We present an optofluidic measurement system that quantifies cell volume, dry mass, and nuclear morphology of neutrophils in high-throughput. While current clinical hematology analyzers can differentiate neutrophils from a blood sample, they do not give other quantitative information beyond their count. In order to better understand the distribution of neutrophil phenotypes in a blood sample, we perform two distinct multivariate measurements. In both measurements, white blood cells are driven through a microfluidic channel and imaged while in flow onto a color camera using a single exposure. In the first measurement, we quantify cell volume, scattering strength, and cell dry mass by combining quantitative phase imaging with dye exclusion cell volumetric imaging. In the second measurement, we quantify cell volume and nuclear morphology using a nucleic acid fluorescent stain. In this way, we can correlate cell volume to other cellular characteristics, which would not be possible using an electrical coulter counter. Unlike phase imaging or cell scattering analysis, the optical coulter counter is capable of quantifying cell volume virtually independent of the cell’s refractive index and unlike optical tomography, measurements are possible on quickly flowing cells, enabling high-throughput.
Semen analysis is widely used as diagnostic tool for assessing male fertility, controlling and managing the animal reproduction. The most important parameters measured in a semen analysis are the morphology and biochemical alterations. For obtaining such information, non-invasive, label-free and non-destructive techniques have to be used. Digital Holography (DH) combined with Raman Spectroscopy (RS) could represent the perfect candidate for a rapid, non-destructive and high-sensitive morphological and biochemical sperm cell analysis. In this study, DH-RS combined approach is used for a complete analysis of single bovine spermatozoa. High-resolution images of bovine sperm have been obtained by DH microscopy from the reconstruction of a single acquired hologram, highlighting in some cases morphological alterations. Quantitative 3D reconstructions of sperm head, both normal and anomalous, have been studied and an unexpected structure of the post-acrosomal region of the head has been detected. Such anomalies have been also confirmed by Raman imaging analysis, suggesting the protein vibrations as associated Raman marker of the defect.
We present an opto-fluidic measurement system that quantifies cell volume, dry mass and nuclear morphology of neutrophils in high-throughput. While current clinical hematology analyzers can differentiate neutrophils from a blood sample, they do not give other quantitative information beyond their count. In order to better understand the distribution of neutrophil phenotypes in a blood sample, we perform two distinct multivariate measurements. In both measurements, white blood cells are driven through a microfluidic channel and imaged while in flow onto a color camera using a single exposure. In the first measurement, we quantify cell volume, scattering strength, and cell dry mass by combining quantitative phase imaging with dye exclusion cell volumetric imaging. In the second measurement, we quantify cell volume and nuclear morphology using a nucleic acid fluorescent stain. In this way, we can correlate cell volume to other cellular characteristics, which would not be possible using an electrical coulter counter. Unlike phase imaging or cell scattering analysis, the optical coulter counter is capable of quantifying cell volume virtually independent of the cell’s refractive index and unlike optical tomography, measurements are possible on quickly flowing cells, enabling high-throughput.
KEYWORDS: Biological research, Digital holography, Microfluidics, 3D metrology, Head, In vitro testing, Holograms, Optical tweezers, Microscopy, 3D modeling
For the monitoring of biological samples, physical parameters such as size, shape and refractive index are of crucial importance. However, up to now the morphological in-vitro analysis of in-vitro cells has been limited to 2D analysis by classical optical microscopy such as phase-contrast or DIC. Here we show an approach that exploits the capability of optical tweezers to trap and put in self-rotation bovine spermatozoa flowing into a microfluidic channel. At same time, digital holographic microscopy allows to image the cell in phase-contrast modality for each different angular position, during the rotation. From the collected information about the cell’s phase-contrast signature, we demonstrate that it is possible to reconstruct the 3D shape of the cell and estimate its volume. The method can open new pathways for rapid measurement of in-vitro cells volume in microfluidic lab-on-a-chip platform, thus having access to 3D shape of the object avoiding tomography microscopy, that is an overwhelmed and very complex approach for measuring 3D shape and biovolume estimation.
We present the possibility to trap cells (mouse fibroblasts, bovine spermatozoa and diatoms), to manage their position and to induce rotation, by using optical tweezers. The aim is to place them in desired positions, in order to record holographic images in a microscope configuration. Then we are able to recover the 3D shape and to calculate the biovolume of the cells starting from the reconstructed quantitative phase maps (QPMs).
Diatoms are monocellular algae responsible of 20-25% of the global oxygen produced by photosynthetic processes. The protoplasm of every single cell is enclosed in an external wall made of porous hydrogenated silica, the frustule. In recent times, many effects related to photonic properties of diatom frustules have been discovered and exploited in applications: light confinement induced by multiple diffraction, frustule photoluminescence applied to chemical and biochemical sensing, photonic-crystal-like behavior of valves and girdles. In present work we show how several techniques (e.g. digital holography) allowed us to retrieve information on light manipulation by diatom single valves in terms of amplitude, phase and polarization, both in air and in a cytoplasmatic environment. Possible applications in optical microsystems of diatom frustules and frustule-inspired devices as active photonic elements are finally envisaged.
Numerical analysis is implemented to investigate biological sample starting from Digital Holographic (DH) recording.
The aim is to improve visualization and detection of cow spermatozoa. Digital holograms are recorded in the off-axis
geometry where optical setup is a Mach-Zehnder interferometer. Then holograms are numerically manipulated to
retrieve, besides the usual Quantitative Phase Map (QPM), Differential Interference Contrast (DIC) visualization.
Furthermore, a new approach, named digital self-referencing holography, is described it's able to accomplish
quantitative phase analysis especially useful for specimen flowing in microfluidic channels.
Digital holography (DH) has been employed in the retrieval of three dimensional images of bull's sperm heads. The
system allows a three dimensional analysis of the sperm morphology by means of a Digital Holographic Microscope
(DHM). Microscopic holography measurements are performed by projecting a magnified image of a microscopic
hologram plane onto a CCD plane. This could constitute the basis of an alternative method for the zoothecnic
industry aimed at the investigation of morphological features and the sorting of the motile sperm cells. Indeed, one
of the main advantages of digital holography consists in its full non-invasivity and in the capability of investigating
the shape of the sperm cells without altering their characteristics. In particular the proposed technique could be
applied to investigate the frequency of aberrant spermatozoa. Until now, in fact, such industrial investigations have
been mainly performed by means of specific painting probes: unfortunately this technique dramatically reduces the
vitality of the sperm cells and can even cause chromosome aberration, making them useless for the zootechnical
applications.
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