Plane Illumination Research Articles

Advancing ovarian folliculometry with selective plane illumination microscopy.

Determination of ovarian status and follicle monitoring are common methods of diagnosing female infertility. We evaluated the suitability of selective plane illumination microscopy (SPIM) for the study of ovarian follicles. The large field of view and fast acquisition speed of our SPIM system enables rendering of volumetric image stacks from intact whole porcine ovarian follicles, clearly visualizing follicular features including follicle volume and average diameter (70 μm–2.5 mm), their spherical asymmetry parameters, size of developing cumulus oophorus complexes (40 μm–110 μm), and follicular wall thickness (90 μm–120 μm). Follicles at all developmental stages were identified. A distribution of the theca thickness was measured for each follicle, and a relationship between these distributions and the stages of follicular development was discerned. The ability of the system to non-destructively generate sub-cellular resolution 3D images of developing follicles, with excellent image contrast and high throughput capacity compared to conventional histology, suggests that it can be used to monitor follicular development and identify structural abnormalities indicative of ovarian ailments. Accurate folliculometric measurements provided by SPIM images can immensely help the understanding of ovarian physiology and provide important information for the proper management of ovarian diseases.

3D Visualization of Developmental Toxicity of 2,4,6-Trinitrotoluene in Zebrafish Embryogenesis Using Light-Sheet Microscopy.

Environmental contamination by trinitrotoluene is of global concern due to its widespread use in military ordnance and commercial explosives. Despite known long-term persistence in groundwater and soil, the toxicological profile of trinitrotoluene and other explosive wastes have not been systematically measured using in vivo biological assays. Zebrafish embryos are ideal model vertebrates for high-throughput toxicity screening and live in vivo imaging due to their small size and transparency during embryogenesis. Here, we used Single Plane Illumination Microscopy (SPIM)/light sheet microscopy to assess the developmental toxicity of explosive-contaminated water in zebrafish embryos and report 2,4,6-trinitrotoluene-associated developmental abnormalities, including defects in heart formation and circulation, in 3D. Levels of apoptotic cell death were higher in the actively developing tissues of trinitrotoluene-treated embryos than controls. Live 3D imaging of heart tube development at cellular resolution by light-sheet microscopy revealed trinitrotoluene-associated cardiac toxicity, including hypoplastic heart chamber formation and cardiac looping defects, while the real time PCR (polymerase chain reaction) quantitatively measured the molecular changes in the heart and blood development supporting the developmental defects at the molecular level. Identification of cellular toxicity in zebrafish using the state-of-the-art 3D imaging system could form the basis of a sensitive biosensor for environmental contaminants and be further valued by combining it with molecular analysis.

Imaging the lung: the old ways and the new.

Our understanding of lung biology can be greatly enhanced by studying embryonic and postnatal lung development, and the perturbations which occur during disease. Imaging techniques provide a unique insight into these processes. A wide variety of imaging techniques have been used to study the lungs at various stages of development and disease, ranging from histological stains to more novel techniques such as single plane illumination microscopy (SPIM), intravital microscopy (IVM), and micro-computed tomography (micro-CT). Each of these tools can be used to elicit different information about the lungs and each has its own unique advantages and disadvantages for pulmonary research. In this review we assess some of the most commonly-used and novel imaging techniques available for lung research today.

πSPIM: high NA high resolution isotropic light-sheet imaging in cell culture dishes.

Light-sheet fluorescence microscopy (LSFM), also termed single plane illumination microscopy (SPIM), enables live cell fluorescence imaging with optical sectioning capabilities superior to confocal microscopy and without any out-of-focus exposure of the specimen. However, the need of two objective lenses, one for light-sheet illumination and one for imaging, imposes geometrical constraints that require LSFM setups to be adapted to the specific needs of different types of specimen in order to obtain optimal imaging conditions. Here we demonstrate the use of an oblique light-sheet configuration adapted to provide the highest possible Gaussian beam enabled resolution in LSFM. The oblique light-sheet configuration furthermore enables LSFM imaging at the surface of a cover slip, without the need of specific sample mounting. In addition, the system is compatible with simultaneous high NA wide-field epi-fluorescence imaging of the specimen contained in a glass-bottom cell culture dish. This prevents cumbersome sample mounting and enables rapid screening of large areas of the specimen followed by high-resolution LSFM imaging of selected cells. We demonstrate the application of this microscope for in toto imaging of endocytosis in yeast, showing for the first time imaging of all endocytic events of a given cell over a period of >5 minutes with sub-second resolution.

Scattering correction through a space-variant blind deconvolution algorithm.

Scattering within biological samples limits the imaging depth and the resolution in microscopy. We present a prior and regularization approach for blind deconvolution algorithms to correct the influence of scattering to increase the imaging depth and resolution. The effect of the prior is demonstrated on a three-dimensional image stack of a zebrafish embryo captured with a selective plane illumination microscope. Blind deconvolution algorithms model the recorded image as a convolution between the distribution of fluorophores and a point spread function (PSF). Our prior uses image information from adjacent z-planes to estimate the unknown blur in tissue. The increased size of the PSF due to the cascading effect of scattering in deeper tissue is accounted for by a depth adaptive regularizer model. In a zebrafish sample, we were able to extend the point in depth, where scattering has a significant effect on the image quality by around 30???m.

Biocompatibility and biodistribution of functionalized carbon nano-onions (f-CNOs) in a vertebrate model.

Functionalized carbon nano-onions (f-CNOs) are of great interest as platforms for imaging, diagnostic and therapeutic applications due to their high cellular uptake and low cytotoxicity. To date, the toxicological effects of f-CNOs on vertebrates have not been reported. In this study, the possible biological impact of f-CNOs on zebrafish during development is investigated, evaluating different toxicity end-points such as the survival rate, hatching rate, and heart beat rate. Furthermore, a bio-distribution study of boron dipyrromethene (BODIPY) functionalized CNOs in zebrafish larvae is performed by utilizing inverted selective plane illumination microscopy (iSPIM), due to its intrinsic capability of allowing for fast 3D imaging. Our in vivo findings indicate that f-CNOs exhibit no toxicity, good biocompatibility (in the concentration range of 5–100 μg mL−1) and a homogenous biodistribution in zebrafish larvae.

EduSPIM: Light Sheet Microscopy in the Museum.

Light Sheet Microscopy in the MuseumLight sheet microscopy (or selective plane illumination microscopy) is an important imaging technique in the life sciences. At the same time, this technique is also ideally suited for community outreach projects, because it produces visually appealing, highly dynamic images of living organisms and its working principle can be understood with basic optics knowledge. Still, the underlying concepts are widely unknown to the non-scientific public. On the occasion of the UNESCO International Year of Light, a technical museum in Dresden, Germany, launched a special, interactive exhibition. We built a fully functional, educational selective plane illumination microscope (eduSPIM) to demonstrate how developments in microscopy promote discoveries in biology.Design Principles of an Educational Light Sheet MicroscopeTo maximize educational impact, we radically reduced a standard light sheet microscope to its essential components without compromising functionality and incorporated stringent safety concepts beyond those needed in the lab. Our eduSPIM system features one illumination and one detection path and a sealed sample chamber. We image fixed zebrafish embryos with fluorescent vasculature, because the structure is meaningful to laymen and visualises the optical principles of light sheet microscopy. Via a simplified interface, visitors acquire fluorescence and transmission data simultaneously.The eduSPIM Design Is Tailored Easily to Fit Numerous ApplicationsThe universal concepts presented here may also apply to other scientific approaches that are communicated to laymen in interactive settings. The specific eduSPIM design is adapted easily for various outreach and teaching activities. eduSPIM may even prove useful for labs needing a simple SPIM. A detailed parts list and schematics to rebuild eduSPIM are provided.

Using Stage- and Slit-Scanning to Improve Contrast and Optical Sectioning in Dual-View Inverted Light Sheet Microscopy (diSPIM).

Dual-view inverted selective plane illumination microscopy (diSPIM) enables high-speed, long-term, four-dimensional (4D) imaging with isotropic spatial resolution. It is also compatible with conventional sample mounting on glass coverslips. However, broadening of the light sheet at distances far from the beam waist and sample-induced scattering degrades diSPIM contrast and optical sectioning. We describe two simple improvements that address both issues and entail no additional hardware modifications to the base diSPIM. First, we demonstrate improved diSPIM sectioning by keeping the light sheet and detection optics stationary, and scanning the sample through the stationary light sheet (rather than scanning the broadening light sheet and detection plane through the stationary sample, as in conventional diSPIM). This stage-scanning approach allows a thinner sheet to be used when imaging laterally extended samples, such as fixed microtubules or motile mitochondria in cell monolayers, and produces finer contrast than does conventional diSPIM. We also used stage-scanning diSPIM to obtain high-quality, 4D nuclear datasets derived from an uncompressed nematode embryo, and performed lineaging analysis to track 97% of cells until twitching. Second, we describe the improvement of contrast in thick, scattering specimens by synchronizing light-sheet synthesis with the rolling, electronic shutter of our scientific complementary metal-oxide-semiconductor (sCMOS) detector. This maneuver forms a virtual confocal slit in the detection path, partially removing out-of-focus light. We demonstrate the applicability of our combined stage- and slit-scanning- methods by imaging pollen grains and nuclear and neuronal structures in live nematode embryos. All acquisition and analysis code is freely available online.

Multimodal embryonic imaging using optical coherence tomography, selective plane illumination microscopy, and optical projection tomography.

The murine model is commonly utilized for studying developmental diseases. Different optical techniques have been developed to image mouse embryos, but each has its own set of limitations and restrictions. In this study, we compare the performance of the well-established technique of optical coherence tomography (OCT) to the relatively new methods of selective plane illumination microscopy (SPIM) and optical projection tomography (OPT) to assess murine embryonic development. OCT can provide label free high resolution images of the mouse embryo, but suffers from light attenuation that limits visualization of deeper structures. SPIM is able to image shallow regions with great detail utilizing fluorescent contrast. OPT can provide superior imaging depth, and can also use fluorescence labels but, it requires samples to be fixed and cleared before imaging. OCT requires no modification of the embryo, and thus, can be used in vivo and in utero. In this study, we compare the efficacy of OCT, SPIM, and OPT for imaging murine embryonic development. The data demonstrate the superior capability of SPIM and OPT for imaging fine structures with high resolution while only OCT can provide structural and functional imaging of live embryos with micrometer scale resolution.

Spatiotemporal mapping of diffusion dynamics and organization in plasma membranes

Imaging fluorescence correlation spectroscopy (FCS) and the related FCS diffusion law have been applied in recent years to investigate the diffusion modes of lipids and proteins in membranes. These efforts have provided new insights into the membrane structure below the optical diffraction limit, new information on the existence of lipid domains, and on the influence of the cytoskeleton on membrane dynamics. However, there has been no systematic study to evaluate how domain size, domain density, and the probe partition coefficient affect the resulting imaging FCS diffusion law parameters. Here, we characterize the effects of these factors on the FCS diffusion law through simulations and experiments on lipid bilayers and live cells. By segmenting images into smaller 7 × 7 pixel areas, we can evaluate the FCS diffusion law on areas smaller than 2 µm and thus provide detailed maps of information on the membrane structure and heterogeneity at this length scale. We support and extend this analysis by deriving a mathematical expression to calculate the mean squared displacement (MSDACF) from the autocorrelation function of imaging FCS, and demonstrate that the MSDACF plots depend on the existence of nanoscopic domains. Based on the results, we derive limits for the detection of domains depending on their size, density, and relative viscosity in comparison to the surroundings. Finally, we apply these measurements to bilayers and live cells using imaging total internal reflection FCS and single plane illumination microscopy FCS.

Abstract 4262: Combined 3D quantitative imaging and 3D cell culture for cancer drug discovery

Abstract Monitoring drug activity in multicellular tumor spheroid in vitro models is an important challenge as these 3D models have demonstrated their major relevance in pharmacological screening and anticancer drugs evaluation (1). Optical microscopy coupled to the selective plane illumination microscopy (SPIM)3D microscopy, and, and automatic automated images processing and quantitative analysis are promising approaches to process optimizes the evaluation of drug response in a 3D cell culture models with, in a high/mediumhigh throughput screening waycapabilities. BIOMIMESYS® used as a 3D cell model is based on hyaluronic to provide an in vivo like environment to the cells. The In this study the effects of the topoisomerase I inhibitor Topotecan were investigated on colon adenocarcinoma HCT-116 3D cell culture. Spheroids were prepared and grown in BIOMIMESYS® used as a 3D cell model isa matrix based on hyaluronic acid tothat provides an in vivo like environment to the cells. was monitored and coupled with mMorphometric parameters and with nuclei fluorescence markers were used to monitor analysis for drug activity. Data provideand to characterization characterize of both cytostatic and cytotoxic effects of this drug withon the basis of both quantitative 3D and imaging of the mechanisms. These data highlight the relevance of combining a biomimetic microenvironment for 3D cell culture recreating the the in vivo features of a tumor cell population biology, together with and high resolution content microscopy-based quantification for drug screening. This biomimetic model provides an innovative platform for the in-depth analysis of tumor development and for the discovery or the characterization of novel therapeutic targets. Ref: 1) Breslin S, O’Driscoll L. Drug Discov Today.18: 240-249 (2013) Citation Format: Guillaume Vidal, Valérie Lobjois, Zied Souguir, Pauline Pannetier, Elise Demange, Jean-Michel Lagarde. Combined 3D quantitative imaging and 3D cell culture for cancer drug discovery. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4262.

Image Cross-Correlation Analysis of Time Varying Flows.

In vivo studies of blood circulation pathologies have great medical relevance and need methods for the characterization of time varying flows at high spatial and time resolution in small animal models. We test here the efficacy of the combination of image correlation techniques and single plane illumination microscopy (SPIM) in characterizing time varying flows in vitro and in vivo. As indicated by numerical simulations and by in vitro experiments on straight capillaries, the complex analytical form of the cross-correlation function for SPIM detection can be simplified, in conditions of interest for hemodynamics, to a superposition of Gaussian components, easily amenable to the analysis of variable flows. The possibility to select a wide field of view with a good spatial resolution along the collection optical axis and to compute the cross-correlation between regions of interest at varying distances on a single time stack of images allows one to single out periodic flow components from spurious peaks on the cross-correlation functions and to infer the duration of each flow component. We apply this cross-correlation analysis to the blood flow in Zebrafish embryos at 4 days after fertilization, measuring the average speed and the duration of the systolic and diastolic phases.

The Secreted Signaling Protein Wnt3 Is Associated with Membrane Domains In Vivo: A SPIM-FCS Study

Wnt3 is a morphogen that activates the Wnt signaling pathway and regulates a multitude of biological processes ranging from cell proliferation and cell fate specification to differentiation over embryonic induction to neural patterning. Recent studies have shown that the palmitoylation of Wnt3 by Porcupine, a membrane-bound O-acyltransferase, plays a significant role in the intracellular membrane trafficking of Wnt3 and subsequently, its secretion in live zebrafish embryos, where chemical inhibition of Porcupine reduced the membrane-bound and secreted fractions of Wnt3 and eventually led to defective brain development. However, the membrane distribution of Wnt3 in cells remains not fully understood. Here, we determine the membrane organization of functionally active Wnt3-EGFP in cerebellar cells of live transgenic zebrafish embryos and the role of palmitoylation in its organization using single plane illumination microscopy-fluorescence correlation spectroscopy (SPIM-FCS), a multiplexed modality of FCS, which generates maps of molecular dynamics, concentration, and interaction of biomolecules. The FCS diffusion law was applied to SPIM-FCS data to study the subresolution membrane organization of Wnt3. We find that at the plasma membrane in vivo, Wnt3 is associated with cholesterol-dependent domains. This association reduces with increasing concentrations of Porcupine inhibitor (C59), confirming the importance of palmitoylation of Wnt3 for its association with cholesterol-dependent domains. Reduction of membrane cholesterol also results in a decrease of Wnt3 association with cholesterol-dependent domains in live zebrafish. This demonstrates for the first time, to our knowledge, in live vertebrate embryos that Wnt3 is associated with cholesterol-dependent domains.

Light-sheet microscopy for everyone? Experience of building an OpenSPIM to study flatworm development.

BackgroundSelective plane illumination microscopy (SPIM a type of light-sheet microscopy) involves focusing a thin sheet of laser light through a specimen at right angles to the objective lens. As only the thin section of the specimen at the focal plane of the lens is illuminated, out of focus light is naturally absent and toxicity due to light (phototoxicity) is greatly reduced enabling longer term live imaging. OpenSPIM is an open access platform (Pitrone et al. 2013 and OpenSPIM.org) created to give new users step-by-step instructions on building a basic configuration of a SPIM microscope, which can in principle be adapted and upgraded to each laboratory’s own requirements and budget. Here we describe our own experience with the process of designing, building, configuring and using an OpenSPIM for our research into the early development of the polyclad flatworm Maritigrella crozieri – a non-model animal.ResultsOur OpenSPIM builds on the standard design with the addition of two colour laser illumination for simultaneous detection of two probes/molecules and dual sided illumination, which provides more even signal intensity across a specimen. Our OpenSPIM provides high resolution 3d images and time lapse recordings, and we demonstrate the use of two colour lasers and the benefits of two color dual-sided imaging. We used our microscope to study the development of the embryo of the polyclad flatworm M. crozieri. The capabilities of our microscope are demonstrated by our ability to record the stereotypical spiral cleavage pattern of M. crozieri with high-speed multi-view time lapse imaging. 3D and 4D (3D + time) reconstruction of early development from these data is possible using image registration and deconvolution tools provided as part of the open source Fiji platform. We discuss our findings on the pros and cons of a self built microscope.ConclusionsWe conclude that home-built microscopes, such as an OpenSPIM, together with the available open source software, such as MicroManager and Fiji, make SPIM accessible to anyone interested in having continuous access to their own light-sheet microscope. However, building an OpenSPIM is not without challenges and an open access microscope is a worthwhile, if significant, investment of time and money. Multi-view 4D microscopy is more challenging than we had expected. We hope that our experience gained during this project will help future OpenSPIM users with similar ambitions.Electronic supplementary materialThe online version of this article (doi:10.1186/s12861-016-0122-0) contains supplementary material, which is available to authorized users.

Selective plane illumination microscopy with a light sheet of uniform thickness formed by an electrically tunable lens.

Light sheet microscopy is a powerful technique for rapid, three-dimensional fluorescence imaging of large specimen such as drosophila and zebrafish embryos. Yet, beam divergence results in a loss of axial resolution at the periphery of the light sheet. Here, we demonstrate how an electrically tunable lens can be utilized to maintain the minimal, diffraction-limited thickness of the light sheet over a wide field of view (>600 µm) at high frame rates (40 fps). This mode of operation is necessary for the application of fluorescence fluctuation spectroscopy in images. Microsc. Res. Tech. 81:924-928, 2018. © 2016 Wiley Periodicals, Inc.

Single-molecule RNA detection at depth by hybridization chain reaction and tissue hydrogel embedding and clearing.

Accurate and robust detection of mRNA molecules in thick tissue samples can reveal gene expression patterns in single cells within their native environment. Preserving spatial relationships while accessing the transcriptome of selected cells is a crucial feature for advancing many biological areas – from developmental biology to neuroscience. However, because of the high autofluorescence background of many tissue samples, it is difficult to detect single-molecule fluorescence in situ hybridization (smFISH) signals robustly in opaque thick samples. Here, we draw on principles from the emerging discipline of dynamic nucleic acid nanotechnology to develop a robust method for multi-color, multi-RNA imaging in deep tissues using single-molecule hybridization chain reaction (smHCR). Using this approach, single transcripts can be imaged using epifluorescence, confocal or selective plane illumination microscopy (SPIM) depending on the imaging depth required. We show that smHCR has high sensitivity in detecting mRNAs in cell culture and whole-mount zebrafish embryos, and that combined with SPIM and PACT (passive CLARITY technique) tissue hydrogel embedding and clearing, smHCR can detect single mRNAs deep within thick (0.5 mm) brain slices. By simultaneously achieving ∼20-fold signal amplification and diffraction-limited spatial resolution, smHCR offers a robust and versatile approach for detecting single mRNAs in situ, including in thick tissues where high background undermines the performance of unamplified smFISH.

A Variational Model for Multiplicative Structured Noise Removal

We consider the problem of restoring images impaired by noise that is simultaneously structured and multiplicative. Our primary motivation for this setting is the selective plane illumination micro...

Model-based shading and lighting controls considering visual comfort and energy use

Dynamic facades with high performance glazing and shading systems have the potential to balance daylighting needs, comfort and energy use, when integrated with lighting and thermal system controls. This paper presents the development and implementation of model-based control (MBC) algorithms for shading and lighting operation, aiming at minimizing lighting energy use while satisfying glare constraints. A fast and reliable lighting-glare model was used to compute real-time interior lighting conditions, lighting energy use and daylight glare probability, for predetermined shade positions (10% increments) at each control time step, based on the readings of two sensors on each building facade. Three visual comfort criteria (DGP, vertical and work plane illuminance) were embedded and compared in the real-time shading control logic, which selects the highest shade position (maximum daylight utilization) that satisfies the visual comfort criteria at each time step. The approach is similar to an optimization scheme for discrete shade positions, using visual comfort constraints as a priority, but here it is applied efficiently with a semi-analytical model in real-time control.The developed MBC strategies were successfully demonstrated in office spaces, controlling shades and electric lighting in real-time, using simple sensor readings as inputs. An innovative, variable control interval logic was also developed and implemented, resulting in reduced shading operation without sacrificing the benefits, creating less disturbance for occupants and extending equipment life. Finally, the developed MBC strategies were evaluated using annual simulation, in terms of frequency of shade movements, annual percentage of working hours with risk of glare, lighting energy use, and fraction of outside view.

Single shot embedded surface plasmon microscopy with vortex illumination.

In previous work we demonstrated how a confocal microscope with a spatial light modulator in the back focal plane could perform accurate measurement of the k-vector of a propagating surface plasmon. This involved forming an embedded interferometer between light incident close to normal incidence (reference beam) and light incident at the angle to excite surface plasmons (sample beam). The signal from the interferometer was extracted by stepping the phase of the reference beam relative to the sample beam using a spatial light modulator; this requires at least 3 phase steps, which limits the speed of operation. To overcome this and extract the same information with a single measurement, we project an azimuthal varying phase between 0 and 2π in the central region of the back focal plane; corresponding to small angles of incidence. This projects a vortex beam as the reference, so that the phase of the reference beam varies with azimuthal angle. By extracting the interference signal from different portions of the reference beam, different phase steps between the reference and the sample are obtained, so all the values required for phase reconstruction can be extracted simultaneously. It is thus possible to obtain the same information with a single shot measurement, at each defocus position, without additional changes to the back focal plane illumination. Results are presented to show that the vortex illuminated sample provides similar results to the phase stepped version, whose values are, in turn, validated with ellipsometry and surface profilometry.

Deep imaging in scattering media with selective plane illumination microscopy.

In most biological tissues, light scattering due to small differences in refractive index limits the depth of optical imaging systems. Two-photon microscopy (2PM), which significantly reduces the scattering of the excitation light, has emerged as the most common method to image deep within scattering biological tissue. This technique, however, requires high-power pulsed lasers that are both expensive and difficult to integrate into compact portable systems. Using a combination of theoretical and experimental techniques, we show that if the excitation path length can be minimized, selective plane illumination microscopy (SPIM) can image nearly as deep as 2PM without the need for a high-powered pulsed laser. Compared to other single-photon imaging techniques like epifluorescence and confocal microscopy, SPIM can image more than twice as deep in scattering media ( ? 10 times the mean scattering length). These results suggest that SPIM has the potential to provide deep imaging in scattering media in situations in which 2PM systems would be too large or costly.