Showing papers on "Photoactivated localization microscopy published in 2009"

Super-Resolution Fluorescence Microscopy

Abstract: Achieving a spatial resolution that is not limited by the diffraction of light, recent developments of super-resolution fluorescence microscopy techniques allow the observation of many biological structures not resolvable in conventional fluorescence microscopy. New advances in these techniques now give them the ability to image three-dimensional (3D) structures, measure interactions by multicolor colocalization, and record dynamic processes in living cells at the nanometer scale. It is anticipated that super-resolution fluorescence microscopy will become a widely used tool for cell and tissue imaging to provide previously unobserved details of biological structures and processes.

Interferometric fluorescent super-resolution microscopy resolves 3D cellular ultrastructure

Abstract: Understanding molecular-scale architecture of cells requires determination of 3D locations of specific proteins with accuracy matching their nanometer-length scale. Existing electron and light microscopy techniques are limited either in molecular specificity or resolution. Here, we introduce interferometric photoactivated localization microscopy (iPALM), the combination of photoactivated localization microscopy with single-photon, simultaneous multiphase interferometry that provides sub-20-nm 3D protein localization with optimal molecular specificity. We demonstrate measurement of the 25-nm microtubule diameter, resolve the dorsal and ventral plasma membranes, and visualize the arrangement of integrin receptors within endoplasmic reticulum and adhesion complexes, 3D protein organization previously resolved only by electron microscopy. iPALM thus closes the gap between electron tomography and light microscopy, enabling both molecular specification and resolution of cellular nanoarchitecture.

Photoactivatable mCherry for high-resolution two-color fluorescence microscopy

Abstract: The reliance of modern microscopy techniques on photoactivatable fluorescent proteins prompted development of mCherry variants that are initially dark but become red fluorescent after violet-light irradiation. Using ensemble and single-molecule characteristics as selection criteria, we developed PAmCherry1 with excitation/emission maxima at 564/595 nm. Compared to other monomeric red photoactivatable proteins, it has faster maturation, better pH stability, faster photoactivation, higher photoactivation contrast and better photostability. Lack of green fluorescence and single-molecule behavior make monomeric PAmCherry1 a preferred tag for two-color diffraction-limited photoactivation imaging and for super-resolution techniques such as one- and two-color photoactivated localization microscopy (PALM). We performed PALM imaging using PAmCherry1-tagged transferrin receptor expressed alone or with photoactivatable GFP-tagged clathrin light chain. Pair correlation and cluster analyses of the resulting PALM images identified < or =200 nm clusters of transferrin receptor and clathrin light chain at < or =25 nm resolution and confirmed the utility of PAmCherry1 as an intracellular probe.

Selective plane illumination microscopy techniques in developmental biology.

Abstract: Selective plane illumination microscopy (SPIM) and other fluorescence microscopy techniques in which a focused sheet of light serves to illuminate the sample have become increasingly popular in developmental studies. Fluorescence light-sheet microscopy bridges the gap in image quality between fluorescence stereomicroscopy and high-resolution imaging of fixed tissue sections. In addition, high depth penetration, low bleaching and high acquisition speeds make light-sheet microscopy ideally suited for extended time-lapse experiments in live embryos. This review compares the benefits and challenges of light-sheet microscopy with established fluorescence microscopy techniques such as confocal microscopy and discusses the different implementations and applications of this easily adaptable technology.

Accuracy and precision in quantitative fluorescence microscopy

Abstract: The light microscope has long been used to document the localization of fluorescent molecules in cell biology research. With advances in digital cameras and the discovery and development of genetically encoded fluorophores, there has been a huge increase in the use of fluorescence microscopy to quantify spatial and temporal measurements of fluorescent molecules in biological specimens. Whether simply comparing the relative intensities of two fluorescent specimens, or using advanced techniques like Forster resonance energy transfer (FRET) or fluorescence recovery after photobleaching (FRAP), quantitation of fluorescence requires a thorough understanding of the limitations of and proper use of the different components of the imaging system. Here, I focus on the parameters of digital image acquisition that affect the accuracy and precision of quantitative fluorescence microscopy measurements.

Far-Field Optical Nanoscopy

Abstract: Diffraction-unlimited imaging is one of the emerging fields in microscopy. In all of these techniques, fluorophore switching is key. The first technique developed is STED, which recently has progressed to video-rate imaging of living cells.

Imaging biological structures with fluorescence photoactivation localization microscopy

Abstract: Fluorescence photoactivation localization microscopy (FPALM) images biological structures with subdiffraction-limited resolution. With repeated cycles of activation, readout and bleaching, large numbers of photoactivatable probes can be precisely localized to obtain a map (image) of labeled molecules with an effective resolution of tens of nanometers. FPALM has been applied to a variety of biological imaging applications, including membrane, cytoskeletal and cytosolic proteins in fixed and living cells. Molecular motions can be quantified. FPALM can also be applied to nonbiological samples, which can be labeled with photoactivatable probes. With emphasis on cellular imaging, we describe here the adaptation of a conventional widefield fluorescence microscope for FPALM and present step-by-step procedures to successfully obtain and analyze FPALM images. The fundamentals of this protocol may also be applicable to users of similar imaging techniques that apply localization of photoactivatable probes to achieve super-resolution. Once alignment of the setup has been completed, data acquisitions can be obtained in approximately 1–30 min and analyzed in approximately 0.5–4 h.

Nanoscale imaging of molecular positions and anisotropies

Abstract: A Polarization Fluorescence Photoactivation Localization Microscopy (P-FPALM) system and method are provided to simultaneously image the localizations and fluorescence anisotropics of large numbers of single molecules within a sample. The system modifies known FPALM systems by adding a polarizing beam splitter. The beam splitter polarizes emissions perpendicular and parallel to an axis in the sample to allow spatially separate imaging of fluorescence emitted from a sample. The system includes lenses and mirrors so that the separate, polarized beams are detected simultaneously. The present invention includes methods of using the system to image localizations and fluorescence anisotropics of single molecules, and methods of using data obtained with the system to predict 3-D orientation of the molecules. The system and method achieve substantially improved lateral resolution within even dense samples over known microscopic imaging techniques, and does not compromise speed or sensitivity.

High-aperture cryogenic light microscopy.

Abstract: We report here the development of instruments and protocols for carrying out high numerical aperture immersion light microscopy on cryogenic specimens. Imaging by this modality greatly increases the lifetimes of fluorescence probes, including those commonly used for protein localization studies, while retaining the ability to image the specimen with high fidelity and spatial resolution. The novel use of a cryogenic immersion fluid also minimizes the refractive index mismatch between the sample and lens, leading to a more efficient coupling of the light from the sample to the image forming system. This enhancement is applicable to both fluorescence and transmitted light microscopy techniques. The design concepts used for the cryogenic microscope can be applied to virtually any existing light-based microscopy technique. This prospect is particularly exciting in the context of 'super-resolution' techniques, where enhanced fluorescence lifetime probes are especially useful. Thus, using this new modality it is now possible to observe dynamic events in a live cell, and then rapidly vitrify the specimen at a specific time point prior to carrying out high-resolution imaging. The techniques described can be used in conjunction with other imaging modalities in correlated studies. We have also developed instrumentation to perform cryo-light imaging together with soft X-ray tomography on the same cryo-fixed specimen as a means of carrying out high content, quantifiable correlated imaging analyses. These methods are equally applicable to correlated light and electron microscopy of frozen biological objects.

Bright Field Microscopy as an Alternative to Whole Cell Fluorescence in Automated Analysis of Macrophage Images

Abstract: Background Fluorescence microscopy is the standard tool for detection and analysis of cellular phenomena. This technique, however, has a number of drawbacks such as the limited number of available fluorescent channels in microscopes, overlapping excitation and emission spectra of the stains, and phototoxicity. Methodology We here present and validate a method to automatically detect cell population outlines directly from bright field images. By imaging samples with several focus levels forming a bright field -stack, and by measuring the intensity variations of this stack over the -dimension, we construct a new two dimensional projection image of increased contrast. With additional information for locations of each cell, such as stained nuclei, this bright field projection image can be used instead of whole cell fluorescence to locate borders of individual cells, separating touching cells, and enabling single cell analysis. Using the popular CellProfiler freeware cell image analysis software mainly targeted for fluorescence microscopy, we validate our method by automatically segmenting low contrast and rather complex shaped murine macrophage cells. Significance The proposed approach frees up a fluorescence channel, which can be used for subcellular studies. It also facilitates cell shape measurement in experiments where whole cell fluorescent staining is either not available, or is dependent on a particular experimental condition. We show that whole cell area detection results using our projected bright field images match closely to the standard approach where cell areas are localized using fluorescence, and conclude that the high contrast bright field projection image can directly replace one fluorescent channel in whole cell quantification. Matlab code for calculating the projections can be downloaded from the supplementary site: http://sites.google.com/site/brightfieldorstaining

Combined non‐linear laser imaging (two‐photon excitation fluorescence microscopy, fluorescence lifetime imaging microscopy, multispectral multiphoton microscopy) in cutaneous tumours: first experiences

Abstract: Background Two-photon excitation (TPE) fluorescence microscopy is a high-resolution laser-scanning imaging technique enabling deep imaging inside biological tissues. TPE microscopy has the triple advantage of offering high spatial resolution (250 nm radially, 800 nm axially), high penetration depth inside skin (200mm ), and low photodamage effects. Further, cells and extracellular matrix intrinsically contain a variety of fluorescent molecules (NADH, tryptophan, keratins, melanin, elastin, cholecalciferol and others), so that biological tissues can be imaged by TPE microscopy without any exogenous probe. The time-resolved analysis of the fluorescence signal, known as fluorescence lifetime imaging microscopy (FLIM), is an additional non-invasive microscopy technique useful to characterize endogenous fluorescence species and their surrounding medium by measuring the mean lifetime of fluorescent emission. Finally, multispectral (MTPE) tissue imaging can also be used to identify different endogenous fluorescent species by measuring their two photon emission spectra. Those techniques offer functional information about the relative quantities of fluorescent molecules, which are correlated with tissue structure in physiological and pathological states. Objective We have decided to apply these three methods at the same time for cutaneous tumors in order to evaluate their possible future use. Method We have analyzed a melanoma and a basal cell carcinoma, with their surrounding healthy skin, to evaluate any difference in healthy skin and neoplasia. The samples were excised during dermatological surgery, then cut, saving some healthy skin in both, to obtain a regular shape, allowing its positioning either with the skin surface parallel to the optical axis (horizontal optical sectioning), or perpendicular (vertical optical sectioning). Conclusion This first result demonstrates that FLIM is effective in discriminating healthy skin from MM, while MTPE is effective in discriminating healthy skin from BCC. Conflicts of interest None declared

Enhanced fluorescence signal in nonlinear microscopy through supplementary fiber-optic light collection

Abstract: Nonlinear microscopy techniques crucially rely on efficient signal detection. Here, we present a ring of large-core optical fibers for epi-collection of fluorescence photons that are not transmitted through the objective and thus normally wasted. Theoretical treatments indicated that such a supplementary fiber-optic light collection system (SUFICS) can provide an up to 4-fold signal gain. In typical in vivo imaging experiments, the fiber-ring channel was brighter than the objective channel down to 800 μm depth, thus providing a gain >2. Moreover, SUFICS reduced noise levels in calcium imaging experiments by about 23%. We recommend SUFICS as a generally applicable, effective add-on to nonlinear microscopes for enhancing fluorescence signals.

Optical microscopy in photosynthesis

Abstract: Emerging as well as the most frequently used optical microscopy techniques are reviewed and image contrast generation methods in a microscope are presented, focusing on the nonlinear contrasts such as harmonic generation and multiphoton excitation fluorescence. Nonlinear microscopy presents numerous advantages over linear microscopy techniques including improved deep tissue imaging, optical sectioning, and imaging of live unstained samples. Nonetheless, with the exception of multiphoton excitation fluorescence, nonlinear microscopy is in its infancy, lacking protocols, users and applications; hence, this review focuses on the potential of nonlinear microscopy for studying photosynthetic organisms. Examples of nonlinear microscopic imaging are presented including isolated light-harvesting antenna complexes from higher plants, starch granules, chloroplasts, unicellular alga Chlamydomonas reinhardtii, and cyanobacteria Leptolyngbya sp. and Anabaena sp. While focusing on nonlinear microscopy techniques, second and third harmonic generation and multiphoton excitation fluorescence microscopy, other emerging nonlinear imaging modalities are described and several linear optical microscopy techniques are reviewed in order to clearly describe their capabilities and to highlight the advantages of nonlinear microscopy.

Ultrahigh resolution imaging of biomolecules by fluorescence photoactivation localization microscopy.

Abstract: Diffraction limits the biological structures that can be imaged by normal light microscopy. However, recently developed techniques are breaking the limits that diffraction poses and allowing imaging of biological samples at the molecular length scale. Fluorescence photoactivation localization microscopy (FPALM) and related methods can now image molecular distributions in fixed and living cells with measured resolution better than 30 nm. Based on localization of single photoactivatable molecules, FPALM uses repeated cycles of activation, localization, and photobleaching, combined with high-sensitivity fluorescence imaging, to identify and localize large numbers of molecules within a sample. Procedures and pitfalls for construction and use of such a microscope are discussed in detail. Representative images of cytosolic proteins, membrane proteins, and other structures, as well as examples of results during acquisition are shown. It is hoped that these details can be used to perform FPALM on a variety of biological samples, to significantly advance the understanding of biological systems.

mhFLIM: Resolution of heterogeneous fluorescence decays in widefield lifetime microscopy

Abstract: Frequency-domain fluorescence lifetime imaging microscopy (FD-FLIM) is a fast and accurate way of measuring fluorescence lifetimes in widefield microscopy. However, the resolution of multiple exponential fluorescence decays has remained beyond the reach of most practical FD-FLIM systems. In this paper we describe the implementation of FD-FLIM using a 40MHz pulse train derived from a supercontinuum source for excitation. The technique, which we term multi-harmonic FLIM (mhFLIM), makes it possible to accurately resolve biexponential decays of fluorophores without any a priori information. The system’s performance is demonstrated using a mixture of spectrally similar dyes of known composition and also on a multiply-labeled biological sample. The results are compared to those obtained from time correlated single photon counting (TCSPC) microscopy and a good level of agreement is achieved. We also demonstrate the first practical application of an algorithm derived by G. Weber [1] for analysing mhFLIM data. Because it does not require nonlinear minimisation, it offers potential for realtime analysis during acquisition.

Improved spatial resolution in fluorescence focal modulation microscopy

Abstract: We show that the focal modulation microscopy (FMM), which combines a spatial phase modulator with confocal microscopy, results in an improvement in spatial resolution. This technique was introduced to increase imaging depth into tissue and rejection of background from a thick scattering object. A theory for image formation in FMM is presented, and the effects of detecting the in-phase modulated fluorescence signal are discussed. Compared with conventional confocal microscopy, the width of the point-spread function for the in-phase fluorescence signal is improved by 16.4%. When applied to saturable fluorescence, the half-width at half-maximum is improved by 33.6%, 50.0%, and 62.9%, at demodulation frequencies 2omega, 4omega, and 8omega, respectively.

Time-gated total internal reflection fluorescence microscopy with a supercontinuum excitation source.

Abstract: We present the instrumental development of a versatile total internal reflection fluorescence lifetime imaging microscopy setup illuminated by a supercontinuum laser source. It enables performing wide-field fluorescence lifetime imaging with subwavelength axial resolution for a large range of fluorophores. The short overall acquisition time and the axial resolution are well suited for dynamic neurobiological applications.

Quantitative fluorescence microscopy techniques.

Abstract: Fluorescence microscopy is a non-invasive technique that allows high resolution imaging of cytoskeletal structures. Advances in the field of fluorescent labelling (e.g., fluorescent proteins, quantum dots, tetracystein domains) and optics (e.g., super-resolution techniques and quantitative methods) not only provide better images of the cytoskeleton, but also offer an opportunity to quantify the complex of molecular events that populate this highly organised, yet dynamic, structure.For instance, fluorescence lifetime imaging microscopy and Forster resonance energy transfer imaging allow mapping of protein-protein interactions; furthermore, techniques based on the measurement of photobleaching kinetics (e.g., fluorescence recovery after photobleaching, fluorescence loss in photobleaching, and fluorescence localisation after photobleaching) permit the characterisation of axonal transport and, more generally, diffusion of relevant biomolecules.Quantitative fluorescence microscopy techniques offer powerful tools for understanding the physiological and pathological roles of molecular machineries in the living cell.

Superresolution imaging in live Caulobacter crescentus cells using photoswitchable enhanced yellow fluorescent protein

Abstract: Recently, photoactivation and photoswitching were used to control single-molecule fluorescent labels and produce images of cellular structures beyond the optical diffraction limit (e.g., PALM, FPALM, and STORM). While previous live-cell studies relied on sophisticated photoactivatable fluorescent proteins, we show in the present work that superresolution imaging can be performed with fusions to the commonly used fluorescent protein EYFP. Rather than being photoactivated, however, EYFP can be reactivated with violet light after apparent photobleaching. In each cycle after initial imaging, only a sparse subset fluorophores is reactivated and localized, and the final image is then generated from the measured single-molecule positions. Because these methods are based on the imaging nanometer-sized single-molecule emitters and on the use of an active control mechanism to produce sparse sub-ensembles, we suggest the phrase "Single-Molecule Active-Control Microscopy" (SMACM) as an inclusive term for this general imaging strategy. In this paper, we address limitations arising from physiologically imposed upper boundaries on the fluorophore concentration by employing dark time-lapse periods to allow single-molecule motions to fill in filamentous structures, increasing the effective labeling concentration while localizing each emitter at most once per resolution-limited spot. We image cell-cycle-dependent superstructures of the bacterial actin protein MreB in live Caulobacter crescentus cells with sub-40-nm resolution for the first time. Furthermore, we quantify the reactivation quantum yield of EYFP, and find this to be 1.6 x 10-6, on par with conventional photoswitchable fluorescent proteins like Dronpa. These studies show that EYFP is a useful emitter for in vivo superresolution imaging of intracellular structures in bacterial cells.

Common-path multimodal optical microscopy.

Abstract: We have developed a common-path multimodal optical microscopy system that is capable of using a single optical source and a single camera to image amplitude, phase, and fluorescence features of a biological specimen. This is achieved by varying either contrast enhancement filters at the Fourier plane and/or neutral density/fluorescence filters in front of the CCD camera. The feasibility of the technique is demonstrated by obtaining brightfield, fluorescence, phase-contrast, spatially filtered, brightfield+fluorescence, phase+fluorescence, and edge-enhanced+fluorescence images of the same Drosophila embryo without the need for image registration and fusion. This comprehensive microscope has the capability of providing both structural and functional information and may be used for applications such as studying live-cell dynamics and in high throughput microscopy and automated microscopy.

Multispot point spread function for multiphoton fluorescence microscopy.

Abstract: We propose and demonstrate an imaging technique capable of generating multiple excitation spot for multiphoton fluorescence microscopy. The point spread function (PSF) is generated by interfering two counterpropagating extended depth of focus beams along the optical axis. At an illumination wavelength of 976 nm and aperture angle of 60°, five distinct nanospots of dimension ≈210 nm is obtained along the optical axis. The resulting PSF has the ability to simultaneously excite multiple planes, and overcomes the sidelobe problem associated with single-photon variant. The proposed multiple-excitation-spot-based-optical imaging technique may find potential application in nanobioimaging and three-dimensional fluorescence microscopy.

Introduction to Theories of Several Super-resolution Fluorescence Microscopy Methods and Recent Advance in The Field

Abstract: In life science research,it is often required to localize proteins in a live cell to a certain accuracy to study their localization-related function. However,due to the Abbe/Rayleigh criteria of light,the widely used wide-field/confocal microscopy can never resolve structures less than 200 nm in diameter. In recent year,different super-resolution microscopy techniques emerge as a result of new fluorescent probes and imaging theories. A full frame to the theories and recent advancements in this field is summarized. The concept of point-spread function of light source in the focal plane and the classical definition of resolution is explained in the first part. The fluorescence single-molecular imaging technique and the equation that defines the localization accuracy of a single molecule is introduced in the second part. Based on these knowledge,super-resolution microscopy methods based on single-molecular imaging technique,such as photoactivated localization microscopy (PALM) and stochastic optical reconstruction microscopy (STORM) is discussed further. On the other hand,by engineering the point spread function of the light source,super-resolution can also be achieved. Two typical methods,stimulated emission depletion (STED) and saturated structure illumination microscopy (SSIM) are explored thereafter. In the end,different methods to extract super-resolutional information along the z axis,and their combinations with the methods to increase xy plane resolution mentioned above are explained. In the end,the limitation to the current super-resolution methods and their future direction are also discussed.

Photoactivation in Fluorescence Microscopy

Abstract: The use of photoactive compounds in microscopy has a long history. Caged compounds have been used for almost forty years, not only to elicit chemical reactions in cells, but also to mark specific cells or regions within cells by photoactivation of fluorescence. During the last seven years, the advent of photoactivatable GFP (PA-GFP) and its successors has opened up a myriad of new applications. All of this work has, of course, been greatly facilitated in live cells through the possibility of genetic labeling that is given by the fluorescent proteins. However, even as more photo-activatable and photo-switchable proteins are discovered, they are still limited in terms of wavelength ranges and photophysical properties. Thus, there has been a resurgence of interest in small organic photoactive molecules for cell biology experiments. In this short introductory overview, we will present the basic concepts of photoactivation and discuss many of the strengths and limitations of various approaches. We will also provide a general description of the kinds of applications for which these probes can be used.

High Resolution Microscopy

Abstract: Carl Zeiss will launch a new product line for high resolution microscopy this year. Thanks to the combination of two techniques in Zeiss microscopes – HR-SIM (High Resolution Structured Illumination Microscopy) and PAL-M (Photoactivated Localization Microscopy) – biomedical scientists are able to examine objects at maximum resolution.

Adding new dimensions to laser-scanning fluorescence microscopy.

Abstract: We describe a novel method of optical imaging by exploiting simple ideas borrowed from pulsed optics. We show that the use of ultrafast pulsed one-photon excitation in laser-scanning fluorescence microscopy dramatically brings together several advantages offered by two widely used present day microscopic techniques, confocal and multi-photon fluorescence microscopy. The method appears as a novel tool in the context of laser-scanning fluorescence microscopy by having a 'built-in' 3D spatial resolution.

Photoswitching microscopy with subdiffraction-resolution

Abstract: High-resolution fluorescence imaging has a vast impact on our understanding of intracellular organization. The key elements for high-resolution microscopy are reversibly photo-switchable fluorophores that can be cycled between a fluorescent and a non-fluorescent (dark) state and can be localized with nanometer accuracy. For example, it has been demonstrated that conventional cyanine dyes (Cy5, Alexa647) can serve as efficient photoswitchable fluorescent probes. We extended this principle for carbocyanines without the need of an activator fluorophore nearby, and named our approach direct stochastic optical reconstruction microscopy (d STORM). Recently, we introduced a general approach for superresolution microscopy that uses commercial fluorescent probes as molecular photoswitches by generating long lived dark states such as triplet states or radical states. Importantly, this concept can be extended to a variety of conventional fluorophores, such as ATTO520, ATTO565, or ATTO655. The generation of non-fluorescent dark states as the underlying principle of superresolution microscopy is generalized under the term photoswitching microscopy, and unlocks a broad spectrum of organic fluorophores for multicolor application. Hereby, this method supplies subdiffraction-resolution of subcellular compartments and can serve as a tool for molecular quantification.

Improving the resolution in total internal reflection fluorescence and phase microscopy

Abstract: III

Fluorescence Microscopy in the Neurosciences

Abstract: Fluorescence is a physical phenomenon in which a particular chemical compound (the fluorochrome) emits light of a particular color very shortly after being hit by light of another color. In this article we provide a brief survey of the physics of fluorescence and the design essentials of modern fluorescence microscopes. We further discuss in a practical sense fluorochromes and the importance of knowing their excitation and emission spectra. Appropriate filtering in single and multiple fluorescence applications is discussed as well, in addition to the problem of false positive results caused by fluorescence cross-talk.

Three Dimensional Super Resolution Fluorescence Imaging of Single Bacterial Cells by Stereo Photoactivated Localization Microscopy

Abstract: Extended abstract of a paper presented at Microscopy and Microanalysis 2009 in Richmond, Virginia, USA, July 26 – July 30, 2009