Light sheet fluorescence microscopy

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Three photon light sheet fluorescence microscopy

Three photon light sheet fluorescence microscopy

In parallel, the geometry used in light-sheet fluorescence microscopy (LSFM) has revolutionized the field of imaging by using a thin sheet of light to optically section samples which are typically transparent. In this technique, fluorescent light emitted by the sample is collected by a detection imaging system that is perpendicular to the illuminated plane. This particular config- uration results in improved contrast and high axial resolution with very short acquisition times because it avoids scanning a focused beam across the field-of-view (FOV) [7]. In addition, as only the plane of interest is illuminated during a single exposure, photo-toxicity is vastly reduced. This makes LSFM very attrac- tive for long term live imaging of biomedical samples [8, 9]. At the same time, the FOV can be increased in LFSM notably by using propagation invariant light fields such as Bessel and Airy beams [10, 11].
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Image analysis in light sheet fluorescence microscopy images of transgenic zebrafish vascular development

Image analysis in light sheet fluorescence microscopy images of transgenic zebrafish vascular development

Abstract. The zebrafish has become an established model to study vas- cular development and disease in vivo. However, despite it now being pos- sible to acquire high-resolution data with state-of-the-art fluorescence microscopy, such as lightsheet microscopy, most data interpretation in pre-clinical neurovascular research relies on visual subjective judgement, rather than objective quantification. Therefore, we describe the develop- ment of an image analysis workflow towards the quantification and de- scription of zebrafish neurovascular development. In this paper we focus on data acquisition by lightsheet fluorescence microscopy, data proper- ties, image pre-processing, and vasculature segmentation, and propose future work to derive quantifications of zebrafish neurovasculature de- velopment.
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Light sheet fluorescence microscopy for neuroscience

Light sheet fluorescence microscopy for neuroscience

with close to isotropic resolution. In this configuration, the detection and col- lection paths are still orthogonal to each other but are placed at a 45 degree angle relative to the sample that is usually placed on a regular microscope slide. The require for orthogonality precludes the use of the highest NA objectives which restrict space, however, if the light sheet is made sufficiently thin, the ax-

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Macro optical trapping for sample confinement in light sheet microscopy

Macro optical trapping for sample confinement in light sheet microscopy

Light sheet fluorescence microscopy (LSFM) or selective plane illumination microscopy (SPIM) uses a thin sheet of light to illuminate a sample, whilst fluorescent images are taken perpendicular to the illuminated plane [1]. This geometry gives LSFM multiple advantages over other types of microscopy: Firstly, the unilluminated part of the sample remains unex- posed to light and cannot be detected. This not only enhances the axial resolution and image contrast, but it also reduces photo-bleaching and phototoxicity to which the sample is exposed. Secondly, the axial resolution of LSFM is mainly determined by the thickness of the light sheet, which is independent of the detection optics. Hence low magnification objectives can be used for a large field of view (FOV) while still achieving good axial resolution. Thirdly, as the whole plane is simultaneously illuminated and imaged, the imaging speed is dramatically enhanced compared to scanning confocal microscopy. These advantages make LSFM suitable for con- structing 3D images of large samples and even long term monitoring of a living sample. This modality can been extended by utilizing more advanced beam shapes, such as the Bessel beam or the Airy beam [2–5].
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Integrating light-sheet imaging with virtual reality to recapitulate developmental cardiac mechanics

Integrating light-sheet imaging with virtual reality to recapitulate developmental cardiac mechanics

Figure 2. Endocardial trabecular network in a transgenic Tg(cmlc2-gfp) zebrafish ventricle at 60 dpf. (A) VR accentuates the invaginating muscu- lar ridges in the apical region. (B–D) VR-LSFM enables navigation through various projections into the branching network. Different views are indi- cated by white arrows (B). (E and F) The conventional (E) 2D raw data and (F) 3D rendering results are limited in revealing the highly trabeculated 2-chambered heart, consisting of an atrium and a ventricle, as the per- spective view is predefined. Scale bar: 100 μm. (G) Quantitative measure- ments of the distance between the ventriculobulbar valve leaflets. dpf, days after fertilization; VR, virtual reality; LSFM, light-sheet fluorescence microscopy. All of these images are shown in pseudocolor.
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PubMedCentral-PMC5752350.pdf

PubMedCentral-PMC5752350.pdf

Chagas disease is a parasitic infection caused by Trypanosoma cruzi, whose motility is not only important for localization, but also for cellular binding and invasion. Current animal models for the study of T. cruzi allow limited observation of parasites in vivo, representing a challenge for understanding parasite behavior during the initial stages of infection in humans. This protozoan has a flagellar stage in both vector and mammalian hosts, but there are no studies describing its motility in vivo.The objective of this project was to establish a live vertebrate zebrafish model to evaluate T. cruzi motility in the vascular system. Transparent zebrafish larvae were injected with fluorescently labeled trypomastigotes and observed using light sheet fluorescence microscopy (LSFM), a noninvasive method to visualize live organisms with high optical resolution. The parasites could be visualized for extended periods of time due to this technique's relatively low risk of photodamage compared to confocal or epifluorescence microscopy. T. cruzi parasites were observed traveling in the circulatory system of live zebrafish in different-sized blood vessels and the yolk. They could also be seen attached to the yolk sac wall and to the atrioventricular valve despite the strong forces associated with heart contractions. LSFM of T. cruzi-inoculated zebrafish larvae is a valuable method that can be used to visualize circulating parasites and evaluate their tropism, migration patterns, and motility in the dynamic environment of the cardiovascular system of a live animal.
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Multimode fibre : light sheet microscopy at the tip of a needle

Multimode fibre : light sheet microscopy at the tip of a needle

Light-sheet fluorescence microscopy has emerged as a powerful platform for 3-D volumetric imaging in the life sciences. Here, we introduce an important step towards its use deep inside biological tissue. Our new technique, based on digital holography, enables delivery of the light-sheet through a multimode optical fibre – an optical element with extremely small footprint, yet permitting complex control of light transport processes within. We show that this approach supports some of the most advanced methods in light-sheet microscopy: by taking advantage of the cylindrical symmetry of the fibre, we facilitate the wavefront engineering methods for generation of both Bessel and structured Bessel beam plane illumination. Finally, we assess the quality of imaging on a sample of fluorescent beads fixed in agarose gel and we conclude with a proof-of-principle imaging of a biological sample, namely the regenerating operculum prongs of Spirobranchus lamarcki.
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Wavefront corrected light sheet microscopy in turbid media

Wavefront corrected light sheet microscopy in turbid media

Light sheet microscopy (LSM) has emerged as a power- ful wide-field fluorescence technique that has demonstrated exceptional high-resolution, high-speed, imaging in a wide variety of applications from developmental biology to colloi- dal studies. 1–4 In particular, it offers powerful capabilities for imaging larger biomedical specimens. Rapid single-axis scanning can create a “thin” two-dimensional light sheet that is then projected into the specimen at 90 to the detection objective axis. Both the Gaussian light sheet (GLS) 5 and Bessel Beam light sheet (BBLS) 6 imaging geometries have emerged as popular choices. Image quality and resolution in LSM are directly linked to the light sheet thickness and its uniformity across the imaged field of view (FOV). Both of these key properties are degraded in the presence of scatter- ing and specimen-induced aberrations. Methods to circum- vent these deleterious effects have included tissue clearing, 7 Bessel light modes, 8 and post-processing background sup- pression. 9 However, to truly extend LSM to a wider range of biomedical samples in their native state requires a significant improvement in overcoming aberrations as and where they arise within the sample. In this letter, we demonstrate how an in situ wavefront correction addresses this key point and allows the reconstruction of the beam profile exactly where one desires within the sample medium. It is important to stress that our method does not require any specialist sample preparation and crucially can be used to significantly improve any form of input light mode used in LSM, includ- ing both Gaussian and Bessel modes.
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Quantitative imaging of cell dynamics in mouse embryos using light sheet microscopy

Quantitative imaging of cell dynamics in mouse embryos using light sheet microscopy

Single/selective-plane illumination, or light-sheet, systems offer several advantages over other fluorescence microscopy methods for live, 3D microscopy. These systems are valuable for studying embryonic development in several animal systems, such as Drosophila, C. elegans and zebrafish. The geometry of the light path in this form of microscopy requires the sample to be accessible from multiple sides and fixed in place so that it can be rotated around a single axis. Popular methods for mounting include hanging the specimen from a pin or embedding it in 1-2% agarose. These methods can be particularly problematic for certain samples, such as post- implantation mouse embryos, that expand significantly in size and are very delicate and sensitive to mounting. To overcome the current limitations and to establish a robust strategy for long-term (24 h) time-lapse imaging of E6.5-8.5 mouse embryos with light-sheet microscopy, we developed and tested a method using hollow agarose cylinders designed to accommodate for embryonic growth, yet provide boundaries to minimize tissue drift and enable imaging in multiple orientations. Here, we report the first 24-h time-lapse sequences of post-implantation mouse embryo development with light-sheet microscopy. We demonstrate that light-sheet imaging can provide both quantitative data for tracking changes in morphogenesis and reveal new insights into mouse embryogenesis. Although we have used this approach for imaging mouse embryos, it can be extended to imaging other types of embryos as well as tissue explants.
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Imaging Flies by Fluorescence Microscopy: Principles, Technologies, and Applications

Imaging Flies by Fluorescence Microscopy: Principles, Technologies, and Applications

Light sheet microscopy: In contrast to the above-mentioned optical sectioning microscopy technologies, in light sheet microscopy, the illumination and detection paths are perpen- dicular to each other (Figure 4B), a concept first introduced in 1993 (Voie et al. 1993). Building on this basic idea, Selective Plane Illumination Microscopy (SPIM) emerged in 2004, fa- cilitating unprecedented imaging speeds at cellular and sub- cellular resolution (Huisken et al. 2004). In light sheet microscopy, the specimen is illuminated by a focused light sheet generated, for example, by a cylindrical lens in the illumination path. In this way, a specific plane (optical sec- tion) of the specimen is selectively and directly illuminated across the entire field of view. All emitted fluorescence signals are collected at once by fast area detectors (e.g., CMOS tech- nology) included in the perpendicularly oriented detection path of a light sheet microscope. Hence, this illumination strategy enables imaging speeds that are multiple orders of magnitude faster than any other optical sectioning micros- copy technology. Due to the fact that only the imaged focal planes are illuminated at any given time, bleaching of fluo- rophores is strongly reduced and phototoxic effects on cells in a life-imaging setup are almost negligible (Icha et al. 2017; Laissue et al. 2017). In addition, the sample can be mounted in a way that allows its rotation around the z-axis to facilitate illumination and imaging from multiple angles (Schmied and Tomancak 2016). In general, light sheet microscopy often requires unconventional approaches to sample mount- ing, breaking away from the “biology on coverslips” paradigm (even though such an arrangement is also possible) [reviewed in Pampaloni et al. (2007)]. Furthermore, it has been suggested that light sheet microscopes should be built around the sample and consequently the sample can be mounted in the most physiological manner compatible with its long-term health [reviewed in Power and Huisken (2017)].
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Between Life and Death: Reducing Phototoxicity in Super-Resolution Microscopy

Between Life and Death: Reducing Phototoxicity in Super-Resolution Microscopy

sheet of light and then detecting the fluorescence perpendic- ular to the direction of sheet propagation (139, 140) (Fig. 5c, ‘Gaussian light sheet’). This confers low phototoxicity as only the part of the sample being imaged is illuminated without the need for non-linear optical processes (which is the case in two-photon microscopy). Indeed, light-sheet mi- croscopy was named the Nature Methods technique of the year in 2014, in part due to its low phototoxicity (141). There are several ways in which light-sheet microscopy schemes can yield super-resolution with reduced phototoxicity. Super- resolution in live samples has been demonstrated using light- sheet microscopy by simply combining this illumination ge- ometry with SRM techniques such as SMLM (142–144) and RESOLFT (145). However, the employed SRM methods still require high-intensity illumination, and thus such compos- ite techniques do not exploit the inherent low phototoxicity of light-sheet imaging. Therefore, a more elegant approach involves illuminating the sample with a light-sheet regime followed by the application of SMLM analytics designed for ultra-high-density datasets, which allows for reduction of the illumination power ((146) and Analytics section, see below). The more widely-explored method for combining SRM and light-sheet microscopy has been the use of novel methods for generating and shaping the light-sheet. Bessel beams have been used to generate thinner light-sheets (147), and these beams have also been extended to incorporate SIM (148). The latter strategy has also been demonstrated on a system with two counterpropagating light-sheets formed us- ing standard Gaussian beams (149). The most radical and live-imaging-friendly light-sheet SRM technique developed to date is lattice light-sheet microscopy (150) (Fig. 5c, ‘Lat- tice light sheet’). This has demonstrated 3D time-lapse super- resolution imaging in both cultured cells and intact model or- ganisms with minimal phototoxicity.
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Greiss, Ferdinand
  

(2017):


	Probing single molecules and binding networks: detection of single molecules on the multi-cellular scale with reflected light-sheet microscopy and probing the cooperativity of binding networks with high-throughput thermoph

Greiss, Ferdinand (2017): Probing single molecules and binding networks: detection of single molecules on the multi-cellular scale with reflected light-sheet microscopy and probing the cooperativity of binding networks with high-throughput thermophoresis. Dissertation, LMU München: Fakultät für Physik

In this study, we developed an automatized measurement platform to quantitatively investigate binding constants in reduced sample volumes of 500 nl and in high throughput with standardized 1,536-well plates. We first used our plat- form to review known concepts and to compare its perfor- mance to commercial solutions. With the high-throughput data acquisition, we then proposed a titration scenario with 256 data points to investigate the formation of a heterotri- meric DNA complex with three-way junction. We screened base pair variations and mismatches within the binding sites. According to the error estimates, reliable binding constants can be given on each reaction step and thus elucidate the thermodynamic properties of the entire system using a single fluorescence dye. A ~2-fold increase in Gibbs free energy was found for the paired binding sites of each species, which is plausible with a homogenous base distribution. Strikingly, we found a coupling effect between independent binding sites that could be explained by the loss in conformational flexibility of the three-way junction in the fully bound state. The energy loss is in the range of ~2 and ~3 kcal/mol for a single-stranded region of three and two bases, respectively.
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Q&A: How can advances in tissue clearing and optogenetics contribute to our understanding of normal and diseased biology?

Q&A: How can advances in tissue clearing and optogenetics contribute to our understanding of normal and diseased biology?

Single-molecule RNA FISH is another labeling option in cleared tissue, first demonstrated in Yang et al. [10]. The ability to label RNAs is crucial to providing a ‘snap-shot’ of cell state at a precise time point. Recent studies have shown that RNA retention can be improved in hydrogel-based clearing methods by adding chemical linkers or an add- itional post-fixation step [26, 27]. However, because it pro- duces a weak signal relative to the background, FISH is only applicable to shallow depths within thick cleared tissue samples [10]. Hybridization chain reaction (HCR) is used to overcome this barrier and can boost the fluorescence signal by an order of magnitude [26–29]. HCR is a non-linear amplification method that uses DNA probes designed to bind the target RNA and trigger self-assembly of fluorophore-conjugated DNA hairpins (Fig. 2a). When HCR is combined with TC, it is possible to detect single mRNA transcripts in 0.5 mm-thick mouse brain tissue (Fig. 2b) [26] and rRNA in sputum samples [30]. As the la- beled RNA photo-bleaches relatively fast, imaging thick cleared tissue benefits from using light sheet microscopy and/or anti-fade buffer [26]. Recently, sequential hybridization and barcoding techniques have enabled the transcriptional profiling of more than 100 genes from thou- sands of single cells, while maintaining their native environ- ment in the tissue or cell culture [31, 32]. Although these results were not obtained in a cleared tissue, they provide the technical framework to achieve highly multiplexed RNA profiling of individual cells in thick cleared tissue. Fur- thermore, the smaller size of the probes facilitates diffusion into deep tissue, thus accelerating the tissue-staining stage in comparison to IHC (Fig. 2c). In addition, applying both FISH and IHC to the same tissue is also feasible when la- beling limitations arise, such as limited antibody availability or RNA probe design difficulties due to sequence homology [33] (Fig. 2c).
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Selective plane illumination microscopy techniques in developmental biology

Selective plane illumination microscopy techniques in developmental biology

Fig. 4. A comparison of light-sheet microscopy techniques. (A) In epifluorescence microscopy, a single objective lens (obj) is used to both illuminate the sample (s) and to collect its fluorescence along the same path. The sample is usually prepared on a glass slide or in a dish. (B-G) In light-sheet-based imaging techniques, by contrast, the sample is illuminated from the side by one or two additional beam paths (B-E), through (F) or along (G) the detection lens. (B) In selective plane illumination microscopy (SPIM), the detection lens is horizontally aligned and immersed into a fluid-filled chamber (ch). The sample is embedded in a transparent gel, immersed in the medium and held from the top. A single cylindrical lens (cyl) is used to form the light-sheet inside the chamber. A stack of images is acquired by moving the sample in a stepwise fashion along the detection axis. Optionally, the sample is turned for complementary data acquisition. (C) In objective-coupled planar illumination microscopy (OCPI), the illumination light is delivered through a fiber and focused by optics that are directly attached to the detection lens. A three-dimensional (3D) image stack is rapidly acquired by moving this arrangement, leaving the sample at rest. (D) Ultramicroscopy was developed to image fixed and cleared samples enclosed in a chamber. Two counter-propagating laser beams are focused into a light-sheet by cylindrical lenses and illuminate the sample simultaneously from both sides, thereby providing a more even illumination in clear tissue. (E) In multidirectional SPIM (mSPIM), the sample is illuminated independently from two sides over a range of angles. Shadowing and scattering (a common problem in live, scattering tissue) are thereby reduced. Three water-dipping objective lenses eliminate the need for any chamber windows. (F) In highly inclined and laminated optical sheet (HILO) microscopy, a single lens is used for both illumination and detection; however, the light-sheet is tilted and intersects the focal plane only in the center of the field of view. (G) The concept of an attachment ring to provide light-sheet illumination could be implemented as an add-on to existing microscopes. Blue arrows indicate the direction of illumination, green arrows indicate the direction of detection.
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Probing neural tissue with airy light sheet microscopy : investigation of imaging performance at depth within turbid media

Probing neural tissue with airy light sheet microscopy : investigation of imaging performance at depth within turbid media

Adult female wild type mice were anaesthetised as described above. Fluorescent beads (Duke Scientific R600, 600nm diameter polystyrene, red fluorescence), diluted 1:50 in PBS, were stereotaxically injected with a volume of 500nL/side bilaterally into the arcuate nucleus as described previously. Mice were culled 2h following bead injection and post-fixed as described above. After clearing, the density of beads was significantly reduced and a further injection was performed on the fixed tissue.

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Dynamic light sheet generation and fluorescence imaging behind turbid media

Dynamic light sheet generation and fluorescence imaging behind turbid media

In our experiments, the excitation beam and the fluorescence signal could reach the camera directly without being scattered. However, in most biological settings, the detection path will not be clear and the signal coming from the sample plane will be aberrated and scattered as well. This would hinder the evaluation of the light sheet using its image on the camera. In this case, this type of optimization using the direct beam can only be performed by weak scattering on the detec- tion side where significant amount of the direct beam reaches the camera as ballistic photons. In such a sce- nario, adaptive optics tailored for the detection arm of light sheet microscopes can be implemented as well [37]. If the detection side is subject to severe scattering, as we assume for many biological applications, alterna- tive approaches using fluorescent probe as feedback signal can be applied. For this purpose, a bright and isolated fluorescent particle can be placed at the sample plane of interest. A fluorescently labelled structure of the specimen itself might act as a source as well. The scattered fluorescence signal from the source can be focused on a single point detector on the back side of the overall turbid media. The wave-front of the illumin- ation can then be optimized to increase the signal at the detector which corresponds to an intensity increase of the excitation at the region of interest. Such methods using the image brightness as a metric for different optimization algorithms have already been used for implementation of adaptive optics in confocal and mul- tiphoton microscopy as compared by Wright et al. [38]. To use a labelled structure in the specimen is surely the better choice than invasively bringing a feedback source into the sample which can also destroy the structures in the ROI. However, the labelling might be then too dense leading to a mixed signal from a bigger area. On the other side, sparse labeling can bleach quickly. In that case, quantum dots would offer practic- ally non-bleaching signal. The proposed method would work for creating a single focus in the sample which can be then scanned within the range of memory effect. To collect signal from a line shaped source instead of a point source can lead to an increase of overall intensity of a light sheet. However, the homogeneity cannot be deduced from the signal increase at the detector since the scattered fluorescence from different parts of the line shaped source would be mixed up. To still ensure homogeneity and create directly a light sheet without scanning, a chain of spectrally different quantum dots can act as feedback source. Many quantum dots can be excited by the same wavelength but will emit fluorescence at a different wavelength. Optimizing on different colors from adjacent spots can lead to a more homogenous intensity increase of light sheet type illumination.
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Light sheet theta microscopy for rapid high-resolution imaging of large biological samples

Light sheet theta microscopy for rapid high-resolution imaging of large biological samples

acrylamide, 0.05% (wt/vol) bisacrylamide, 4% paraformalde- hyde (PFA), 1× phosphate-buffered saline (PBS), deionized water, and 0.25% thermal initiation VA-044 (Wako Chemicals, Boston, MA, USA; NC0632395). All ani- mal procedures were followed according to Institu- tional Animal Care and Use Committee (IACUC) guidelines. For whole brain clearing, transcardiac per- fusion was performed with 20 mL HM solution, followed by overnight incubation at 4 °C. The rat brain was perfused with 4% PFA, post-fixed for 16 h, and then frozen in isopentane for storage. The frozen brain was thawed at room temperature in PBS buffer, then sliced and incubated in HM solution overnight at 4 °C. The human brain tissue was incubated in 4% PFA for ~ 2 days, followed by incubation in HM solu- tion overnight at 4 °C. All the perfused tissues were de-gassed and then stored at 37 °C for 3–4 h for hydrogel polymerization. The tissues were cleared by incubating (with shaking) in clearing buffer (4% (wt/ vol) sodium dodecyl sulfate (SDS), 0.2 M boric acid, pH 8.5) at 37 °C until clear (2–3 weeks). Afterwards, the tissues were washed with 0.2 M boric acid buffer (pH 8.5) with 0.1% Triton X-100 for up to 24 h. The cleared tissue was labeled with DAPI (1 μg/mL final concentration) and/or the blood vessel marker tomato lectin (Vector Labs, Burlingame, CA, USA; FL-1171) by incubating in the labeling solution for 3–4 days. After washing with the buffered solution (0.2 M boric acid buffer, pH 7.5, 0.1% Triton X-100), the tissue was transferred into 85–87% glycerol solution in graded fashion (i.e. 25%, 50%, 65%, and finally 87%) for final clearing and imaging. For uniform tissue expansion (4–4.5× uniformly), a Thy1-eYFP mouse brain slice (250 μm, perfused and fixed with 4% PFA and sliced with vibratome) was gelled and digested following the protein retention expansion microscopy (proExM) protocol [34]. The sample was stored in 1× PBS before changing the buffer to 65% glycerol (with 2.5 mg/mL 1,4-diazabicyclo[2.2.2]octane (DABCO)) for the LSTM imaging. All imaging experiments were performed with an effective light sheet thickness of 2–5 μm.
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Fluorescence Microscopy of Nicotinic Acetylcholine Receptors

Fluorescence Microscopy of Nicotinic Acetylcholine Receptors

imaging. 48 h post-transfection cells were fixed, washed with 1% phosphate buffered saline (PBS), and imaged in PBS at room temperature after excitation with a 488nm laser. Images were spectrally unmixed against control spectra for mEGFP and background controls. When transfected with only α4-mEGFP and β2 plasmid DNA, mouse e17 cortical neurons express α4-mEGFPβ2 with a transfection efficiency of approximately 10% (figure 3.1). When α5-mEGFP is transfected with unlabeled α4 and β2 subunits, very low intensity of α5-mEGFP fluorescence is seen. In addition to reduced fluorescence, a cell death rate of over 50% is observed. Unlike α4-mEGFP, α5-mEGFP exhibits low intensity fluorescence that is confined to the cell bodies (see figure 3.1B). Interestingly, when a red fluorescent α4-mCherry is transfected with green fluorescent α5-mEGFP and unlabeled β2, all detected fluorescence, not just that from α5-mEGFP, is confined to the cell body (figure 3.1D). This is not true when fluorescent α4 subunits are transfected with unlabeled β2 subunits alone (figure 3.1C).
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Applications of Oxygen-Carrying Micro/Nanobubbles: a Potential Approach to Enhance Photodynamic Therapy and Photoacoustic Imaging

Applications of Oxygen-Carrying Micro/Nanobubbles: a Potential Approach to Enhance Photodynamic Therapy and Photoacoustic Imaging

Figure 3 shows an MNB containing oxygen with a photosensitizer drug incorporated in the shell. These MNBs can be used for PAI and PDT, and are more beneficial owing to the oxygen in the core. This yields the benefit of a dual modality imaging technique that shows anatomical, functional, and molecular imaging. Similar MNBs can be used for the enhancement of PDT in the targeted area. As shown in figure 3, MNBs can be intravenously injected into the tumors and when laser light is applied, the photosensitizer will be activated. This will improve targeting therapy for tumors and enhance the effectiveness due to the release of the oxygen from MNB simultaneously. More availability of oxygen will help in downregulation of the HIF-1α protein and higher ROS generation. Furthermore, MNBs can be co-administered with other nanocarriers to increase the volume of oxygen in the target area, thereby increasing the efficacy of PTD. MNBs can be applied to multifunctional targeting and they can be tailored to the requirement.
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4 Dimensional light sheet microscopy to elucidate shear stress modulation of cardiac trabeculation

4 Dimensional light sheet microscopy to elucidate shear stress modulation of cardiac trabeculation

4D cardiac SPIM imaging with synchronization algorithm. We have integrated our in-house 4D SPIM imaging system with postprocess- ing synchronization (Supplemental Methods) to visualize the dynamic cardiac architecture with high axial resolution (Supplemental Figure 1). Using the SPIM technique, we scanned 300 sections from the ros- tral to the caudal end of the zebrafish heart. Each section was captured with 300 x-y planes (frames) at 10-ms exposure time per frame via a sCMOS camera (Hamamatsu Photonics). The thickness of the light sheet was tuned to approximately 5 μm to provide a high axial (z axis) resolution for adequate reconstruction of the 3D cardiac morphol- ogy, and the Z scanning was set to 1 μm for lossless digital sampling according to the Nyquist sampling principle. To synchronize with the cardiac cycle, we determined the cardiac periodicity on a frame-to- frame basis by comparing the pixel intensity from the smallest during peak systole to the largest ventricular volume during end diastole (61, 62). The reconstructed 4D images were processed by Amira software (Supplemental Video 8).
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