RESUMO
The complex environment of biological cells and tissues has motivated development of three-dimensional (3D) imaging in both light and electron microscopies. To this end, one of the primary tools in fluorescence microscopy is that of computational deconvolution. Wide-field fluorescence images are often corrupted by haze due to out-of-focus light, i.e., to cross-talk between different object planes as represented in the 3D image. Using prior understanding of the image formation mechanism, it is possible to suppress the cross-talk and reassign the unfocused light to its proper source post facto. Electron tomography based on tilted projections also exhibits a cross-talk between distant planes due to the discrete angular sampling and limited tilt range. By use of a suitably synthesized 3D point spread function, we show here that deconvolution leads to similar improvements in volume data reconstructed from cryoscanning transmission electron tomography (CSTET), namely a dramatic in-plane noise reduction and improved representation of features in the axial dimension. Contrast enhancement is demonstrated first with colloidal gold particles and then in representative cryotomograms of intact cells. Deconvolution of CSTET data collected from the periphery of an intact nucleus revealed partially condensed, extended structures in interphase chromatin.
Assuntos
Tomografia com Microscopia Eletrônica/métodos , Aumento da Imagem/métodos , Imageamento Tridimensional , Microscopia Eletrônica de Transmissão e Varredura/métodos , Algoritmos , Linhagem Celular , Secções Congeladas , Coloide de Ouro , HumanosRESUMO
Four-dimensional fluorescence microscopy--which records 3D image information as a function of time--provides an unbiased way of tracking dynamic behavior of subcellular components in living samples and capturing key events in complex macromolecular processes. Unfortunately, the combination of phototoxicity and photobleaching can severely limit the density or duration of sampling, thereby limiting the biological information that can be obtained. Although widefield microscopy provides a very light-efficient way of imaging, obtaining high-quality reconstructions requires deconvolution to remove optical aberrations. Unfortunately, most deconvolution methods perform very poorly at low signal-to-noise ratios, thereby requiring moderate photon doses to obtain acceptable resolution. We present a unique deconvolution method that combines an entropy-based regularization function with kernels that can exploit general spatial characteristics of the fluorescence image to push the required dose to extreme low levels, resulting in an enabling technology for high-resolution in vivo biological imaging.
Assuntos
Entropia , Imageamento Tridimensional/métodos , Microscopia de Fluorescência/métodos , Razão Sinal-Ruído , Algoritmos , Animais , Linhagem Celular , Modelos Moleculares , Modelos Teóricos , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Conformação Proteica , Reprodutibilidade dos Testes , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
The organization and the mechanisms of condensation of mitotic chromosomes remain unsolved despite many decades of efforts. The lack of resolution, tight compaction, and the absence of function-specific chromatin labels have been the key technical obstacles. The correlation between DNA sequence composition and its contribution to the chromosome-scale structure has been suggested before; it is unclear though if all DNA sequences equally participate in intra- or inter-chromatin or DNA-protein interactions that lead to formation of mitotic chromosomes and if their mitotic positions are reproduced radially. Using high-resolution fluorescence microscopy of live or minimally perturbed, fixed chromosomes in Drosophila embryonic cultures or tissues expressing MSL3-GFP fusion protein, we studied positioning of specific MSL3-binding sites. Actively transcribed, dosage compensated Drosophila genes are distributed along the euchromatic arm of the male X chromosome. Several novel features of mitotic chromosomes have been observed. MSL3-GFP is always found at the periphery of mitotic chromosomes, suggesting that active, dosage compensated genes are also found at the periphery of mitotic chromosomes. Furthermore, radial distribution of chromatin loci on mitotic chromosomes was found to be correlated with their functional activity as judged by core histone modifications. Histone modifications specific to active chromatin were found peripheral with respect to silent chromatin. MSL3-GFP-labeled chromatin loci become peripheral starting in late prophase. In early prophase, dosage compensated chromatin regions traverse the entire width of chromosomes. These findings suggest large-scale internal rearrangements within chromosomes during the prophase condensation step, arguing against consecutive coiling models. Our results suggest that the organization of mitotic chromosomes is reproducible not only longitudinally, as demonstrated by chromosome-specific banding patterns, but also radially. Specific MSL3-binding sites, the majority of which have been demonstrated earlier to be dosage compensated DNA sequences, located on the X chromosomes, and actively transcribed in interphase, are positioned at the periphery of mitotic chromosomes. This potentially describes a connection between the DNA/protein content of chromatin loci and their contribution to mitotic chromosome structure. Live high-resolution observations of consecutive condensation states in MSL3-GFP expressing cells could provide additional details regarding the condensation mechanisms.
Assuntos
Cromossomos de Insetos/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila/genética , Mitose , Proteínas Nucleares/metabolismo , Fatores de Transcrição/metabolismo , Animais , Células Cultivadas , Cromossomos de Insetos/ultraestrutura , Drosophila/crescimento & desenvolvimento , Proteínas de Drosophila/genética , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Histonas/metabolismo , Masculino , Microscopia de Fluorescência , Proteínas Nucleares/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Fatores de Transcrição/genética , Transcrição Gênica , Cromossomo X/metabolismoRESUMO
Live fluorescence microscopy has the unique capability to probe dynamic processes, linking molecular components and their localization with function. A key goal of microscopy is to increase spatial and temporal resolution while simultaneously permitting identification of multiple specific components. We demonstrate a new microscope platform, OMX, that enables subsecond, multicolor four-dimensional data acquisition and also provides access to subdiffraction structured illumination imaging. Using this platform to image chromosome movement during a complete yeast cell cycle at one 3D image stack per second reveals an unexpected degree of photosensitivity of fluorophore-containing cells. To avoid perturbation of cell division, excitation levels had to be attenuated between 100 and 10,000× below the level normally used for imaging. We show that an image denoising algorithm that exploits redundancy in the image sequence over space and time allows recovery of biological information from the low light level noisy images while maintaining full cell viability with no fading.
Assuntos
Microscopia de Fluorescência/métodos , Algoritmos , Animais , Sobrevivência Celular , Drosophila melanogaster/citologia , Saccharomyces cerevisiae/citologia , SoftwareRESUMO
We model the effect of depth dependent spherical aberration caused by a refractive index mismatch between the mounting and immersion mediums in a 3D structured illumination microscope (SIM). We first derive a forward model that takes into account the effect of the depth varying aberrations on both the illumination and the detection processes. From the model, we demonstrate that depth dependent spherical aberration leads to loss of signal only due to its effect on the detection response of the system, while its effect on illumination leads to phase shifts between orders that can be handled computationally in the reconstruction process. Further, by using the model, we provide guidelines for optical corrections of aberrations with different complexities, and explain how the proposed corrections simplify the forward model. Finally, we show that it is possible to correct both illumination and detection aberrations using a deformable mirror only on the detection path of the microscope.
Assuntos
Artefatos , Aumento da Imagem/instrumentação , Lentes , Iluminação/instrumentação , Microscopia/instrumentação , Nefelometria e Turbidimetria/instrumentação , Simulação por Computador , Desenho Assistido por Computador , Desenho de Equipamento , Análise de Falha de Equipamento , Luz , Modelos Biológicos , Reprodutibilidade dos Testes , Espalhamento de Radiação , Sensibilidade e EspecificidadeRESUMO
We have developed "Microscope-Cockpit" (Cockpit), a highly adaptable open source user-friendly Python-based Graphical User Interface (GUI) environment for precision control of both simple and elaborate bespoke microscope systems. The user environment allows next-generation near instantaneous navigation of the entire slide landscape for efficient selection of specimens of interest and automated acquisition without the use of eyepieces. Cockpit uses "Python-Microscope" (Microscope) for high-performance coordinated control of a wide range of hardware devices using open source software. Microscope also controls complex hardware devices such as deformable mirrors for aberration correction and spatial light modulators for structured illumination via abstracted device models. We demonstrate the advantages of the Cockpit platform using several bespoke microscopes, including a simple widefield system and a complex system with adaptive optics and structured illumination. A key strength of Cockpit is its use of Python, which means that any microscope built with Cockpit is ready for future customisation by simply adding new libraries, for example machine learning algorithms to enable automated microscopy decision making while imaging.
RESUMO
We address the problem of computational representation of image formation in 3D widefield fluorescence microscopy with depth varying spherical aberrations. We first represent 3D depth-dependent point spread functions (PSFs) as a weighted sum of basis functions that are obtained by principal component analysis (PCA) of experimental data. This representation is then used to derive an approximating structure that compactly expresses the depth variant response as a sum of few depth invariant convolutions pre-multiplied by a set of 1D depth functions, where the convolving functions are the PCA-derived basis functions. The model offers an efficient and convenient trade-off between complexity and accuracy. For a given number of approximating PSFs, the proposed method results in a much better accuracy than the strata based approximation scheme that is currently used in the literature. In addition to yielding better accuracy, the proposed methods automatically eliminate the noise in the measured PSFs.
Assuntos
Imageamento Tridimensional/métodos , Microscopia de Fluorescência/métodos , Algoritmos , Biofísica/métodos , Processamento de Imagem Assistida por Computador , Microscopia/métodos , Modelos Estatísticos , Óptica e Fotônica , Análise de Componente Principal , Reprodutibilidade dos Testes , SoftwareRESUMO
Rapid cryopreservation of biological specimens is the gold standard for visualizing cellular structures in their true structural context. However, current commercial cryo-fluorescence microscopes are limited to low resolutions. To fill this gap, we have developed cryoSIM, a microscope for 3D super-resolution fluorescence cryo-imaging for correlation with cryo-electron microscopy or cryo-soft X-ray tomography. We provide the full instructions for replicating the instrument mostly from off-the-shelf components and accessible, user-friendly, open-source Python control software. Therefore, cryoSIM democratizes the ability to detect molecules using super-resolution fluorescence imaging of cryopreserved specimens for correlation with their cellular ultrastructure.
RESUMO
Dual-axis electron microscopic tomography minimizes the missing wedge-induced resolution loss by taking two complementary tilt data sets of the same target along two orthogonal axes. The potential of this powerful approach has been hampered by the practical challenges inherent in finding the original targets that are dramatically displaced due to non-eucentric specimen rotation. Not only is the manual search for the original targets time consuming and tedious but the added dose during manual searching is uncontrollable. We have developed a hierarchical alignment scheme that allows tomographic data to be collected from an arbitrary number of target sites in one grid orientation and then to find and collect orthogonal data sets with little or no user intervention. Inspired by the successful multi-scale mapping in Leginon, our alignment is performed in three levels to gradually pinpoint the original targets. At the lowest level the grid lattice is used to determine the rotation angle and translational shift resulting from specimen rotation via auto- and cross-correlative analysis of a pair of atlas maps constructed before and after specimen rotation. The target locations are further refined at the next level using a pair of smaller atlas maps. The final refinement of target positions is done by aligning the target contained image tiles. Given the batch processing nature of this hierarchical alignment, multiple targets are initially selected in a group and then sequentially acquired. Upon completion of the data collection on all the targets along the first axis and after specimen rotation, the hierarchical alignment is performed to relocate the original targets. The data collection is then resumed on these targets for the second axis. Therefore, only one specimen rotation is needed for collecting multiple dual-axis tomographic data sets. The experiment of acquiring 20S Proteasomes dual-axis tomographic data sets in vitreous ice at 86,000x CCD magnification on our FEI Tecnai Polara TF30 electron microscope has suggested that the developed scheme is very robust. The extra doses for finding and centering the original targets are almost negligible. This scheme has been integrated into UCSF Tomography software suite that can be downloaded at www.msg.ucsf.edu/tomography free for academic use.
Assuntos
Microscopia Crioeletrônica/métodos , Tomografia com Microscopia Eletrônica/métodosRESUMO
Chromosome organization inside the nucleus is not random but rather is determined by a variety of factors, including interactions between chromosomes and nuclear components such as the nuclear envelope or nuclear matrix. Such interactions may be critical for proper nuclear organization, chromosome partitioning during cell division, and gene regulation. An important, but poorly documented subset, includes interactions between specific chromosomal regions. Interactions of this type are thought to be involved in long-range promoter regulation by distant enhancers or locus control regions and may underlie phenomena such as transvection. Here, we used an in vivo microscopy assay based on Lac Repressor/operator recognition to show that Mcp, a polycomb response element from the Drosophila bithorax complex, is able to mediate physical interaction between remote chromosomal regions. These interactions are tissue specific, can take place between multiple Mcp elements, and seem to be stable once established. We speculate that this ability to interact may be part of the mechanism through which Mcp mediates its regulatory function in the bithorax complex.
Assuntos
Pareamento Cromossômico/genética , Cromossomos/metabolismo , Drosophila/genética , Elementos de Resposta , Transportadores de Cassetes de Ligação de ATP/genética , Animais , Cromossomos/genética , Drosophila/citologia , Proteínas de Drosophila/genética , Proteínas do Olho/genética , Regiões Operadoras Genéticas , Proteínas Repressoras/metabolismoRESUMO
A new type of wide-field fluorescence microscopy is described, which produces 100-nm-scale spatial resolution in all three dimensions, by using structured illumination in a microscope that has two opposing objective lenses. Illumination light is split by a grating and a beam splitter into six mutually coherent beams, three of which enter the specimen through each objective lens. The resulting illumination intensity pattern contains high spatial frequency components both axially and laterally. In addition, the emission is collected by both objective lenses coherently, and combined interferometrically on a single camera, resulting in a detection transfer function with axially extended support. These two effects combine to produce near-isotropic resolution. Experimental images of test samples and biological specimens confirm the theoretical predictions.
Assuntos
Aumento da Imagem/instrumentação , Imageamento Tridimensional/instrumentação , Lentes , Microscopia/instrumentação , Nanotecnologia/instrumentação , Desenho de Equipamento , Análise de Falha de Equipamento , Microscopia/métodos , Nanotecnologia/métodos , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
Structured illumination microscopy is a method that can increase the spatial resolution of wide-field fluorescence microscopy beyond its classical limit by using spatially structured illumination light. Here we describe how this method can be applied in three dimensions to double the axial as well as the lateral resolution, with true optical sectioning. A grating is used to generate three mutually coherent light beams, which interfere in the specimen to form an illumination pattern that varies both laterally and axially. The spatially structured excitation intensity causes normally unreachable high-resolution information to become encoded into the observed images through spatial frequency mixing. This new information is computationally extracted and used to generate a three-dimensional reconstruction with twice as high resolution, in all three dimensions, as is possible in a conventional wide-field microscope. The method has been demonstrated on both test objects and biological specimens, and has produced the first light microscopy images of the synaptonemal complex in which the lateral elements are clearly resolved.
Assuntos
Algoritmos , Aumento da Imagem/métodos , Interpretação de Imagem Assistida por Computador/métodos , Imageamento Tridimensional/métodos , Iluminação/métodos , Microscopia de Fluorescência/métodos , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
Malaria parasites utilize a short N-terminal amino acid motif termed the Plasmodium export element (PEXEL) to export an array of proteins to the host erythrocyte during blood stage infection. Using immunoaffinity chromatography and mass spectrometry, insight into this signal-mediated trafficking mechanism was gained by discovering that the PEXEL motif is cleaved and N-acetylated. PfHRPII and PfEMP2 are two soluble proteins exported by Plasmodium falciparum that were demonstrated to undergo PEXEL cleavage and N-acetylation, thus indicating that this N-terminal processing may be general to many exported soluble proteins. It was established that PEXEL processing occurs upstream of the brefeldin A-sensitive trafficking step in the P. falciparum secretory pathway, therefore cleavage and N-acetylation of the PEXEL motif occurs in the endoplasmic reticulum (ER) of the parasite. Furthermore, it was shown that the recognition of the processed N-terminus of exported proteins within the parasitophorous vacuole may be crucial for protein transport to the host erythrocyte. It appears that the PEXEL may be defined as a novel ER peptidase cleavage site and a classical N-acetyltransferase substrate sequence.
Assuntos
Plasmodium/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas de Protozoários/metabolismo , Animais , Cromatografia de Afinidade , Retículo Endoplasmático/metabolismo , Espectrometria de Massas , Modelos Biológicos , Sinais Direcionadores de ProteínasRESUMO
BACKGROUND: Meiotic pairing is essential for the proper orientation of chromosomes at the metaphase plate and their subsequent disjunction during anaphase I. In male Drosophila melanogaster, meiosis occurs in the absence of recombination or a recognizable synaptonemal complex (SC). Due to limitations in available cytological techniques, the early stages of homologous chromosome pairing in male Drosophila have not been observed, and the mechanisms involved are poorly understood. RESULTS: Chromosome tagging with GFP-Lac repressor protein allowed us to track, for the first time, the behavior of meiotic chromosomes at high resolution, live, at all stages of male Drosophila meiosis. Homologous chromosomes pair throughout the euchromatic regions in spermatogonia and during the early phases of spermatocyte development. Extensive separation of homologs and sister chromatids along the chromosome arms occurs in mid-G2, several hours before the first meiotic division, and before the G2/M transition. Centromeres, on the other hand, show complex association patterns, with specific homolog pairing taking place in mid-G2. These changes in chromosome pairing parallel changes in large-scale chromosome organization. CONCLUSIONS: Our results suggest that widespread interactions along the euchromatin are required for the initiation, but not the maintenance, of meiotic pairing of autosomes in male Drosophila. We propose that heterochromatic associations, or chromatid entanglement, may be responsible for the maintenance of homolog association during late G2. Our data also suggest that the formation of chromosome territories in the spermatocyte nucleus may play an active role in ensuring the specificity of meiotic pairing in late prophase by disrupting interactions between nonhomologous chromosomes.
Assuntos
Pareamento Cromossômico/fisiologia , Drosophila melanogaster/fisiologia , Meiose/fisiologia , Espermatócitos/fisiologia , Anáfase , Animais , Células Cultivadas , Cromátides/fisiologia , Segregação de Cromossomos/fisiologia , Drosophila melanogaster/genética , Feminino , Genes Reporter , Proteínas de Fluorescência Verde , Imageamento Tridimensional , Óperon Lac , Proteínas Luminescentes/genética , Masculino , Metáfase , Microscopia de Fluorescência , Modelos Genéticos , Fotomicrografia/métodos , Prófase , Recombinação Genética , Espermatócitos/ultraestrutura , Espermatogônias/ultraestrutura , Complexo Sinaptonêmico/fisiologiaRESUMO
Trabecular bone structure and bone density contribute to the strength of bone and are important in the study of osteoporosis. Wavelets are a powerful tool in characterizing and quantifying texture in an image. The purpose of this study was to validate wavelets as a tool in computing trabecular bone thickness directly from gray-level images. To this end, eight cylindrical cores of vertebral trabecular bone were imaged using 3-T magnetic resonance imaging (MRI) and micro-computed tomography (microCT). Thickness measurements of the trabecular bone from the wavelet-based analysis were compared with standard 2D structural parameters analogous to bone histomorphometry (MR images) and direct 3D distance transformation methods (microCT images). Additionally, bone volume fraction was determined using each method. The average difference in trabecular thickness between the wavelet and standard methods was less than the size of 1 pixel size for both MRI and microCT analysis. A correlation (R) of .94 for microCT measurements and that of .52 for MRI were found for the bone volume fraction. Based on these results, we conclude that wavelet-based methods deliver results comparable with those from established MR histomorphometric measurements. Because the wavelet transform is more robust with respect to image noise and operates directly on gray-level images, it could be a powerful tool for computing structural bone parameters from MR images acquired using high resolution and thus limited signal scenarios.
Assuntos
Densidade Óssea/fisiologia , Densitometria/métodos , Interpretação de Imagem Assistida por Computador/métodos , Imageamento Tridimensional/métodos , Imageamento por Ressonância Magnética/métodos , Coluna Vertebral/anatomia & histologia , Coluna Vertebral/fisiologia , Algoritmos , Humanos , Aumento da Imagem/métodos , Técnicas In Vitro , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Tomografia Computadorizada por Raios XRESUMO
We have developed a color barcode labeling strategy for use with fluorescence in situ hybridization that enables the discrimination of multiple, identically labeled loci. Barcode labeling of chromosomes provides long-range path information and allows structural analysis at a scale and resolution beyond what was previously possible. Here, we demonstrate the use of a three-color, 13-probe barcode for the structural analysis of Drosophila chromosome 2L in blastoderm stage embryos. We observe the chromosome to be strongly polarized in the Rabl orientation and for some loci to assume defined positions relative to the nuclear envelope. Our analysis indicates packing approximately 15- to 28-fold above the 30-nm fiber, which varies along the chromosome in a pattern conserved across embryos. Using a clustering implementation based on rigid body alignment, our analysis suggests that structures within each embryo represent a single population and are effectively modeled as oriented random coils confined within nuclear boundaries. We also found an increased similarity between homologous chromosomes that have begun to pair. Chromosomes in embryos at equivalent developmental stages were found to share structural features and nuclear localization, although size-related differences that correlate with the cell cycle also were observed. The methodology and tools we describe provide a direct means for identifying developmental and cell type-specific features of higher order chromosome and nuclear organization.
Assuntos
Cromossomos/metabolismo , Cor , Drosophila melanogaster/citologia , Drosophila melanogaster/genética , Hibridização in Situ Fluorescente/instrumentação , Hibridização in Situ Fluorescente/métodos , Interfase , Animais , Núcleo Celular/metabolismo , Análise por Conglomerados , Drosophila melanogaster/embriologiaRESUMO
Full resolution electron microscopic tomographic (EMT) reconstruction of large-scale tilt series requires significant computing power. The desire to perform multiple cycles of iterative reconstruction and realignment dramatically increases the pressing need to improve reconstruction performance. This has motivated us to develop a distributed multi-GPU (graphics processing unit) system to provide the required computing power for rapid constrained, iterative reconstructions of very large three-dimensional (3D) volumes. The participating GPUs reconstruct segments of the volume in parallel, and subsequently, the segments are assembled to form the complete 3D volume. Owing to its power and versatility, the CUDA (NVIDIA, USA) platform was selected for GPU implementation of the EMT reconstruction. For a system containing 10 GPUs provided by 5 GTX295 cards, 10 cycles of SIRT reconstruction for a tomogram of 4096(2) × 512 voxels from an input tilt series containing 122 projection images of 4096(2) pixels (single precision float) takes a total of 1845 s of which 1032 s are for computation with the remainder being the system overhead. The same system takes only 39 s total to reconstruct 1024(2) × 256 voxels from 122 1024(2) pixel projections. While the system overhead is non-trivial, performance analysis indicates that adding extra GPUs to the system would lead to steadily enhanced overall performance. Therefore, this system can be easily expanded to generate superior computing power for very large tomographic reconstructions and especially to empower iterative cycles of reconstruction and realignment.
Assuntos
Tomografia com Microscopia Eletrônica/estatística & dados numéricos , Algoritmos , Animais , Centrossomo/ultraestrutura , Redes de Comunicação de Computadores , Gráficos por Computador , Drosophila/ultraestrutura , Processamento de Imagem Assistida por Computador/estatística & dados numéricos , Imageamento Tridimensional/estatística & dados numéricosRESUMO
A three-dimensional wide-field image of a small fluorescent bead contains more than enough information to accurately calculate the wavefront in the microscope objective back pupil plane using the phase retrieval technique. The phase-retrieved wavefront can then be used to set a deformable mirror to correct the point-spread function (PSF) of the microscope without the use of a wavefront sensor. This technique will be useful for aligning the deformable mirror in a widefield microscope with adaptive optics and could potentially be used to correct aberrations in samples where small fluorescent beads or other point sources are used as reference beacons. Another advantage is the high resolution of the retrieved wavefont as compared with current Shack-Hartmann wavefront sensors. Here we demonstrate effective correction of the PSF in 3 iterations. Starting from a severely aberrated system, we achieve a Strehl ratio of 0.78 and a greater than 10-fold increase in maximum intensity.
RESUMO
Photoactivated localization microscopy (PALM) and related fluorescent biological imaging methods are capable of providing very high spatial resolutions (up to 20 nm). Two major demands limit its widespread use on biological samples: requirements for photoactivatable/photoconvertible fluorescent molecules, which are sometimes difficult to incorporate, and high background signals from autofluorescence or fluorophores in adjacent focal planes in three-dimensional imaging which reduces PALM resolution significantly. We present here a high-resolution PALM method utilizing conventional EGFP as the photoconvertible fluorophore, improved algorithms to deal with high levels of biological background noise, and apply this to imaging higher order chromatin structure. We found that the emission wavelength of EGFP is efficiently converted from green to red when exposed to blue light in the presence of reduced riboflavin. The photon yield of red-converted EGFP using riboflavin is comparable to other bright photoconvertible fluorescent proteins that allow <20 nm resolution. We further found that image pre-processing using a combination of denoising and deconvolution of the raw PALM images substantially improved the spatial resolution of the reconstruction from noisy images. Performing PALM on Drosophila mitotic chromosomes labeled with H2AvD-EGFP, a histone H2A variant, revealed filamentous components of â¼70 nm. This is the first observation of fine chromatin filaments specific for one histone variant at a resolution approximating that of conventional electron microscope images (10-30 nm). As demonstrated by modeling and experiments on a challenging specimen, the techniques described here facilitate super-resolution fluorescent imaging with common biological samples.
Assuntos
Cromossomos de Insetos/química , Cromossomos de Insetos/genética , Drosophila/genética , Histonas/química , Microscopia de Fluorescência/métodos , Mitose , Animais , Estruturas Cromossômicas , Drosophila/química , Proteínas de Fluorescência Verde/química , Microscopia de Fluorescência/instrumentaçãoRESUMO
In a forward genetic screen for mutations that destabilize the neuromuscular junction, we identified a novel long isoform of Drosophila ankyrin2 (ank2-L). We demonstrate that loss of presynaptic Ank2-L not only causes synapse disassembly and retraction but also disrupts neuronal excitability and NMJ morphology. We provide genetic evidence that ank2-L is necessary to generate the membrane constrictions that normally separate individual synaptic boutons and is necessary to achieve the normal spacing of subsynaptic protein domains, including the normal organization of synaptic cell adhesion molecules. Mechanistically, synapse organization is correlated with a lattice-like organization of Ank2-L, visualized using extended high-resolution structured-illumination microscopy. The stabilizing functions of Ank2-L can be mapped to the extended C-terminal domain that we demonstrate can directly bind and organize synaptic microtubules. We propose that a presynaptic Ank2-L lattice links synaptic membrane proteins and spectrin to the underlying microtubule cytoskeleton to organize and stabilize the presynaptic terminal.