13 research outputs found
Lithium niobate micromachining for the fabrication of microfluidic droplet generators
In this paper, we present the first microfluidic junctions for droplet generation directly engraved on lithium niobate crystals by micromachining techniques, preparatory to a fully integrated opto-microfluidics lab-on-chip system. In particular, laser ablation technique and the mechanical micromachining technique are exploited to realise microfluidic channels in T-and cross junction configurations. The quality of both lateral and bottom surfaces of the channels are therefore compared together with a detailed study of their roughness measured by means of atomic force microscopy in order to evaluate the final performance achievable in an optofluidic device. Finally, the microfluidics performances of these water-in-oil droplets generators are investigated depending on these micromachining techniques, with particular focus on a wide range of droplet generation rates
Integrated Opto-Microfluidic Lab-on-a-Chip in Lithium Niobate for Droplet Generation and Sensing
In the last decades microfluidics has gained an increasing interest by the scientific community
due to its capability of manipulating liquids on the microscale. In particular droplet
microfluidics technology holds great promise due to its precise control on very small volumes
of fluid: droplets can be coalesced, mixed and sorted, employed either as micro chemical
reactors or as carriers of biological samples.
This features, in combination with fast analysis tools, allow for the realization of Lab-on-a-
Chips (LOCs), miniaturized and portable devices able to perform chemical, biological,
environmental or medical analyses where laboratory facilities lack.
Nevertheless, in most cases, sensing inside LOCs is performed by external optical stages
somehow added to the microfluidic chip. One of the hurdle towards the effective employment
of such systems is indeed the complete integration between the microfluidic stage
and the optical one. Often extended systems such as microscope objectives and fast CCD
cameras have been used to detect droplets inside microfluidic channels nullifying the efforts
spent to decrease the dimensions of LOCs and their related advantages.
In this work the first opto-microfluidic Lab-on-a-Chip (LOC) for both generation and
detection of droplets, entirely integrated in lithium niobate is presented.
The main elements of the LOC are a passive droplet generator, where water in oil droplets
are produced by the cross-flow of immiscible phases, and two waveguides on the surface
of the crystal able to illuminate droplets perpendicularly to their flow and to collect the
transmitted intensity.
The realization of single mode channel waveguides at a wavelength of 632.8nm on lithium
niobate by titanium in-diffusion is achieved and the obtained waveguides are characterized
by Rutherford Backscattering Spectrometrxy (RBS), Secondary Ion Mass Spectrometry
(SIMS) and near field measurements.
A deep investigation on the applicability of lithium niobate on the field of microfluidics is
carried out through a study of its wettability. In addition a functionalization procedure to
improve its hydrophobicity is defined.
Various techniques to engrave the microfluidic channels directly on the crystal are taken
into account. In particular the ablation by a femtosecond pulsed laser at a wavelength of
800nm is widely investigated by optical microscopy and atomic force microscopy (AFM)
in order to define the best process parameters to get the lower average roughness of the
channel walls (Ra˜50nm). Channels engraved with a dicing saw are also characterized
showing to be the best solution for optofluidic applications due to their extremely low
average roughness (Ra = (6÷7) nm).
New techniques for sealing the channels engraved on the surface of the crystal are described
with a particular care to the device durability and its applicability to different purposes.
Passive droplet generators with a T-junction geometry obtained by laser ablation in lithium
niobate are shown to generate droplets in a wide range of frequencies (10÷1000Hz)
and with a very sharp distribution of droplets volumes (sigma < 3%). Their performances are
characterized employing an optical microscopy setup and the experimental data are discussed
with respect to the theoretical models reported in literature. Discrepancies from the
theory at low values of the capillary number (Ca < 3*10ˆ-3) are highlighted and described
by means of an empirical law.
Finally the coupling of the waveguides to the microfluidic stage is discussed showing
how it can be used to count and trigger the droplets during their flow, achieving better
performances than the standard optical microscopy setup.
The ultimate configuration of the presented LOC prototype is characterized by two crossing
channels obtained by mechanical dicing with three inlet branches and one outlet branch.
Three phases are flown together (oil, water and a saline solution) and alternating droplets
of pure water and saline solution are produced in oil. The transmitted intensity from the
waveguide is shown to be sensitive to the refractive index of the solution with a sensitivity
of dn = 2*10ˆ{-3} in the range n = [1.339, 1.377].
This is the first example of a Lab-On-a-Chip for real time droplet counting and refractive
index sensing, completely integrated in lithium niobate.Negli ultimi decenni la microfluidica ha riscosso un crescente interesse presso la comunitÃ
scientifica grazie alla sua capacità di manipolare liquidi su scala micrometrica. In particolare
la microfluidica a gocce è particolarmente promettente per la possibilità di controllare
volumi di fluido molto ridotti: le gocce possono essere unite, mescolate e selezionate,
utilizzate come microreattori chimici o per trasportare campioni biologici.
Queste peculiarità , unite a strumenti di analisi rapidi, permettono di realizzare i cosiddetti
Lab-on-a-Chip (LOC), dispositivi miniaturizzati e portatili capaci di condurre analisi
chimiche, biologiche, ambientali o mediche in mancanza di veri e propri laboratori.
Ciononostante, nella maggior parte dei casi, la rilevazione all’interno dei Lab-on-a-Chip
viene praticata da stadi esterni di ottica affiancati al chip microfluidico. Infatti uno degli
ostacoli maggiori verso l’effettivo utilizzo di questi sistemi è il raggiungimento di una completa
integrazione tra lo stadio microfluidico e quello ottico. Spesso sistemi ingombranti
come gli obiettivi di un microscopio o fotocamere veloci sono utilizzate per rilevare gocce
all'interno di canali microfluidici, vanificando gli sforzi fatti per ridurre le dimensioni dei
LOC e i vantaggi ad essi collegati.
In questo lavoro viene presentato il primo Lab-on-a-Chip opto-microfluidico per la generazione
e la rilevazione di gocce, interamente integrato in niobato di litio.
I principali elementi del LOC sono un generatore di gocce passivo, dove vengono prodotte
gocce di acqua in olio attraverso l’incontro tra flussi di fasi immiscibili, e due guide d’onda
sulla superficie del cristallo capaci di illuminare le gocce perpendicolarmente alla direzione
in cui scorrono e raccoglierne l’intensità trasmessa.
Vengono mostrate la realizzazione di guide a canale monomodo alla lunghezza d’onda di
632.8nm in niobato di litio per diffusione di titanio e la loro caratterizzazione attraverso
le tecniche di Rutherford Backscattering Spectrometry (RBS), Secondary Ion Mass Spectrometry
(SIMS) e near field.
Un’indagine approfondita sull'applicabilità del niobato di litio in campo microfluidico viene
svolta attraverso lo studio delle sue proprietà di bagnabilità . In aggiunta viene definita
una procedura di funzionalizzazione per aumentarne l'idrofobicità .
Vengono prese in considerazione varie tecniche per scavare i canali direttamente sul cristallo.
In particolare l’ablazione per mezzo di un laser impulsato al femtosecondo ad una lunghezza
d’onda di 800nm viene caratterizzata tramite microscopia ottica e microscopia a forza
atomica (AFM) per stabilire i migliori parametri del processo al fine di ottenere la minima
rugosità media possibile (Ra˜50 nm). Vengono inoltre caratterizzati canali scavati
con una lama autolucidante che risultano essere la migliore soluzione nell'impiego in optofluidica
per la loro rugosità estremamente ridotta (Ra = (6÷7) nm).
Sono state inoltre sperimentate nuove tecniche per la chiusura dei canali, prestando particolare
attenzione alla durevolezza del dispositivo e alla possibile applicazione in diversi
campi.
Si mostra come i generatori passivi di gocce con geometria a "T" (T-junction) ottenuti per
ablazione laser in niobato di litio siano in grado di generare gocce in un ampia gamma di frequenze
(10÷1000 Hz) e con una distribuzione di lunghezze estremamente piccata (sigma < 3%).
Ne vengono caratterizzate le prestazioni con l’impiego di un sistema di microscopia ottica
e i dati sperimentali vengono discussi confrontandoli con i modelli teorici riportati in letteratura.
Le discrepanze tra la teoria e i dati sperimentali a bassi valori del numero di
capillarità (Ca < 3*10ˆ-3) vengono evidenziate e descritte per mezzo di una legge empirica.
Infine viene affrontato l’accoppiamento delle guide d’onda allo stadio microfluidico mostrando
come il chip possa essere sfruttato per misurare il tempo di passaggio delle gocce,
ottenendo risultati migliori rispetto al classico sistema di microscopia ottica.
La configurazione finale del LOC è caratterizzata da due canali che si intersecano, ottenuti
per lavorazione meccanica, con tre rami di immissione e uno di uscita. Tre fasi vengono
flussate contemporaneamente (olio, acqua e una soluzione salina) in modo da ottenere gocce
alternate di acqua e di soluzione salina in olio. Si mostra come l’intensità trasmessa dalla
guida d’onda dipenda dall'indice di rifrazione della soluzione salina con una sensibilità di
dn = 2*10ˆ-3 nel range di valori n = [1.339, 1.377].
Il dispositivo presentato è il primo esempio di Lab-On-a-Chip per il conteggio e la misure
dell’indice di rifrazione di gocce in tempo reale, completamente integrato in niobato di litio
Integrated Opto-Microfluidic Lab-on-a-Chip in Lithium Niobate for Droplet Generation and Sensing
In the last decades microfluidics has gained an increasing interest by the scientific community
due to its capability of manipulating liquids on the microscale. In particular droplet
microfluidics technology holds great promise due to its precise control on very small volumes
of fluid: droplets can be coalesced, mixed and sorted, employed either as micro chemical
reactors or as carriers of biological samples.
This features, in combination with fast analysis tools, allow for the realization of Lab-on-a-
Chips (LOCs), miniaturized and portable devices able to perform chemical, biological,
environmental or medical analyses where laboratory facilities lack.
Nevertheless, in most cases, sensing inside LOCs is performed by external optical stages
somehow added to the microfluidic chip. One of the hurdle towards the effective employment
of such systems is indeed the complete integration between the microfluidic stage
and the optical one. Often extended systems such as microscope objectives and fast CCD
cameras have been used to detect droplets inside microfluidic channels nullifying the efforts
spent to decrease the dimensions of LOCs and their related advantages.
In this work the first opto-microfluidic Lab-on-a-Chip (LOC) for both generation and
detection of droplets, entirely integrated in lithium niobate is presented.
The main elements of the LOC are a passive droplet generator, where water in oil droplets
are produced by the cross-flow of immiscible phases, and two waveguides on the surface
of the crystal able to illuminate droplets perpendicularly to their flow and to collect the
transmitted intensity.
The realization of single mode channel waveguides at a wavelength of 632.8nm on lithium
niobate by titanium in-diffusion is achieved and the obtained waveguides are characterized
by Rutherford Backscattering Spectrometrxy (RBS), Secondary Ion Mass Spectrometry
(SIMS) and near field measurements.
A deep investigation on the applicability of lithium niobate on the field of microfluidics is
carried out through a study of its wettability. In addition a functionalization procedure to
improve its hydrophobicity is defined.
Various techniques to engrave the microfluidic channels directly on the crystal are taken
into account. In particular the ablation by a femtosecond pulsed laser at a wavelength of
800nm is widely investigated by optical microscopy and atomic force microscopy (AFM)
in order to define the best process parameters to get the lower average roughness of the
channel walls (Ra˜50nm). Channels engraved with a dicing saw are also characterized
showing to be the best solution for optofluidic applications due to their extremely low
average roughness (Ra = (6÷7) nm).
New techniques for sealing the channels engraved on the surface of the crystal are described
with a particular care to the device durability and its applicability to different purposes.
Passive droplet generators with a T-junction geometry obtained by laser ablation in lithium
niobate are shown to generate droplets in a wide range of frequencies (10÷1000Hz)
and with a very sharp distribution of droplets volumes (sigma < 3%). Their performances are
characterized employing an optical microscopy setup and the experimental data are discussed
with respect to the theoretical models reported in literature. Discrepancies from the
theory at low values of the capillary number (Ca < 3*10ˆ-3) are highlighted and described
by means of an empirical law.
Finally the coupling of the waveguides to the microfluidic stage is discussed showing
how it can be used to count and trigger the droplets during their flow, achieving better
performances than the standard optical microscopy setup.
The ultimate configuration of the presented LOC prototype is characterized by two crossing
channels obtained by mechanical dicing with three inlet branches and one outlet branch.
Three phases are flown together (oil, water and a saline solution) and alternating droplets
of pure water and saline solution are produced in oil. The transmitted intensity from the
waveguide is shown to be sensitive to the refractive index of the solution with a sensitivity
of dn = 2*10ˆ{-3} in the range n = [1.339, 1.377].
This is the first example of a Lab-On-a-Chip for real time droplet counting and refractive
index sensing, completely integrated in lithium niobate.Negli ultimi decenni la microfluidica ha riscosso un crescente interesse presso la comunitÃ
scientifica grazie alla sua capacità di manipolare liquidi su scala micrometrica. In particolare
la microfluidica a gocce è particolarmente promettente per la possibilità di controllare
volumi di fluido molto ridotti: le gocce possono essere unite, mescolate e selezionate,
utilizzate come microreattori chimici o per trasportare campioni biologici.
Queste peculiarità , unite a strumenti di analisi rapidi, permettono di realizzare i cosiddetti
Lab-on-a-Chip (LOC), dispositivi miniaturizzati e portatili capaci di condurre analisi
chimiche, biologiche, ambientali o mediche in mancanza di veri e propri laboratori.
Ciononostante, nella maggior parte dei casi, la rilevazione all’interno dei Lab-on-a-Chip
viene praticata da stadi esterni di ottica affiancati al chip microfluidico. Infatti uno degli
ostacoli maggiori verso l’effettivo utilizzo di questi sistemi è il raggiungimento di una completa
integrazione tra lo stadio microfluidico e quello ottico. Spesso sistemi ingombranti
come gli obiettivi di un microscopio o fotocamere veloci sono utilizzate per rilevare gocce
all'interno di canali microfluidici, vanificando gli sforzi fatti per ridurre le dimensioni dei
LOC e i vantaggi ad essi collegati.
In questo lavoro viene presentato il primo Lab-on-a-Chip opto-microfluidico per la generazione
e la rilevazione di gocce, interamente integrato in niobato di litio.
I principali elementi del LOC sono un generatore di gocce passivo, dove vengono prodotte
gocce di acqua in olio attraverso l’incontro tra flussi di fasi immiscibili, e due guide d’onda
sulla superficie del cristallo capaci di illuminare le gocce perpendicolarmente alla direzione
in cui scorrono e raccoglierne l’intensità trasmessa.
Vengono mostrate la realizzazione di guide a canale monomodo alla lunghezza d’onda di
632.8nm in niobato di litio per diffusione di titanio e la loro caratterizzazione attraverso
le tecniche di Rutherford Backscattering Spectrometry (RBS), Secondary Ion Mass Spectrometry
(SIMS) e near field.
Un’indagine approfondita sull'applicabilità del niobato di litio in campo microfluidico viene
svolta attraverso lo studio delle sue proprietà di bagnabilità . In aggiunta viene definita
una procedura di funzionalizzazione per aumentarne l'idrofobicità .
Vengono prese in considerazione varie tecniche per scavare i canali direttamente sul cristallo.
In particolare l’ablazione per mezzo di un laser impulsato al femtosecondo ad una lunghezza
d’onda di 800nm viene caratterizzata tramite microscopia ottica e microscopia a forza
atomica (AFM) per stabilire i migliori parametri del processo al fine di ottenere la minima
rugosità media possibile (Ra˜50 nm). Vengono inoltre caratterizzati canali scavati
con una lama autolucidante che risultano essere la migliore soluzione nell'impiego in optofluidica
per la loro rugosità estremamente ridotta (Ra = (6÷7) nm).
Sono state inoltre sperimentate nuove tecniche per la chiusura dei canali, prestando particolare
attenzione alla durevolezza del dispositivo e alla possibile applicazione in diversi
campi.
Si mostra come i generatori passivi di gocce con geometria a "T" (T-junction) ottenuti per
ablazione laser in niobato di litio siano in grado di generare gocce in un ampia gamma di frequenze
(10÷1000 Hz) e con una distribuzione di lunghezze estremamente piccata (sigma < 3%).
Ne vengono caratterizzate le prestazioni con l’impiego di un sistema di microscopia ottica
e i dati sperimentali vengono discussi confrontandoli con i modelli teorici riportati in letteratura.
Le discrepanze tra la teoria e i dati sperimentali a bassi valori del numero di
capillarità (Ca < 3*10ˆ-3) vengono evidenziate e descritte per mezzo di una legge empirica.
Infine viene affrontato l’accoppiamento delle guide d’onda allo stadio microfluidico mostrando
come il chip possa essere sfruttato per misurare il tempo di passaggio delle gocce,
ottenendo risultati migliori rispetto al classico sistema di microscopia ottica.
La configurazione finale del LOC è caratterizzata da due canali che si intersecano, ottenuti
per lavorazione meccanica, con tre rami di immissione e uno di uscita. Tre fasi vengono
flussate contemporaneamente (olio, acqua e una soluzione salina) in modo da ottenere gocce
alternate di acqua e di soluzione salina in olio. Si mostra come l’intensità trasmessa dalla
guida d’onda dipenda dall'indice di rifrazione della soluzione salina con una sensibilità di
dn = 2*10ˆ-3 nel range di valori n = [1.339, 1.377].
Il dispositivo presentato è il primo esempio di Lab-On-a-Chip per il conteggio e la misure
dell’indice di rifrazione di gocce in tempo reale, completamente integrato in niobato di litio
Lithium Niobate Micromachining for the Fabrication of Microfluidic Droplet Generators
In this paper, we present the first microfluidic junctions for droplet generation directly engraved on lithium niobate crystals by micromachining techniques, preparatory to a fully integrated opto-microfluidics lab-on-chip system. In particular, laser ablation technique and the mechanical micromachining technique are exploited to realise microfluidic channels in T- and cross junction configurations. The quality of both lateral and bottom surfaces of the channels are therefore compared together with a detailed study of their roughness measured by means of atomic force microscopy in order to evaluate the final performance achievable in an optofluidic device. Finally, the microfluidics performances of these water-in-oil droplets generators are investigated depending on these micromachining techniques, with particular focus on a wide range of droplet generation rates
Brain malformations associated to Aldh7a1 gene mutations: Report of a novel homozygous mutation and literature review
Background: The ALDH7A1 gene is known to be responsible for autosomal recessive pyridoxine-dependent epilepsy (OMIM 266100). The phenotypic spectrum of ALDH7A1 mutations is very heterogeneous ranging from refractory epilepsy and neurodevelopmental delay, to multisystem neonatal disorder. Aim: The present study aims at describing the phenotype associated with a novel homozygous ALDH7A1 mutation and the spectrum of brain malformations associated with pyridoxine-dependent epilepsy. Methods: We conducted a literature review on the Internet database Pubmed (up to November 2017) searching for ALDH7A1 mutations associated with brain malformations and brain MRI findings. Results: We present the case of two siblings, children of related parents. The proband presented neonatal focal seizures not responding to conventional antiepileptic drugs. Electroencephalography showed a suppression burst pattern and several multifocal ictal patterns, responsive to pyridoxine. Brain MRI was normal. Molecular analysis by targeted next-generation sequencing panel for epileptic encephalopathy disclosed a homozygous missense mutation of ALDH7A1. The same mutation was then found in a stored sample of DNA from peripheral blood of an older sister dead 3 years earlier. This girl presented a complex brain malformation diagnosed with a foetal MRI and had neonatal refractory seizures with suppression burst pattern. She died at 6 months of age. Literature review: The brain abnormalities most frequently reported in pyridoxine-dependent epilepsy include: agenesia/hypoplasia of the corpus callosum, not specific white matter abnormalities, large cisterna magna, ventriculomegaly, haemorrhages, cerebellum hypoplasia/dysplasia, and, more rarely, dysplasia of the brainstem and hydrocephalus. Discussion and conclusions: ALDH7A1 mutations have been associated to different brain abnormalities, documented by MRI only in few cases. The study cases expand the clinical spectrum of ALDH7A1 associated conditions, suggesting to look for ALDH7A1 mutations not only in classical phenotypes but also in patients with brain malformations, mainly if there is a response to a pyridoxine trial
Integrated opto-microfluidics platforms in lithium niobate crystals for sensing applications
In micro-analytical chemistry and biology applications, droplet microfluidic technology holds great promise for efficient lab-on-chip systems where higher levels of integration of different stages on the same platform is constantly addressed. The possibility of integration of opto-microfluidic functionalities in lithium niobate (LiNbO3) crystals is presented. Microfluidic channels were directly engraved in a LiNbO3 substrate by precision saw cutting, and illuminated by optical waveguides integrated on the same substrate. The morphological characterization of the microfluidic channel and the optical response of the coupled optical waveguide were tested. In particular, the results indicate that the optical properties of the constituents dispersed in the fluid flowing in the microfluidic channel can be monitored in situ, opening to new compact optical sensor prototypes based on droplets generation and optical analysis of the relative constituents
Management of hypertensive emergencies: a practical approach
none9noBackground: Acute increases of high blood pressure values, usually described as ‘hypertensive crises’, ‘hypertensive urgencies’ or ‘hypertensive emergencies’, are common causes of patients’ presentation to emergency departments. Owing to the lack of ad hoc randomized clinical trials, current recommendations/suggestions for treatment of these patients are not evidenced-based and, therefore, the management of acute increases of blood pressure values represent a clinical challenge. However, an improved understanding of the underlying pathophysiology has changed radically the approach to management of the patients presenting with these conditions in recent years. Accordingly, it has been proposed to abandon the terms ‘hypertensive crises’ and ‘hypertensive urgencies’, and restrict the focus to ‘hypertensive emergencies’. Aims and Methods: Starting from these premises, we aimed at systematically review all available studies (years 2010-2020) to garner information on the current management of hypertensive emergencies, in order to develop a novel symptoms- and evidence-based streamlined algorithm for the assessment and treatment of these patients. Results and Conclusions: In this educational review we proposed the BARKH-based algorithm for a quick identification of hypertensive emergencies and associated acute organ damage, to allow the patients with hypertensive emergencies to receive immediate treatment in a proper setting.restrictedRossi G.P.; Rossitto G.; Maifredini C.; Barchitta A.; Bettella A.; Latella R.; Ruzza L.; Sabini B.; Seccia T.M.Rossi, G. P.; Rossitto, G.; Maifredini, C.; Barchitta, A.; Bettella, A.; Latella, R.; Ruzza, L.; Sabini, B.; Seccia, T. M
Integrated optics on Lithium Niobate for sensing applications
In micro-analytical chemistry and biology applications, optofluidic technology holds great promise for creating efficient lab-on-chip systems where higher levels of integration of different stages on the same platform is constantly addressed. Therefore, in this work the possibility of integrating opto-microfluidic functionalities in lithium niobate (LiNbO3) crystals is presented. In particular, a T-junction droplet generator is directly engraved in a LiNbO3 substrate by means of laser ablation process and optical waveguides are realized in the same material by exploiting the Titanium in-diffusion approach. The coupling of these two stages as well as the realization of holographic gratings in the same substrate will allow creating new compact optical sensor prototypes, where the optical properties of the droplets constituents can be monitored
Application of muon tomography to detect radioactive sources hidden in scrap metal containers2013 3rd International Conference on Advancements in Nuclear Instrumentation, Measurement Methods and their Applications (ANIMMA)
none13The accidental melting of radioactive sources hidden inside metal scrap containers can produce severe environmental harm. Modern melting facilities are equipped with portals measuring radiation levels. Nonetheless, sources can pass undiscovered when shielded inside shells of high density material, such as lead. From time to time indeed some radioactive sources pass undetected through the controls at foundries entrance. Once melted they caused enormous damages to the steel mills, contaminating all the production line. The muon tomography technique allows to discriminate high- Z materials measuring multiple scattering of cosmic ray muons inside matter. Therefore this technique can be used to analyze a truck container searching for high-density source shields. We report here the results about simulation studies of a muon tomography portal. Within the Mu-Steel European project we developed the prototype design, the three-dimensional images reconstruction software and the high density material identification algorithm. MonteCarlo simulation was validated with data from a large volume demonstrator (~11 m3) built using spare muon drift-time chambers of the CMS high energy physics experiment operating at the Large Hadron Collider at CERN.noneMatteo Furlan;Andrea Rigoni;Sara Vanini;Gianni Zumerle;Paolo Checchia;Ludovico Cossutta;Giacomo Bettella;Pietro Zanuttigh;Piero Calvini;Luca Dassa;Antonietta Donzella;Germano Bonomi;Aldo ZenoniFurlan, Matteo; RIGONI GAROLA, Andrea; Vanini, Sara; Zumerle, Gianni; Checchia, Paolo; Ludovico, Cossutta; Bettella, Giacomo; Zanuttigh, Pietro; Piero, Calvini; Luca, Dassa; Antonietta, Donzella; Germano, Bonomi; Aldo, Zenon