Daniele Pirone

Italian National Research Council | CNR · Institute of Applied Sciences & Intelligent Systems

This study presents an innovative methodology to analyze a blood sample from a healthy donor, providing a quantitative characterization of white blood cells (WBCs). It aims to evaluate the effectiveness of holographic quantitative phase imaging (QPI) flow cytometry (FC) in examining phase-contrast maps at the cellular level, thereby enabling the id...

Gold standard imaging modalities in biological field are based on fluorescence signals providing high specificity and high resolution. Recently, Fluorescence Microscopy has been combined with microfluidics to develop instrumentations called Imaging Flow Cytometers, high-throughput tools that supply bright-field, darkfield and multiple-channels fluo...

The introduction of holo-tomographic flow cytometry has unlocked the possibility of imaging the 3D refractive index (RI) distribution of suspended cells while they flow and rotate in microfluidic streams. Similarly, approaches that optically trap and rotate the samples can image them in suspension conditions. Great effort has been spent in developi...

In this paper we show the application of In-Flow Holographic Tomography for the reconstruction of the 3D refractive index of marine phytoplankton. The relevance of this biological class is linked to their primary role in the life pyramid and their action as balance regulators of marine environment, whose health has been seriously threatened by anth...

Nowadays, radiotherapy (RT) is a consolidated treatment for the cancer care. In fact, ionizing radiations are employed in over 50% of cancer patients. However, some side effects are correlated with RT, such as the radiation-induced lymphopenia (RIL). RIL is due to circulating lymphocytes (LCs) that pass through the irradiation field. LCs are the mo...

Liquid biopsy, intended as the detection of circulating tumor cells (CTCs) in hematic specimens, is an emerging tool for both early cancer detection and estimation of prognosis. Herein, the strength of quantitative phase imaging (QPI) is investigated to achieve effective distinction of ovarian cancer (OC) from other blood cell populations based on...

Lab-on-a-Chip microfluidic devices present an innovative and cost-effective platform in the current trend of miniaturization and simplification of imaging flow cytometry; they are excellent candidates for high-throughput single-cell analysis. In such microfluidic platforms, cell tracking becomes a fundamental tool for investigating biophysical proc...

An intriguing field of research has recently emerged in which biological cells can be demonstrated to behave as photonics devices, such as optical microlenses. This research highlights yeast cells as promising candidates for engineering biolenses with customizable focal properties. Typically serving as positive biolenses due to their quasi‐spherica...

Accumulation of bioavailable heavy metals in aquatic environment poses a serious threat to marine communities and human health due to possible trophic transfers through the food chain of toxic, non-degradable, exogenous pollutants. Copper (Cu) is one of the most spread heavy metals in water, and can severely affect primary producers at high doses....

Breast cancer is one of the most spread and monitored pathologies in high-income countries. After breast biopsy, histological tissue is stored in paraffin, sectioned and mounted. Conventional inspection of tissue slides under benchtop light microscopes involves paraffin removal and staining, typically with H&E. Then, expert pathologists are called...

Lysosomes are the terminal end of catabolic pathways in the cell, as well as signaling centers performing important functions such as the recycling of macromolecules, organelles, and nutrient adaptation. The importance of lysosomes in human health is supported by the fact that the deficiency of most lysosomal genes causes monogenic diseases called...

Graphene is considered a possible drug deliver in nanomedicine for its mechanical, physical and chemical characteristics. Thus, studying graphene biocompatibility is pivotal to contribute to the modern nano-therapy science. The coexistence between cells and graphene should be analysed using non-invasive technologies and thus Quantitative Phase Imag...

Breast cancer is one of the most spread and monitored pathologies in high-income countries. After breast biopsy, histological tissue is stored in paraffin, sectioned and mounted. Conventional inspection of tissue slides under benchtop light microscopes involves paraffin removal and staining, typically with H&E. Then, expert pathologists are called...

In‐flow phase‐contrast tomography provides a 3D refractive index of label‐free cells in cytometry systems. Its major limitation, as with any quantitative phase imaging approach, is the lack of specificity compared to fluorescence microscopy, thus restraining its huge potentialities in single‐cell analysis and diagnostics. Remarkable results in intr...

To efficiently tackle certain tumor types, finding new biomarkers for rapid and complete phenotyping of cancer cells is highly demanded. This is especially the case for the most common pediatric solid tumor of the sympathetic nervous system, namely, neuroblastoma (NB). Liquid biopsy is in principle a very promising tool for this purpose, but usuall...

Image-based identification of circulating tumor cells in microfluidic cytometry condition is one of the most challenging perspectives in the Liquid Biopsy scenario. Here we show a machine learning-powered tomographic phase imaging flow cytometry system capable to provide high-throughput 3D phase-contrast tomograms of each single cell. In fact, we s...

Tomographic phase microscopy (TPM) in flow cytometry is one of the most promising computational imaging techniques for the quantitative 3-dimensional (3D) analysis of unstained single cells. Continuous cells’ flow, combined with the stain-free mode, can assure the high-throughput collection of quantitative and informative 3D data. TPM promises to a...

The most recent discoveries in the biochemical field are highlighting the increasingly important role of lipid droplets (LDs) in several regulatory mechanisms in living cells. LDs are dynamic organelles and therefore their complete characterization in terms of number, size, spatial positioning and relative distribution in the cell volume can shed l...

The three-dimensional version of Zernike polynomials are used to encode single-cell phase contrast tomograms, allowing an efficient tomographic data representation, compression and reconstruction with negligible information loss.

MicroLenses Array are ubiquitous in current technologies because of miniaturization needs. Applications ranges from high-resolution endoscopes to OLED. Here, a fabrication\actuation principle exploiting ferroelectric crystal is presented and a new paradigm on bio-lenses described.

Nowadays, label-free Imaging flow cytometry at single-cell level is considered the stepforward lab-on-a-chip technology to address challenges in clinical diagnostics, biology, life sciences and healthcare. In this framework, digital holography...

In digital holography (DH) modality for lab on chip application the cells passing through the field of view (FOV) of the microscope can be detected and analyzed even if they are flowing at different depths. In fact, in-focus imaging of each cell can be easily retrieved thanks to the ability of DH to obtain numerical focus ex-post the recording proc...

Identifying drug-resistant cancer cells is of fundamental importance to afford disease and find the most effective therapies for the patients. Recently, label-free imaging flow cytometry has been deeply investigated in cell recognition. In particular, the combination of flow cytometry and machine learning allows for achieving high accuracy in cell...

In recent years, intracellular LDs have been discovered to play an important role in several pathologies. Therefore, detection of LDs would provide an in-demand diagnostic tool if coupled with flow-cytometry to give significant statistical analysis and especially if the diagnosis is made in full non-invasive mode. Here we combine the experimental r...

Patterned microstructures are commonly used for investigating cells proliferation, guiding cell fate and promoting adhesion and differentiation. Analyzing the behavior of cells layered onto functionalized micropatterned substrates ideally requires large Field of View (FoV), depth of focus, and spatial resolution. Managing the trade-off among these...

Live cells act as biological lenses and can be employed as real‐world optical components in bio‐hybrid systems. Imaging at nanoscale, optical tweezers, lithography and also photonic waveguiding are some of the already proven functionalities, boosted by the advantage that cells are fully biocompatible for intra‐body applications. So far, various cel...

Phase-contrast tomographic flow cytometry combines quantitative 3D analysis of unstained single cells and high-throughput. A crucial issue of this method is the storage and management of the huge amount of 3D tomographic data. Here we show an effective quasi lossless compression of tomograms data through 3D Zernike descriptors, unlocking data manag...

Fourier ptychographic microscopy probes label-free samples from multiple angles and achieves super resolution phase-contrast imaging according to a synthetic aperture principle. Thus, it is particularly suitable for high-resolution imaging of tissue slides over a wide field of view. Recently, in order to make the optical setup robust against misali...

Tomographic flow cytometry by digital holography is an emerging imaging modality capable of collecting multiple views of moving and rotating cells with the aim of recovering their refractive index distribution in 3D. Although this modality allows us to access high-resolution imaging with high-throughput, the huge amount of time-lapse holographic im...

Fourier Ptychographic Microscopy (FPM) is a powerful bioimaging tool. Here we show numerical Multi-Look FPM and GAN-based reconstruction of biological samples to get rid of system misalignments and hone the FPM use in clinical practice.

A method is discussed to retrieve the unknown 3D positions and rolling angles from the digital holograms of flowing cells for reconstructing the spatial distribution of their refractive index through the tomographic flow cytometry tool.

The huge amount of phase maps to be numerically retrieved from digital holograms is the actual bottleneck of the high-throughput holographic flow cytometry. An end-to-end neural network is discussed to speed up the holographic processing.

Quantitative Phase Imaging (QPI) has gained popularity because it can avoid the staining step, which in some cases is difficult or impossible. However, QPI does not provide the well-known specificity to various parts of the cell (e.g., organelles, membrane). Here we show a novel computational segmentation method based on statistical inference that...

The most recent discoveries in the biochemical field are highlighting the increasingly important role of lipid droplets (LDs) in several regulatory mechanisms in living cells. LDs are dynamic organelles and therefore their complete characterization in terms of number, size, spatial positioning and relative distribution in the cell volume can shed l...

Digital holography in microscopy plays an important role in optical imaging technique thanks to its noninvasive, label free, quantitative and high-resolution approach. In particular DH allows to obtain the imaging of 3D volume by using the whole complex field of the scattered radiation by the sample by means of numerical reconstruction methods. Tha...

Single-cell phase-contrast tomography promises to become decisive for studying 3D intracellular structures in biology. It involves probing cells with light at wide angles, which unfortunately requires complex systems. Here we show an intriguing concept based on an inherent natural process for plants biology, i.e., dehydration, allowing us to easily...

Holographic Tomography (HT) is an emerging label-free technique for microscopic bioimaging applications, that allows reconstructing the three-dimensional (3D) refractive index (RI) distribution of biological specimens. Recently, an in-flow HT technique has been proposed in which multiple digital holograms are recorded at different viewing angles ar...

The label-free single cell analysis by machine and Deep Learning, in combination with digital holography in transmission microscope configuration, is becoming a powerful framework exploited for phenotyping biological samples. Usually, quantitative phase images of cells are retrieved from the reconstructed complex diffraction patterns and used as in...

We investigate the ability of machine learning to provide an accurate classification of cancer cell in microfluidics when only raw digital holograms are used as input data. Comparison among different learning strategies is addressed.

The loss of water content in plant cells results in the increasing of refractive index difference between organelles and background, thus allowing high contrast digital holographic observation of intracellular dynamics.

Holographic tomography allows the 3D mapping of the refractive index of biological samples thanks to reconstruction methods based on the knowledge of illumination directions or rotation angles of the imaged sample. Recently, phase contrast tomographic flow cytometry by digital holography has been demonstrated to reconstruct the three-dimensional re...

We show that dehydrating process can be effective for improving the phase-contrast in bioimaging, thus permitting a better observation of plant cells with the scope of learning more about cellular dynamics.