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Papers by Tommaso Alterini

Research paper thumbnail of Medidas hiperespectrales de fondo de ojo: una nueva herramienta para el diagnóstico oftalmológico

Research paper thumbnail of Hyperspectral fundus camera with sensitivity beyond the visible range: a pilot study

Investigative Ophthalmology & Visual Science, 2018

Research paper thumbnail of High-fidelity imaging with Bessel-beam light-sheet microscopy for whole-brain structural and functional studies

Brain, 2017

Bessel beam have attractive properties like their propagation invariance and “self-healing” capab... more Bessel beam have attractive properties like their propagation invariance and “self-healing” capabilities. Here we present significant advantages of Bessel illumination in structural and functional imaging in light-sheet microscopy compared to conventional Gaussian beam illumination.

Research paper thumbnail of Multispectral retinography in healthy adult population

Translational Biophotonics: Diagnostics and Therapeutics

Research paper thumbnail of Dynamic Wavefront Coding Using a Deformable Lens

Research paper thumbnail of Functional Imaging of Zebrafish Neuronal Activity by Bessel Beam Light-Sheet Microscopy

One of the most exciting challenges of neurosciences in the last few years is the real-time recor... more One of the most exciting challenges of neurosciences in the last few years is the real-time recording of neuronal activity with single cell resolution across the entire brain. Thanks to the use of optical methods, together with animal models in which the whole encephalon is optically accessible, this goal is getting within reach. In this work, we use a transgenic zebrafish line expressing the genetically encoded calcium indicator GCaMP6s in which binding of calcium ions leads to an increase in the emitted fluorescence of the reporter. GCaMP6s is the most sensitive calcium reporter within the genetically encoded calcium indicator family and allows us to record zebrafish larva neuronal activity with a high signal-to-noise ratio and single neuron resolution. To record the fluorescence emitted by the GCaMP6s reporter we use a custom-made confocal light-sheet microscope (LSM), in which the sample is illuminated with a thin sheet of light and the detection optical axis is perpendicular to...

Research paper thumbnail of Hyperspectral eye fundus imaging with extended spectral range towards the near infrared

Clinical and Preclinical Optical Diagnostics II

Eye fundus photography routinely used in clinical practice is restricted to color imaging of the ... more Eye fundus photography routinely used in clinical practice is restricted to color imaging of the retina. In the last years, hyperspectral imaging has shown to be a powerful tool for the spectral analysis of biological tissue. In this study, we present a fully custom-made fast hyperspectral fundus camera based on light emitting diodes (LED) with 15 different wavelengths of emission and with extended spectral sensitivity towards the near infrared (NIR) (from 400 nm to 1300 nm), which allows imaging deeper retinal layers, including the choroid, than current clinical devices. These new features will be very useful for a better understanding of ocular diseases as well as aiding in their diagnosis.

Research paper thumbnail of Corrigendum: Bessel Beam Illumination Reduces Random and Systematic Errors in Quantitative Functional Studies Using Light-Sheet Microscopy

Frontiers in Cellular Neuroscience

Research paper thumbnail of Nuove tecniche di transgenesi e imaging: neuro e nefro-applicazioni. Parte 2

Giornale di Clinica Nefrologica e Dialisi

New techniques of transgenesis and imaging: applications in Neurology and Nephrology. Part II Str... more New techniques of transgenesis and imaging: applications in Neurology and Nephrology. Part II Strengthened by the analysis of genetic engineering approaches and strategies related to transgenic animal models and of the most cutting-edge imaging techniques, this article will be centered on the impact that such applications have and will have in research and, as a consequence, in the clinical area. In order to show how identical techniques can be applied in different contexts for the study of different organs and tissues, in this issue the applications of these technologies in the anatomical and functional studies of distinct organs such as the kidney and the central nervous system will be analyzed. In particular, concrete examples about the kidney and regenerative nephrology will be exploited to show how different techniques can lead to similar, or diametrically opposite data, depending on the approach used to reach the specific research or diagnostic goal.

Research paper thumbnail of Increasing sensitivity and accuracy of brain-wide quantitative studies in light-sheet microscopy

Light-sheet microscopy (LSM) has proven a useful tool in neuroscience to image whole brains with ... more Light-sheet microscopy (LSM) has proven a useful tool in neuroscience to image whole brains with high frame rates at cellular resolution. LSM is employed either in combination with tissue clearing to reconstruct the cyto-architecture over the entire mouse brain or with intrinsically transparent samples like zebrafish larvae for functional imaging. Inherently to LSM, however, residual opaque objects cause stripe artifacts, which obscure features of interest and, during functional imaging, modulate fluorescence variations related to neuronal activity. Here, we report how Bessel beams reduce streaking artifacts and produce high-fidelity structural data. Furthermore, using Bessel beams, we demonstrate a fivefold increase in sensitivity to calcium transients and a 20 fold increase in accuracy in the detection of activity correlations in functional imaging. Our results demonstrate the contamination of data by systematic and random errors through Gaussian illumination and furthermore quantify the increase in fidelity of such data when using Bessel beams.

Research paper thumbnail of Bessel Beam Illumination Reduces Random and Systematic Errors in Quantitative Functional Studies Using Light-Sheet Microscopy

Frontiers in Cellular Neuroscience, Sep 20, 2018

Light-sheet microscopy (LSM), in combination with intrinsically transparent zebrafish larvae, is ... more Light-sheet microscopy (LSM), in combination with intrinsically transparent zebrafish larvae, is a method of choice to observe brain function with high frame rates at cellular resolution. Inherently to LSM, however, residual opaque objects cause stripe artifacts, which obscure features of interest and, during functional imaging, modulate fluorescence variations related to neuronal activity. Here, we report how Bessel beams reduce streaking artifacts and produce high-fidelity quantitative data demonstrating a fivefold increase in sensitivity to calcium transients and a 20-fold increase in accuracy in the detection of activity correlations in functional imaging. Furthermore, using principal component analysis, we show that measurements obtained with Bessel beams are clean enough to reveal in one-shot experiments correlations that can not be averaged over trials after stimuli as is the case when studying spontaneous activity. Our results not only demonstrate the contamination of data by systematic and random errors through conventional Gaussian illumination and but,furthermore, quantify the increase in fidelity of such data when using Bessel beams.

Research paper thumbnail of Fast visible and extended near-infrared multispectral fundus camera

Journal of Biomedical Optics

We present a multispectral fundus camera that performs fast imaging of the ocular posterior pole ... more We present a multispectral fundus camera that performs fast imaging of the ocular posterior pole in the visible and near-infrared (400 to 1300 nm) wavelengths through 15 spectral bands, using a flashlight source made of light-emitting diodes, and CMOS and InGaAs cameras. We investigate the potential of this system for visualizing occult and overlapping structures of the retina in the unexplored wavelength range beyond 900 nm, in which radiation can penetrate deeper into the tissue. Reflectance values at each pixel are also retrieved from the acquired images in the analyzed spectral range. The available spectroscopic information and the visualization of retinal structures, specifically the choroidal vasculature and drusen-induced retinal pigment epithelium degeneration, which are hardly visible in conventional color fundus images, underline the clinical potential of this system as a new tool for ophthalmic diagnosis.

Research paper thumbnail of High-fidelity functional and structural whole-brain imaging with Bessel-beam light-sheet microscopy (Conference Presentation)

Neural Imaging and Sensing

Light-sheet microscopy (LSM) has proven a useful tool in neuroscience and is particularly well su... more Light-sheet microscopy (LSM) has proven a useful tool in neuroscience and is particularly well suited to image the entire brain with high frame rates at single cell resolution. On the one hand, LSM is employed in combination with tissue clearing methods like CLARITY which allows for the reconstruction of neuronal or vascular anatomy over cm-sized samples. On the other hand, LSM has been paired with intrinsically transparent samples for real-time recording of neuronal activity with single cell resolution across the entire brain, using calcium indicators like GCaMP6. Despite its intrinsic advantages in terms of high imaging speed and reduced photobleaching, LSM is very sensitive to residual opaque objects present in the sample, which cause dark horizontal stripes in the collected images. In the best case, these artefacts obscure the features of interest in structural imaging; in the worst case, dynamic shadowing introduced by red blood cells significantly alters the fluorescence signal variations related to neuronal activity. We show how the use of Bessel beams in LSM can dramatically reduce such artefacts even in conventional one-sided illumination schemes, thanks to their “self-healing” properties. On the functional side, Bessel-beam LSM allows recording neuronal activity traces without any disturbing flickering caused by the movement of red blood cells. On the structural side, our proposed method is capable of obtaining anatomical information across the entire volume of whole mouse brains allowing tracing blood vessels and neuronal projections also in poorly cleared specimens.

Research paper thumbnail of New techniques of transgenesis and imaging: applications in Neurology and Nephrology. Part I

Giornale Di Tecniche Nefrologiche E Dialitiche, Jun 30, 2014

New techNiques of traNsgeNesis aNd imagiNg: applicatioNs iN Neurology aNd Nephrology. part i Abst... more New techNiques of traNsgeNesis aNd imagiNg: applicatioNs iN Neurology aNd Nephrology. part i Abstract. The emerging techniques and applications related to transgenic animals, together with the large progresses in the development of imaging systems finalized at ensuring a high-level resolution, allow nowadays to analyze the finer details of a wide range of pathophysiological phenomena. The combination of the new methods applicable in the two fields, for instance, has made possible not only to explore biological processes in vivo as they occur, but also to analyze them at a whole organ level. At the same time, it is possible to create reconstructions in two or three dimensions of the entire organ, or of its specific functional units. A fourth temporal dimension can also be added thanks to the analysis in time-lapse or to the acquisition of data in continuous. The purpose of this first of two contributions is to review the latest innovations in the fields of microscopy and transgenesis, with particular attention to how these innovations have been implemented, and which benefits derive from their evolution. The possible applications and advantages of using these systems will further be analyzed and evaluated in more detail in the next issue of this survey.

Research paper thumbnail of Medidas hiperespectrales de fondo de ojo: una nueva herramienta para el diagnóstico oftalmológico

Research paper thumbnail of Hyperspectral fundus camera with sensitivity beyond the visible range: a pilot study

Investigative Ophthalmology & Visual Science, 2018

Research paper thumbnail of High-fidelity imaging with Bessel-beam light-sheet microscopy for whole-brain structural and functional studies

Brain, 2017

Bessel beam have attractive properties like their propagation invariance and “self-healing” capab... more Bessel beam have attractive properties like their propagation invariance and “self-healing” capabilities. Here we present significant advantages of Bessel illumination in structural and functional imaging in light-sheet microscopy compared to conventional Gaussian beam illumination.

Research paper thumbnail of Multispectral retinography in healthy adult population

Translational Biophotonics: Diagnostics and Therapeutics

Research paper thumbnail of Dynamic Wavefront Coding Using a Deformable Lens

Research paper thumbnail of Functional Imaging of Zebrafish Neuronal Activity by Bessel Beam Light-Sheet Microscopy

One of the most exciting challenges of neurosciences in the last few years is the real-time recor... more One of the most exciting challenges of neurosciences in the last few years is the real-time recording of neuronal activity with single cell resolution across the entire brain. Thanks to the use of optical methods, together with animal models in which the whole encephalon is optically accessible, this goal is getting within reach. In this work, we use a transgenic zebrafish line expressing the genetically encoded calcium indicator GCaMP6s in which binding of calcium ions leads to an increase in the emitted fluorescence of the reporter. GCaMP6s is the most sensitive calcium reporter within the genetically encoded calcium indicator family and allows us to record zebrafish larva neuronal activity with a high signal-to-noise ratio and single neuron resolution. To record the fluorescence emitted by the GCaMP6s reporter we use a custom-made confocal light-sheet microscope (LSM), in which the sample is illuminated with a thin sheet of light and the detection optical axis is perpendicular to...

Research paper thumbnail of Hyperspectral eye fundus imaging with extended spectral range towards the near infrared

Clinical and Preclinical Optical Diagnostics II

Eye fundus photography routinely used in clinical practice is restricted to color imaging of the ... more Eye fundus photography routinely used in clinical practice is restricted to color imaging of the retina. In the last years, hyperspectral imaging has shown to be a powerful tool for the spectral analysis of biological tissue. In this study, we present a fully custom-made fast hyperspectral fundus camera based on light emitting diodes (LED) with 15 different wavelengths of emission and with extended spectral sensitivity towards the near infrared (NIR) (from 400 nm to 1300 nm), which allows imaging deeper retinal layers, including the choroid, than current clinical devices. These new features will be very useful for a better understanding of ocular diseases as well as aiding in their diagnosis.

Research paper thumbnail of Corrigendum: Bessel Beam Illumination Reduces Random and Systematic Errors in Quantitative Functional Studies Using Light-Sheet Microscopy

Frontiers in Cellular Neuroscience

Research paper thumbnail of Nuove tecniche di transgenesi e imaging: neuro e nefro-applicazioni. Parte 2

Giornale di Clinica Nefrologica e Dialisi

New techniques of transgenesis and imaging: applications in Neurology and Nephrology. Part II Str... more New techniques of transgenesis and imaging: applications in Neurology and Nephrology. Part II Strengthened by the analysis of genetic engineering approaches and strategies related to transgenic animal models and of the most cutting-edge imaging techniques, this article will be centered on the impact that such applications have and will have in research and, as a consequence, in the clinical area. In order to show how identical techniques can be applied in different contexts for the study of different organs and tissues, in this issue the applications of these technologies in the anatomical and functional studies of distinct organs such as the kidney and the central nervous system will be analyzed. In particular, concrete examples about the kidney and regenerative nephrology will be exploited to show how different techniques can lead to similar, or diametrically opposite data, depending on the approach used to reach the specific research or diagnostic goal.

Research paper thumbnail of Increasing sensitivity and accuracy of brain-wide quantitative studies in light-sheet microscopy

Light-sheet microscopy (LSM) has proven a useful tool in neuroscience to image whole brains with ... more Light-sheet microscopy (LSM) has proven a useful tool in neuroscience to image whole brains with high frame rates at cellular resolution. LSM is employed either in combination with tissue clearing to reconstruct the cyto-architecture over the entire mouse brain or with intrinsically transparent samples like zebrafish larvae for functional imaging. Inherently to LSM, however, residual opaque objects cause stripe artifacts, which obscure features of interest and, during functional imaging, modulate fluorescence variations related to neuronal activity. Here, we report how Bessel beams reduce streaking artifacts and produce high-fidelity structural data. Furthermore, using Bessel beams, we demonstrate a fivefold increase in sensitivity to calcium transients and a 20 fold increase in accuracy in the detection of activity correlations in functional imaging. Our results demonstrate the contamination of data by systematic and random errors through Gaussian illumination and furthermore quantify the increase in fidelity of such data when using Bessel beams.

Research paper thumbnail of Bessel Beam Illumination Reduces Random and Systematic Errors in Quantitative Functional Studies Using Light-Sheet Microscopy

Frontiers in Cellular Neuroscience, Sep 20, 2018

Light-sheet microscopy (LSM), in combination with intrinsically transparent zebrafish larvae, is ... more Light-sheet microscopy (LSM), in combination with intrinsically transparent zebrafish larvae, is a method of choice to observe brain function with high frame rates at cellular resolution. Inherently to LSM, however, residual opaque objects cause stripe artifacts, which obscure features of interest and, during functional imaging, modulate fluorescence variations related to neuronal activity. Here, we report how Bessel beams reduce streaking artifacts and produce high-fidelity quantitative data demonstrating a fivefold increase in sensitivity to calcium transients and a 20-fold increase in accuracy in the detection of activity correlations in functional imaging. Furthermore, using principal component analysis, we show that measurements obtained with Bessel beams are clean enough to reveal in one-shot experiments correlations that can not be averaged over trials after stimuli as is the case when studying spontaneous activity. Our results not only demonstrate the contamination of data by systematic and random errors through conventional Gaussian illumination and but,furthermore, quantify the increase in fidelity of such data when using Bessel beams.

Research paper thumbnail of Fast visible and extended near-infrared multispectral fundus camera

Journal of Biomedical Optics

We present a multispectral fundus camera that performs fast imaging of the ocular posterior pole ... more We present a multispectral fundus camera that performs fast imaging of the ocular posterior pole in the visible and near-infrared (400 to 1300 nm) wavelengths through 15 spectral bands, using a flashlight source made of light-emitting diodes, and CMOS and InGaAs cameras. We investigate the potential of this system for visualizing occult and overlapping structures of the retina in the unexplored wavelength range beyond 900 nm, in which radiation can penetrate deeper into the tissue. Reflectance values at each pixel are also retrieved from the acquired images in the analyzed spectral range. The available spectroscopic information and the visualization of retinal structures, specifically the choroidal vasculature and drusen-induced retinal pigment epithelium degeneration, which are hardly visible in conventional color fundus images, underline the clinical potential of this system as a new tool for ophthalmic diagnosis.

Research paper thumbnail of High-fidelity functional and structural whole-brain imaging with Bessel-beam light-sheet microscopy (Conference Presentation)

Neural Imaging and Sensing

Light-sheet microscopy (LSM) has proven a useful tool in neuroscience and is particularly well su... more Light-sheet microscopy (LSM) has proven a useful tool in neuroscience and is particularly well suited to image the entire brain with high frame rates at single cell resolution. On the one hand, LSM is employed in combination with tissue clearing methods like CLARITY which allows for the reconstruction of neuronal or vascular anatomy over cm-sized samples. On the other hand, LSM has been paired with intrinsically transparent samples for real-time recording of neuronal activity with single cell resolution across the entire brain, using calcium indicators like GCaMP6. Despite its intrinsic advantages in terms of high imaging speed and reduced photobleaching, LSM is very sensitive to residual opaque objects present in the sample, which cause dark horizontal stripes in the collected images. In the best case, these artefacts obscure the features of interest in structural imaging; in the worst case, dynamic shadowing introduced by red blood cells significantly alters the fluorescence signal variations related to neuronal activity. We show how the use of Bessel beams in LSM can dramatically reduce such artefacts even in conventional one-sided illumination schemes, thanks to their “self-healing” properties. On the functional side, Bessel-beam LSM allows recording neuronal activity traces without any disturbing flickering caused by the movement of red blood cells. On the structural side, our proposed method is capable of obtaining anatomical information across the entire volume of whole mouse brains allowing tracing blood vessels and neuronal projections also in poorly cleared specimens.

Research paper thumbnail of New techniques of transgenesis and imaging: applications in Neurology and Nephrology. Part I

Giornale Di Tecniche Nefrologiche E Dialitiche, Jun 30, 2014

New techNiques of traNsgeNesis aNd imagiNg: applicatioNs iN Neurology aNd Nephrology. part i Abst... more New techNiques of traNsgeNesis aNd imagiNg: applicatioNs iN Neurology aNd Nephrology. part i Abstract. The emerging techniques and applications related to transgenic animals, together with the large progresses in the development of imaging systems finalized at ensuring a high-level resolution, allow nowadays to analyze the finer details of a wide range of pathophysiological phenomena. The combination of the new methods applicable in the two fields, for instance, has made possible not only to explore biological processes in vivo as they occur, but also to analyze them at a whole organ level. At the same time, it is possible to create reconstructions in two or three dimensions of the entire organ, or of its specific functional units. A fourth temporal dimension can also be added thanks to the analysis in time-lapse or to the acquisition of data in continuous. The purpose of this first of two contributions is to review the latest innovations in the fields of microscopy and transgenesis, with particular attention to how these innovations have been implemented, and which benefits derive from their evolution. The possible applications and advantages of using these systems will further be analyzed and evaluated in more detail in the next issue of this survey.