Differential effects of coverslip-induced hypoxia and cobalt chloride mimetic hypoxia on cellular stress, metabolism, and nuclear structure.

Hypoxia has profound effects on cell physiology, both in normal or pathological settings like cancer. In this study, we asked whether a variant of coverslip-induced hypoxia that recapitulates the conditions found in the tumor microenvironment would elicit similar cellular responses compared to the well established model of cobalt chloride-induced hypoxia. Comparable levels of nuclear HIF-1α were observed after 24 h of coverslip-induced hypoxia or cobalt chloride treatment in CAL-27 oral squamous carcinoma cells. However, cellular stress levels assessed by reactive oxygen species production and lipid droplet accumulation were markedly increased in coverslip-induced hypoxia compared to cobalt chloride treatment. Conversely, mitochondrial ATP production sharply decreased after coverslip-induced hypoxia but was preserved in the presence of cobalt chloride. Coverslip-induced hypoxia also had profound effects in nuclear organization, assessed by changes in nuclear dry mass distribution, whereas these effects were much less marked after cobalt chloride treatment. Taken together, our results show that coverslip-induced hypoxia effects on cell physiology and structure are more pronounced than mimetic hypoxia induced by cobalt chloride treatment. Considering also the simplicity of coverslip-induced hypoxia, our results therefore underscore the usefulness of this method to recapitulate in vitro the effects of hypoxic microenvironments encountered by cells in vivo.

Real-time phase retrieval in division of aperture microscopy with the transport of intensity equation.

The transport of intensity equation (TIE) allows to recover the phase of a microscopy sample from differently focused intensity measures along the axial direction of its optical field. In the present work, we propose a cost-effective technique for snapshot phase retrieval with TIE. The optics of a commercially available camera is replaced with a doublet system consisting of a microscope objective and a lenslet array with an extra lens mask attached to it. The system allows to obtain, in real-time and with no mechanical shift of either the sample or the sensor, the in-focus as well as a defocused image of the sample. From these two sub-aperture images, the intensity derivative term in TIE can then be approximated after image rectification. Phase is then retrieved for static as well as dynamic samples over the common view area. Validation experiments are presented.

Contributions of viral oncogenes of HPV-18 and hypoxia to oxidative stress and genetic damage in human keratinocytes.

Infection with high-risk human papillomaviruses like HPV-16 and HPV-18 is highly associated with the development of cervical and other cancers. Malignant transformation requires viral oncoproteins E5, E6 and E7, which promote cell proliferation and increase DNA damage. Oxidative stress and hypoxia are also key factors in cervical malignant transformation. Increased levels of reactive species of oxygen (ROS) and nitrogen (RNS) are found in the hypoxic tumor microenvironment, promoting genetic instability and invasiveness. In this work, we studied the combined effect of E5, E6 and E7 and hypoxia in increasing oxidative stress and promoting DNA damage and nuclear architecture alterations. HaCaT cells containing HPV-18 viral oncogenes (HaCaT E5/E6/E7-18) showed higher ROS levels in normoxia and higher levels of RNS in hypoxia compared to HaCaT parental cells, as well as higher genetic damage in hypoxia as measured by γH2AX and comet assays. In hypoxia, HaCaT E5/E6/E7-18 increased its nuclear dry mass and both cell types displayed marked heterogeneity in nuclear dry mass distribution and increased nuclear foci. Our results show contributions of both viral oncogenes and hypoxia to oxidative stress, DNA damage and altered nuclear architecture, exemplifying how an altered microenvironment combines with oncogenic transformation to promote tumor progression.

Snapshot polarimetric imaging in multi-view microscopy.

Polarimetric imaging allows for the vector nature of optical information across a scene to be obtained, with recent applications ranging from remote sensing to microscopy. In polarimetric microscopy in particular, different polarization states are conventionally achieved under time-division multiplexing strategies and are mainly subject to static phenomena. In the present work, we propose a cost-effective technique for polarization sensing with the possibility of real-time imaging microscopy. By modifying a commercial camera and replacing the conventional lens with an optical system that integrates a microscope objective and a lenslet array with a polarization mask, linear Stokes parameters can be obtained in a snapshot. The proposed scheme is robust against misalignment and suitable for handling video sequences of microscopic samples. To the best of our knowledge, this is the first report on combining multi-view sensing and polarization imaging for applications to microscopy.

Fully invariant generalized Hough transform by out-of-focus multiview sensing with pupil array.

Recognition of geometrical shapes in real-time and fully invariant (i.e., invariant under changes in position, scale, and orientation) is a demanding task in automated image analysis. In particular, the generalized Hough transform (GHT) is a well-known algorithm for the recognition of complex patterns out of edge binary images even with disconnected boundaries or corrupted by noise. In this work we present a space multiplexed optical implementation of the GHT which, by exploiting the redundancy derived from multiview sensing of a two-dimensional image and its out- of-focus capture with an adequate pupil array, allows us to obtain in a single shot the GHT of this image invariant to target shift, scale, and orientation. Experimental validation of the working principle is presented, along with an assessment of the robustness of the system against noise in the input.