Light-sheet microscopy: a tutorial

This paper is intended to give a comprehensive review of light-sheet (LS) microscopy from an optics perspective. As such, emphasis is placed on the advantages that LS microscope configurations present, given the degree of freedom gained by uncoupling the excitation and detection arms. The new imaging properties are first highlighted in terms of optical parameters and how these have enabled several biomedical applications. Then, the basics are presented for understanding how a LS microscope works. This is followed by a presentation of a tutorial for LS microscope designs, each working at different resolutions and for different applications. Then, based on a numerical Fourier analysis and given the multiple possibilities for generating the LS in the microscope (using Gaussian, Bessel, and Airy beams in the linear and nonlinear regimes), a systematic comparison of their optical performance is presented. Finally, based on advances in optics and photonics, the novel optical implementations possible in a LS microscope are highlighted.Peer ReviewedPostprint (published version

STED imaging performance estimation by means of Fourier transform analysis

Due to relatively high powers used in STED, biological samples may be affected by the illumination in the process of image acquisition. Similarly, the performance of the system may be limited by the sample itself. Optimization of the STED parameters taking into account the sample itself is therefore a complex task as there is no clear methodology that can determine the image improvement in an objective and quantitative manner. In this work, a method based on Fourier transform formalism is presented to analyze the performance of a STED system. The spatial frequency distribution of pairs of confocal and STED images are compared to obtain an objective parameter, the Azimuth Averaged Spectral Content Spread (AASCS), that is related to the performance of the system in which the sample is also considered. The method has been first tested on samples of beads, and then applied to cell samples labeled with multiple fluorescent dyes. The results show that a single parameter, the AASCS, can be used to determine the optimal settings for STED image acquisition in an objective way, only by using the information provided by the images from the sample themselves. The AASCS also helps minimize the depletion power, for better preservation of the samples.Peer ReviewedPostprint (published version

High-Resolution morphological approach to analyse elastic laminae injuries of the ascending aorta in a murine model of Marfan Syndrome

In Marfan syndrome, the tunica media is disrupted, which leads to the formation of ascending aortic aneurysms. Marfan aortic samples are histologically characterized by the fragmentation of elastic laminae. However, conventional histological techniques using transverse sections provide limited information about the precise location, progression and 3D extension of the microstructural changes that occur in each lamina. We implemented a method using multiphoton excitation fluorescence microscopy and computational image processing, which provides high-resolution en-face images of segmented individual laminae from unstained whole aortic samples. We showed that internal elastic laminae and successive 2(nd) laminae are injured to a different extent in murine Marfan aortae; in particular, the density and size of fenestrae changed. Moreover, microstructural injuries were concentrated in the aortic proximal and convex anatomical regions. Other parameters such as the waviness and thickness of each lamina remained unaltered. In conclusion, the method reported here is a useful, unique tool for en-face laminae microstructure assessment that can obtain quantitative three-dimensional information about vascular tissue. The application of this method to murine Marfan aortae clearly shows that the microstructural damage in elastic laminae is not equal throughout the thickness of the tunica media and in the different anatomical regions of the ascending aorta

Deep and Clear Optical Imaging of Thick Inhomogeneous Samples

Inhomogeneity in thick biological specimens results in poor imaging by light microscopy, which deteriorates as the focal plane moves deeper into the specimen. Here, we have combined selective plane illumination microscopy (SPIM) with wavefront sensor adaptive optics (wao). Our waoSPIM is based on a direct wavefront measure using a Hartmann-Shack wavefront sensor and fluorescent beads as point source emitters. We demonstrate the use of this waoSPIM method to correct distortions in three-dimensional biological imaging and to improve the quality of images from deep within thick inhomogeneous samples

Large-area biomolecule nanopatterns on diblock copolymer surfaces for cell adhesion studies

Cell membrane receptors bind to extracellular ligands, triggering intracellular signal transduction pathways that result in specific cell function. Some receptors require to be associated forming clusters for effective signaling. Increasing evidences suggest that receptor clustering is subjected to spatially controlled ligand distribution at the nanoscale. Herein we present a method to produce in an easy, straightforward process, nanopatterns of biomolecular ligands to study ligand–receptor processes involving multivalent interactions. We based our platform in self-assembled diblock copolymers composed of poly(styrene) (PS) and poly(methyl methacrylate) (PMMA) that form PMMA nanodomains in a closed-packed hexagonal arrangement. Upon PMMA selective functionalization, biomolecular nanopatterns over large areas are produced. Nanopattern size and spacing can be controlled by the composition of the block-copolymer selected. Nanopatterns of cell adhesive peptides of different size and spacing were produced, and their impact in integrin receptor clustering and the formation of cell focal adhesions was studied. Cells on ligand nanopatterns showed an increased number of focal contacts, which were, in turn, more matured than those found in cells cultured on randomly presenting ligands. These findings suggest that our methodology is a suitable, versatile tool to study and control receptor clustering signaling and downstream cell behavior through a surface-based ligand patterning technique

Tomato fruit carotenoid biosynthesis is adjusted to actual ripening progression by a light-dependent mechanism

Carotenoids are isoprenoid compounds that are essential for plants to protect the photosynthetic apparatus against excess light. They also function as health-promoting natural pigments that provide colors to ripe fruit, promoting seed dispersal by animals. Work in Arabidopsis thaliana unveiled that transcription factors of the phytochrome-interacting factor (PIF) family regulate carotenoid gene expression in response to environmental signals (i.e. light and temperature), including those created when sunlight reflects from or passes though nearby vegetation or canopy (referred to as shade). Here we show that PIFs use a virtually identical mechanism to modulate carotenoid biosynthesis during fruit ripening in tomato (Solanum lycopersicum). However, instead of integrating environmental information, PIF-mediated signaling pathways appear to fulfill a completely new function in the fruit. As tomatoes ripen, they turn from green to red due to chlorophyll breakdown and carotenoid accumulation. When sunlight passes through the flesh of green fruit, a self-shading effect within the tissue maintains high levels of PIFs that directly repress the master gene of the fruit carotenoid pathway, preventing undue production of carotenoids. This effect is attenuated as chlorophyll degrades, causing degradation of PIF proteins and boosting carotenoid biosynthesis as ripening progresses. Thus, shade signaling components may have been co-opted in tomato fruit to provide information on the actual stage of ripening (based on the pigment profile of the fruit at each moment) and thus finely coordinate fruit color change. We show how this mechanism may be manipulated to obtain carotenoid-enriched fruits.Peer ReviewedPostprint (published version

Spontaneous functional recovery after focal damage in neuronal cultures

Damage in biological neuronal networks triggers a complex functional reorganization whose mechanisms are still poorly understood. To delineate this reorganization process, here we investigate the functional alterations of in vitro rat cortical circuits following localized laser ablation. The analysis of the functional network configuration before and after ablation allowed us to quantify the extent of functional alterations and the characteristic spatial and temporal scales along recovery. We observed that damage precipitated a fast rerouting of information flow that restored network's communicability in about 15 min. Functional restoration was led by the immediate neighbors around trauma but was orchestrated by the entire network. Our in vitro setup exposes the ability of neuronal circuits to articulate fast responses to acute damage, and may serve as a proxy to devise recovery strategies in actual brain circuits. Moreover, this biological setup can become a benchmark to empirically test network theories about the spontaneous recovery in dynamical networks

GCAP neuronal calcium sensor proteins mediate photoreceptor cell death in the rd3 mouse model of LCA12 congenital blindness by involving endoplasmic reticulum stress

Loss-of-function mutations in the retinal degeneration 3 (RD3) gene cause inherited retinopathy with impaired rod and cone function and fast retinal degeneration in patients and in the natural strain of rd3 mice. The underlying physiopathology mechanisms are not well understood. We previously proposed that guanylate cyclase-activating proteins (GCAPs) might be key Ca2+-sensors mediating the physiopathology of this disorder, based on the demonstrated toxicity of GCAP2 when blocked in its Ca2+-free form at photoreceptor inner segments. We here show that the retinal degeneration in rd3 mice is substantially delayed by GCAPs ablation. While the number of retinal photoreceptor cells is halved in 6 weeks in rd3 mice, it takes 8 months to halve in rd3/rd3 GCAPs−/− mice. Although this substantial morphological rescue does not correlate with recovery of visual function due to very diminished guanylate cyclase activity in rd3 mice, it is very informative of the mechanisms underlying photoreceptor cell death. By showing that GCAP2 is mostly in its Ca2+-free-phosphorylated state in rd3 mice, we infer that the [Ca2+]i at rod inner segments is permanently low. GCAPs are therefore retained at the inner segment in their Ca2+-free, guanylate cyclase activator state. We show that in this conformational state GCAPs induce endoplasmic reticulum (ER) stress, mitochondrial swelling, and cell death. ER stress and mitochondrial swelling are early hallmarks of rd3 retinas preceding photoreceptor cell death, that are substantially rescued by GCAPs ablation. By revealing the involvement of GCAPs-induced ER stress in the physiopathology of Leber's congenital amaurosis 12 (LCA12), this work will aid to guide novel therapies to preserve retinal integrity in LCA12 patients to expand the window for gene therapy intervention to restore vision

Nanopatterns of surface-bound ephrinB1 produce multivalent ligand-receptor interactions that tune EphB2 receptor clustering

Here we present a nanostructured surface able to produce multivalent interactions between surface-bound ephrinB1 ligands and membrane EphB2 receptors. We created ephrinB1 nanopatterns of regular size (<30 nm in diameter) by using self-assembled diblock copolymers. Next, we used a statistically enhanced version of the Number and Brightness technique, which can discriminate with molecular sensitivity the oligomeric states of diffusive species to quantitatively track the EphB2 receptor oligomerization process in real time. The results indicate that a stimulation using randomly distributed surface-bound ligands was not sufficient to fully induce receptor aggregation. Conversely, when nanopatterned onto our substrates, the ligands effectively induced a strong receptor oligomerization. This presentation of ligands improved the clustering efficiency of conventional ligand delivery systems, as it required a 9-fold lower ligand surface coverage and included faster receptor clustering kinetics compared to traditional cross-linked ligands. In conclusion, nanostructured diblock copolymers constitute a novel strategy to induce multivalent ligand-receptor interactions leading to a stronger, faster, and more efficient receptor activation, thus providing a useful strategy to precisely tune and potentiate receptor responses. The efficiency of these materials at inducing cell responses can benefit applications such as the design of new bioactive materials and drug-delivery systems

Synaptic phosphorylated a-synuclein in dementia with Lewy bodies

Dementia with Lewy bodies is characterized by the accumulation of Lewy bodies and Lewy neurites in the CNS, both of which are composed mainly of aggregated a-synuclein phosphorylated at Ser129. Although phosphorylated a-synuclein is believed to exert toxic effects at the synapse in dementia with Lewy bodies and other a-synucleinopathies, direct evidence for the precise synaptic localization has been difficult to achieve due to the lack of adequate optical microscopic resolution to study human synapses. In the present study we applied array tomography, a microscopy technique that combines ultrathin sectioning of tissue with immunofluorescence allowing precise identification of small structures, to quantitatively investigate the synaptic phosphorylated a-synuclein pathology in dementia with Lewy bodies. We performed array tomography on human brain samples from five patients with dementia with Lewy bodies, five patients with Alzheimer’s disease and five healthy control subjects to analyse the presence of phosphorylated a-synuclein immunoreactivity at the synapse and their relationship with synapse size. Main analyses were performed in blocks from cingulate cortex and confirmed in blocks from the striatum of cases with dementia with Lewy bodies. A total of 1 318 700 single pre- or post-synaptic terminals were analysed. We found that phosphorylated a-synuclein is present exclusively in dementia with Lewy bodies cases, where it can be identified in the form of Lewy bodies, Lewy neurites and small aggregates (50.16 mm3). Between 19% and 25% of phosphorylated a-synuclein deposits were found in presynaptic terminals mainly in the form of small aggregates. Synaptic terminals that co-localized with small aggregates of phosphorylated a-synuclein were significantly larger than those that did not. Finally, a gradient of phosphorylated a-synuclein aggregation in synapses (pre4pre + post4post-synaptic) was observed. These results indicate that phosphorylated a-synuclein is found at the presynaptic terminals of dementia with Lewy bodies cases mainly in the form of small phosphorylated a-synuclein aggregates that are associated with changes in synaptic morphology. Overall, our data support the notion that pathological phosphorylated a-synuclein may disrupt the structure and function of the synapse in dementia with Lewy bodies.Peer ReviewedPostprint (author's final draft