Martina Mugnano

Tomographic flow cytometry by digital holography

High-throughput single-cell analysis is a challenging task. Label-free tomographic phase microscopy is an excellent candidate to perform this task. However, in-line tomography is very difficult to implement in practice because it requires a complex set-up for rotating the sample and examining the cell along several directions. We demonstrate that by exploiting the random rolling of cells while they are flowing along a microfluidic channel, it is possible to obtain in-line phase-contrast tomography, if smart strategies for wavefront analysis are adopted. In fact, surprisingly, a priori knowledge of the three-dimensional position and orientation of rotating cells is no longer needed because this information can be completely retrieved through digital holography wavefront numerical analysis. This approach makes continuous-flow cytotomography suitable for practical operation in real-world, single-cell analysis and with a substantial simplification of the optical system; that is, no mechanical scanning or multi-direction probing is required. A demonstration is given for two completely different classes of biosamples: red blood cells and diatom algae. An accurate characterization of both types of cells is reported, despite their very different nature and material content, thus showing that the proposed method can be extended by adopting two alternate strategies of wavefront analysis to many classes of cells.

Investigation on dynamics of red blood cells through their behavior as biophotonic lenses

Abstract. The possibility to adopt biological matter as photonic optical elements can open scenarios in biophotonics research. Recently, it has been demonstrated that a red blood cell (RBC) can be seen as an optofluidic microlens by showing its imaging capability as well as its focal tunability. Moreover, correlation between an RBC’s morphology and its behavior as a refractive optical element has been established and its exploitation for biomedical diagnostic purposes has been foreseen. In fact, any deviation from the healthy RBC morphology can be seen as additional aberration in the optical wavefront passing through the cell. By this concept, accurate localization of focal spots of RBCs can become very useful in the blood disorders identification. We investigate the three-dimensional positioning of such focal spots over time for samples with two different osmolarity conditions, i.e., when they assume discocyte and spherical shapes, respectively. We also demonstrate that a temporal variation of an RBC’s focal points along the optical axis is correlated to the temporal fluctuations in the RBC’s thickness maps. Furthermore, we show a sort of synchronization of the whole erythrocytes ensemble.

Nanomechanics of a fibroblast suspended using point-like anchors reveal cytoskeleton formation

In an attempt to better elucidate the material–cytoskeleton crosstalk during the initial stage of cell adhesion, here we report how suspended cells anchored to point-like bonds are able to assemble their cytoskeleton when subjected to mechanical stress. The combination of holographic optical tweezers and digital holography gives the cell footholds for adhesion and mechanical stimulation, and at the same time, acts as a label-free, force-revealing system over time, detecting the cell nanomechanical response in the pN range. To confirm the formation of the cytoskeleton structures after the stimulation, a fluorescence imaging system was added as a control. The strategy here proposed portends broad applicability to investigate the correlation between the forces applied to cells and their cytoskeleton assembly process in this or other complex configurations with multiple anchor points.

Biolens behavior of RBCs under optically-induced mechanical stress: Study of RBC-Biolens Deformations by Digital Holography

In this work, the optical behavior of Red Blood Cells (RBCs) under an optically‐induced mechanical stress was studied. Exploiting the new findings concerning the optical lens‐like behavior of RBCs, the variations of the wavefront refracted by optically‐deformed RBCs were further investigated. Experimental analysis have been performed through the combination of digital holography and numerical analysis based on Zernike polynomials, while the biological lens is deformed under the action of multiple dynamic optical tweezers. Detailed wavefront analysis provides comprehensive information about the aberrations induced by the applied mechanical stress. By this approach it was shown that the optical properties of RBCs in their discocyte form can be affected in a different way depending on the geometry of the deformation. In analogy to classical optical testing procedures, optical parameters can be correlated to a particular mechanical deformation. This could open new routes for analyzing cell elasticity by examining optical parameters instead of direct but with low resolution strain analysis, thanks to the high sensitivity of the interferometric tool. Future application of this approach could lead to early detection and diagnosis of blood diseases through a single‐step wavefront analysis for evaluating different cells elasticity.

Monitoring cell morphology during necrosis and apoptosis by quantitative phase imaging

Cellular morphology changes and volume alterations play significant roles in many biological processes and they are mirrors of cell functions. In this paper, we propose the Digital Holographic microscope (DH) as a non-invasive imaging technique for a rapid and accurate extraction of morphological information related to cell death. In particular, we investigate the morphological variations that occur during necrosis and apoptosis. The study of necrosis is extremely important because it is often associated with unwarranted loss of cells in human pathologies such as ischemia, trauma, and some forms of neurodegeneration; therefore, a better elucidation in terms of cell morphological changes could pave the way for new treatments. Also, apoptosis is extremely important because it’s involved in cancer, both in its formation and in medical treatments. Because the inability to initiate apoptosis enhances tumour formation, current cancer treatments target this pathway. Within this framework, we have developed a transmission off-axis DH apparatus integrated with a micro incubator for investigation of living cells in a temperature and CO2 controlled environment. We employ DH to analyse the necrosis cell death induced by laser light (wavelength 473 nm, light power 4 mW). We have chosen as cellular model NIH 3T3 mouse embryonic fibroblasts because their adhesive features such as morphological changes, and the time needed to adhere and spread have been well characterized in the literature. We have monitored cell volume changes and morphological alterations in real time in order to study the necrosis process accurately and quantitatively. Cell volume changes were evaluated from the measured phase changes of light transmitted through cells. Our digital holographic experiments showed that after exposure of cells to laser light for 90-120 min., they swell and then take on a balloon-like shape until the plasma membrane ruptures and finally the cell volume decreases. Furthermore, we present a preliminary study on the variation of morphological parameters in case of cell apoptosis induced by exposure to 10 μM cadmium chloride. We employ the same cell line, monitoring the process for 18 hours. In the vast group of environmental pollutants, the toxic heavy metal cadmium is considered a likely candidate as a causative agent of several types of cancers. Widely distributed and used in industry, and with a broad range of target organs and a long half-life (10-30 years) in the human body, this element has been long known for its multiple adverse effects on human health, through occupational or environmental exposure. In apoptosis, we measure cell volume decrease and cell shrinking. Both data of apoptosis and necrosis were analysed by means of a Sigmoidal Statistical Distribution function, which allows several quantitative data to be established, such as swelling and cell death time, flux of intracellular material from inside to outside the cell, initial and final volume versus time. In addition, we can quantitatively study the cytoplasmatic granularity that occurs during necrosis. As a future application, DH could be employed as a non-invasive and label-free method to distinguish between apoptosis and necrosis in terms of morphological parameters.

Lab on chip optical imaging of biological sample by quantitative phase microscopy

Quantitative imaging and three dimensional (3D) morphometric analysis of flowing and not-adherent cells is an important aspect for diagnostic purposes at Lab on Chip scale. Diagnostics tools need to be quantitative, label-free and, as much as possible, accurate. In recent years digital holography (DH) has been improved to be considered as suitable diagnostic method in several research field. In this paper we demonstrate that DH can be used for retrieving 3D morphometric data for sorting and diagnosis aims. Several techniques exist for 3D morphological study as optical coherent tomography and confocal microscopy, but they are not the best choice in case of dynamic events as flowing samples. Recently, a DH approach, based on shape from silhouette algorithm (SFS), has been developed for 3D shape display and calculation of cells biovolume. Such approach, adopted in combination with holographic optical tweezers (HOT) was successfully applied to cells with convex shape. Unfortunately, it’s limited to cells with convex surface as sperm cells or diatoms. Here, we demonstrate an improvement of such procedure. By decoupling thickness information from refractive index ones and combining this with SFS analysis, 3D shape of concave cells is obtained. Specifically, the topography contour map is computed and used to adjust the 3D shape retrieved by the SFS algorithm. We prove the new procedure for healthy red blood cells having a concave surface in their central region. Experimental results are compared with theoretical model.

Tomographic flow cytometry of circulating human breast adenocarcinoma cells

We demonstrate the non-invasive investigation of circulating human breast adenocarcinoma cells in microfluidic environment by implementing the full-angle tomographic phase microscopy (TPM). The proposed approach lies in a completely passive optical system, i.e. avoiding mechanical scanning or multi-direction probing of the sample and exploiting the engineered rolling of cells while they are flowing along a microfluidic channel.

In vitro cytotoxicity evaluation of cadmium by label-free holographic microscopy

Among all environmental pollutants, the toxic heavy metal cadmium is considered as a human carcinogen. Cadmium may induce cell death by apoptosis in various cell types, although the underlying mechanisms are still unclear. In this paper we show how a label-free digital holography (DH)-based technique is able to quantify the evolution of key biophysical parameters of cells during the exposure to cadmium for the first time. Murine embryonic fibroblasts NIH 3T3 are chosen here as cellular model for studying the cadmium effects. The results demonstrate that DH is able to retrieve the temporal evolution of different key parameters such as cell volume, projected area, cell thickness and dry mass, thus providing a full quantitative characterization of the cell physical behaviour during cadmium exposure. Our results show that the label-free character of the technique would allow biologists to perform systematic and reliable studies on cell death process induced by cadmium and we believe that more in general this can be easily extended to others heavy metals, thus avoiding the time-consuming, expensive and invasive label-based procedures used nowadays in the field. In fact, pollution by heavy metals is severe issue that needs rapid and reliable methods to be settled.

Label-Free Optical Marker for Red-Blood-Cell Phenotyping of Inherited Anemias

The gold-standard methods for anemia diagnosis are complete blood counts and peripheral-smear observations. However, these do not allow for a complete differential diagnosis as that requires biochemical assays, which are label-dependent techniques. On the other hand, recent studies focus on label-free quantitative phase imaging (QPI) of blood samples to investigate blood diseases by using video-based morphological methods. However, when sick cells are very similar to healthy ones in terms of morphometric features, identification of a blood disease becomes challenging even with QPI. Here, we introduce a label-free optical marker (LOM) to detect red-blood-cell (RBC) phenotypes, demonstrating that a single set of all-optical parameters can clearly identify a signature directly related to an erythrocyte disease through modeling each RBC as a biological lens. We tested this novel biophotonic analysis by proving that several inherited anemias, such as iron-deficiency anemia, thalassemia, hereditary spherocytosis, and congenital dyserythropoietic anemia, can be identified and sorted, thus opening a novel route for blood diagnosis on a completely different concept based on LOMs.

Computational tomographic phase microscopy

We investigate a computational-based approach in tomographic flow cytometry by digital holography to characterize self-rolling cells in microfluidic channels. Different experimental validations, by employing red blood cells (RBCs) and diatoms, are made.