Zeno Lavagnino

Live-cell 3D super-resolution imaging in thick biological samples

We demonstrate three-dimensional (3D) super-resolution live-cell imaging through thick specimens (50–150 μm), by coupling far-field individual molecule localization with selective plane illumination microscopy (SPIM). The improved signal-to-noise ratio of selective plane illumination allows nanometric localization of single molecules in thick scattering specimens without activating or exciting molecules outside the focal plane. We report 3D super-resolution imaging of cellular spheroids.

Two-photon excitation selective plane illumination microscopy (2PE-SPIM) of highly scattering samples: characterization and application

In this work we report the advantages provided by two photon excitation (2PE) implemented in a selective plane illumination microscopy (SPIM) when imaging thick scattering samples. In particular, a detailed analysis of the effects induced on the real light sheet excitation intensity distribution is performed. The comparison between single-photon and two-photon excitation profiles shows the reduction of the scattering effects and sample-induced aberrations provided by 2PE-SPIM. Furthermore, uniformity of the excitation distribution and the consequent improved image contrast is shown when imaging scattering phantom samples in depth by 2PE-SPIM. These results show the advantages of 2PE-SPIM and suggest how this combination can further enhance the SPIM performance. Phantom samples have been designed with optical properties compatible with biological applications of interest.

Rpl13a small nucleolar RNAs regulate systemic glucose metabolism.

Small nucleolar RNAs (snoRNAs) are non-coding RNAs that form ribonucleoproteins to guide covalent modifications of ribosomal and small nuclear RNAs in the nucleus. Recent studies have also uncovered additional non-canonical roles for snoRNAs. However, the physiological contributions of these small RNAs are largely unknown. Here, we selectively deleted four snoRNAs encoded within the introns of the ribosomal protein L13a (Rpl13a) locus in a mouse model. Loss of Rpl13a snoRNAs altered mitochondrial metabolism and lowered reactive oxygen species tone, leading to increased glucose-stimulated insulin secretion from pancreatic islets and enhanced systemic glucose tolerance. Islets from mice lacking Rpl13a snoRNAs demonstrated blunted oxidative stress responses. Furthermore, these mice were protected against diabetogenic stimuli that cause oxidative stress damage to islets. Our study illuminates a previously unrecognized role for snoRNAs in metabolic regulation.

4D (x-y-z-t) imaging of thick biological samples by means of Two-Photon inverted Selective Plane Illumination Microscopy (2PE-iSPIM)

In the last decade light sheet fluorescence microscopy techniques, such as selective plane illumination microscopy (SPIM), has become a well established method for developmental biology. However, conventional SPIM architectures hardly permit imaging of certain tissues since the common sample mounting procedure, based on gel embedding, could interfere with the sample morphology. In this work we propose an inverted selective plane microscopy system (iSPIM), based on non-linear excitation, suitable for 3D tissue imaging. First, the iSPIM architecture provides flexibility on the sample mounting, getting rid of the gel-based mounting typical of conventional SPIM, permitting 3D imaging of hippocampal slices from mouse brain. Moreover, all the advantages brought by two photon excitation (2PE) in terms of reduction of scattering effects and contrast improvement are exploited, demonstrating an improved image quality and contrast compared to single photon excitation. The system proposed represents an optimal platform for tissue imaging and it smooths the way to the applicability of light sheet microscopy to a wider range of samples including those that have to be mounted on non-transparent surfaces.

Two-photon fluorescence excitation within a light sheet based microscopy architecture

Light-sheet microscopy, such as ultramicroscopy, single plane illumination microscopy (SPIM) [1] and digital scanned laser microscopy (DSLM) [2], represents a useful tool for biological investigations of thick samples. Such techniques have been found particularly useful in developmental biology applications since they provide the capability to perform fast imaging of living samples reducing photobleaching effects. The high signal to noise ratio and the intrinsic optical sectioning capability provided by SPIM suggest this technique as the best choice for imaging of thick scattering samples. Nevertheless, imaging in depth of large samples suffers from a decreasing in the image quality due to scattering effects. Two photon excitation microscopy [3] became a popular tool to perform imaging in turbid media since it improves the penetration depth capability and it reduces the image quality degradation due to scattering [4] and light matter interactions. Therefore, two photon excitation within the light sheet illumination scheme has been exploited in order to reduce scattering effects due to light-sample interactions. In this work two photon excitation imaging in SPIM scheme has been performed in order to achieve an improvement in the penetration depth while imaging living biological samples.

Targeting cellular calcium homeostasis to prevent cytokine-mediated beta cell death

Pro-inflammatory cytokines are important mediators of islet inflammation, leading to beta cell death in type 1 diabetes. Although alterations in both endoplasmic reticulum (ER) and cytosolic free calcium levels are known to play a role in cytokine-mediated beta cell death, there are currently no treatments targeting cellular calcium homeostasis to combat type 1 diabetes. Here we show that modulation of cellular calcium homeostasis can mitigate cytokine- and ER stress-mediated beta cell death. The calcium modulating compounds, dantrolene and sitagliptin, both prevent cytokine and ER stress-induced activation of the pro-apoptotic calcium-dependent enzyme, calpain, and partly suppress beta cell death in INS1E cells and human primary islets. These agents are also able to restore cytokine-mediated suppression of functional ER calcium release. In addition, sitagliptin preserves function of the ER calcium pump, sarco-endoplasmic reticulum Ca2+-ATPase (SERCA), and decreases levels of the pro-apoptotic protein thioredoxin-interacting protein (TXNIP). Supporting the role of TXNIP in cytokine-mediated cell death, knock down of TXNIP in INS1-E cells prevents cytokine-mediated beta cell death. Our findings demonstrate that modulation of dynamic cellular calcium homeostasis and TXNIP suppression present viable pharmacologic targets to prevent cytokine-mediated beta cell loss in diabetes.

Role of Scattering and Nonlinear Effects in the Illumination and the Photobleaching Distribution Profiles

Nonlinear optical scanning microscopy has become a useful tool for living tissue imaging. Biological tissues are highly scattering media and this leads to an exponential attenuation of the excitation intensity as the light travels into the sample. While performing imaging of biological scattering tissues in nonlinear excitation regime, the localization of the maximum two-photon excitation (2PE) intensity was found to shift closer to the surface and the 2PE imaging depth limit appears strongly limited by near-surface fluorescence. In this work, we computed the illumination and the photobleaching distribution to characterize the effects induced by scattering. An experimental test has been carried out by imaging, with 0.9 NA objective, thick scattering fluorescent immobile sample (polyelectrolyte gel) as a phantom for biological samples. Results confirm that under these conditions no photobleaching effects due to scattering occur close to the surface.

Non-linear effects and role of scattering in multiphoton imaging of thick biological samples

Non linear optical scanning microscopy has became a useful tool for living tissue imaging. Biological tissues are highly scattering media and this leads to an exponential attenuation of the excitation intensity as the light travels into the sample. While performing imaging of biological scattering tissues in non linear excitation regime, the localization of the maximum 2PE intensity was found to shift closer to the surface1 and the 2PE imaging depth limit appears strongly limited by near surface fluorescence.2 In this work we computed the illumination and the photobleaching distribution3 in order to characterize the effects induced by scattering. The simulations have been performed for different scattering coefficients and different focus depth. An experimental test has been carried out by imaging, with 0.9 numerical aperture objective, thick scattering fluorescent immobile sample (polyelectrolyte gel). Results confirm that under these conditions no photobleaching effects due to scattering occur close to the surface.

Regulation of islet glucagon secretion: Beyond calcium

The islet of Langerhans plays a key role in glucose homeostasis through regulated secretion of the hormones insulin and glucagon. Islet research has focused on the insulin‐secreting β‐cells, even though aberrant glucagon secretion from α‐cells also contributes to the aetiology of diabetes. Despite its importance, the mechanisms controlling glucagon secretion remain controversial. Proper α‐cell function requires the islet milieu, where β‐ and δ‐cells drive and constrain α‐cell dynamics. The response of glucagon to glucose is similar between isolated islets and that measured in vivo, so it appears that the glucose dependence requires only islet‐intrinsic factors and not input from blood flow or the nervous system. Elevated intracellular free Ca2+ is needed for α‐cell exocytosis, but interpreting Ca2+ data is tricky since it is heterogeneous among α‐cells at all physiological glucose levels. Total Ca2+ activity in α‐cells increases slightly with glucose, so Ca2+may serve a permissive, rather than regulatory, role in glucagon secretion. On the other hand, cAMP is a more promising candidate for controlling glucagon secretion and is itself driven by paracrine signalling from β‐ and δ‐cells. Another pathway, juxtacrine signalling through the α‐cell EphA receptors, stimulated by β‐cell ephrin ligands, leads to a tonic inhibition of glucagon secretion. We discuss potential combinations of Ca2+, cAMP, paracrine and juxtacrine factors in the regulation of glucagon secretion, focusing on recent data in the literature that might unify the field towards a quantitative understanding of α‐cell function.

diSPIM Allows Three-dimensional Characterization of Calcium Activity in Intact Islets of Langerhans.

Blood glucose homeostasis is required for normal physiology of many living organisms. Within this framework, the islet of Langerhans plays a fundamental role in the regulation of glycemic levels. This micro-organ is ~100 μm in diameter and is responsible for the secretion of insulin and its counter regulatory hormone, glucagon. When blood glucose increases above normal levels, insulin is secreted by β-cells to restore the equilibrium. If blood glucose decreases, then glucagon is secreted by α-cells to release glucose stores. Because of the therapeutic success of insulin replacement therapy for the management of diabetes, the majority of islet research has focused on insulin and β-cells. This has led to a well-characterized consensus model of glucose stimulated insulin secretion, based around membrane depolarization and calcium influx. In recent years, it has become evident that α-cells and glucagon dysfunction also contribute to the pathophysiology of diabetes, specifically exacerbating hyperglycemia [1]. There is not yet a consensus model for glucose regulation of glucagon secretion, and intracellular and intercellular mechanisms related to glucagon secretion, including the roles of calcium and cAMP, have not been fully characterized. A major impediment in the study of α-cells is a lack of reliable methods to measure the dynamic properties of α-cells within the intact islet of Langerhans [2]. A noninvasive, reliable microscopy technique able to provide three-dimensional imaging of the whole intact islet is crucial, since isolated α-cells demonstrate different behavior than those within intact islets. Light-sheet microscopy is a powerful tool for fast, 3D imaging of thick scattering samples. More recently, diSPIM [3] was demonstrated to yield isotropic resolution in x,y,z by collecting the information from both sides of the illumination and detection pathways.