According to the International agency for Research on Cancer, cadmium (Cd) is considered as a human carcinogen. Cadmium may induce cell death by apoptosis in various cell types, although the underlying mechanisms are still unclear. Nowadays, the cytotoxic potential of heavy metals is commonly evaluated by different cellular endpoints as reactive oxygen species formation, cell viability or cell death. Heavy metals cytotoxicity testing is based on in-vitro methods such as MTT assay, for the colorimetric detection of mitochondrial activity, propidium iodide-staining of DNA, as cell death marker, fluorometric detection of ROS generation to evaluate the stress response and colorimetric detection of cytokine secretion for the inflammatory reaction by ELISA method. In this work, we present a label-free digital holography (DH) based technique as an in-vitro cytotoxicity assay, which overcomes the limitations of conventional in vitro test based on color or fluorescence read outs. In particular, we show how DH is able to quantify the evolution of key biophysical parameters of cells during the exposure to cadmium. 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. This demonstrates DH as an elegant label-free tool for heavy metals toxicity analysis.
Azopolymer materials belong to family special materials, which are subject to photo-isomerization when illuminated by appropriate light wavelength. Optical characterization of azopolymer materials is interesting because they can be patterned when illuminated by coherent polarized light with potentially interesting applications in the biotechnology, photonic elements, molding templates, etch masks and micro-nanochannels. The interference lithography is an excellent tool to trigger the isomerization reaction on the material. During this work, switchable patterns were fabricated by means of a well established holographic set-up: surface relief gratings (SRGs) were realized with Lloyd’s mirror system. Moreover, optical characterization of the material was performed, starting from a commercial one and using a new way to analyse SRGs by means of Digital Holography Microscopy, to determine relevant parameters for the realization of the patterns with different shape and size. Some preliminary results of the influence of such patterns on the cell behavior were shown.
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.
TIRDHM is a technique that allows to analyse the phase change of microscopical sections produced on the prism surface due to material attached on the top. Therefore, due to the evanescence waves properties we can analyse quantitatively the properties and specific morphology located to few nanometers on the top of surface contact. In this work, we study and present an alternative method to off-axis configuration to record and analyse the microscopical phase object information in Total Internal Reflection dispensing with the use of reference arm.
Optical vortices are employed in optical trapping applications for their ability to set microparticles into rotation. Devil’s Vortex Lenses have high diffraction efficiency and it is possible to take advantage of their particular volumetric focal structure to design versatile and efficient optical tweezers. In this communication, we report a simple design procedure, involving arrays of Devil’s Vortex-Lenses implemented in a programmable Spatial Light Modulator, for generating spatial distributions of optical vortices. In our approach, the preferred position and topological charge value can be assigned to each vortex in the structure, tuning the desired angular momentum. We have demonstrated the generation of 3D optical vortex distributions through arrays of Devil’s Vortex-Lenses, including configurations with charges and momenta of opposite sign. Our experimental results present an excellent agreement with the simulations we have developed.