressed fluorescent marker photoactivatable monomeric (m)Cherry (PA-mCherry1) in combination with near diffraction-limited confinement of photoactivation using two-photon illumination and 3D localization methods allowed us to investigate a variety of cellular structures at <50 nm lateral and <100 nm axial resolution. Compared to existing methods, we have substantially reduced excitation and bleaching of unlocalized markers, which allows us to use 3D PALM imaging with high localization density in thick structures. Our 3D localization algorithms, which are based on cross-correlation, do not rely on idealized noise models or specific optical configurations. This allows instrument design to be flexible. By generating appropriate fusion constructs and expressing them in Cos7 cells, we could image invaginations of the nuclear membrane, vimentin fibrils, the mitochondrial network and the endoplasmic reticulum at depths of greater than 8 µm.


Asunto(s)
Microscopía de Fluorescencia por Excitación Multifotónica/métodos , Transporte de Proteínas/fisiología , Algoritmos , Animales , Células COS , Chlorocebus aethiops , Imagenología Tridimensional , Microscopía Confocal , Microscopía de Interferencia , Fotones
20.
Opt Express ; 17(16): 13671-6, 2009 Aug 03.
Artículo en Inglés | MEDLINE | ID: mdl-19654775

RESUMEN

Our simulations show a copropagating pair of laser pulses polarized in two different directions can selectively excite clockwise or counterclockwise molecular rotation in a gas of linear molecules. The resulting birefringence of the gas rotates on a femtosecond timescale and shows a periodic revival structure. The total duration of the pulse pair can be subpicosecond, allowing molecular alignment at the high densities and temperatures necessary to create a transient spinning waveplate.


Asunto(s)
Rayos Láser , Modelos Químicos , Dispositivos Ópticos , Pinzas Ópticas , Transductores , Simulación por Computador , Diseño Asistido por Computadora , Diseño de Equipo , Análisis de Falla de Equipo , Luz , Rotación , Dispersión de Radiación
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