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.
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.
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.