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The article presents the use of an interference microscope, using a Mirau objective for the study of the early adhesion process of osteoblast-like bone cells, using the phase shifting technique. The process is carried out on surgical stainless steel surfaces of interest for the development of bone implants. Experimental phase maps are directly rela...
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... a Mirau interferometer, the light originating from the source goes through a partially reflecting mirror which acts as the reference surface; the transmitted beam is reflected by the sample to later interfere with the reference beam. This simple design allows the manufacture of objectives with the interferometer in its interior ( Fig. 1), which makes the system very stable under mechanical or thermal disturbances (Hariharan, 2003). This compact interferometer is used generally in the study of polished surfaces or those with small varia- tions in their topography (Ge & Kobayashi, 2006). This way, cells like the osteoblasts which are thin and with refractive indices ...
Context 2
... comes from a tungsten lamp with an interferential filter centered at λg 546,1 nm wavelength, and a spectral bandwidth of 10 nm. The trinocular head of the microscope allows for simultane- ously obtaining an image for the observer and another for a digital camera, which allows for control of the system and the acquisition of digital interferograms (Fig. 1). To obtain the optical phase, the phase shifting technique is used. This shifting is carried out by means of a piezoelectric transducer that replaces the traditional support of objectives in the mi- croscope (Gâsvik, 2002;Hariharan, 2003). The control of the piezoelectric system carries out by means of a frequency-to- voltage circuit ...
Citations
... This compact interferometer is commonly used in the study of polished surfaces or those with small variations in their topography 22 . Cells such as osteoblasts, which are thin and have refractive indices similar to that of their surrounding environment, can thus be suitably studied with this device 23 . ...
In this work, we introduce an opto-geometric parameter for the morphologic characterization of cell populations during
their early adhesion process. Using measurements for a cell population of the maximal optical phase for each cell and its
substrate-contact-surface, we show, experimentally, that a relationship exists between these variables. This connection is
primarily associated with morphological cell characteristics. It is also shown that using the relationship obtained above,
we can derive a morphological parameter, which, for the cell populations studied, results in a monodisperse Gaussiansize
distribution, which would allow for the use of regular statistical variables. This result is in total contrast with the
polydisperse distribution obtained if only the contact surface area between cell and substrate is used. In addition, optical
phase measurements where accomplished by phase shifted interferometry using a Mirau-type interference microscope.
The cellular system studied consisted of Osteoblast-like cells, plated on 316VM medical-grade stainless steel polished
surfaces. These cell populations were studied within the same culture conditions of cell type, plating time and substrate
roughness conditions. The existence of a relationship between maximal optical phase and substrate contact area agrees
entirely with the accepted spreading model for cell adhesion; in particular, considering the close link between the optical
phase time change and cell thickness reduction.
Keywords: Optical phase, Cell adhesion, Interference Microscopy, Cell morphology, Quantitative phase microscopy.
... ALP production was measured in cells adherent to the Ti alloy control and TiSiN/Ti alloy substrates at 28 days using a quantitative assay based on p-nitrophenyl phosphate (p-NPP from Sigma#N-1891).500 l of p-NPP at a concentration of 1.0 mg/ml in 0.1 M glycine buffer was added to each well containing a Ti substrate and shaken at 37 • C for 60' then the reaction was neutralised by the addition of 500 l1 M NaOH per well. Sample sets were compared to a standard curve prepared from 10 M/ml stock solution of p-NPP diluted in 0.1 M glycine buffer to create 8 standards ranging from p-NPP dilutions of 1/5 to 1/640.The optical density of wells containing test samples and standards was read spectrophotometrically at 405 nm and the mean value for each test sample normalised against the differentiated TCPS value to give p-NPP mM/cm 2 [15][16][17][18]. ...
Surface modifications of metallic implants are important in order to protect the underlying metals from the harsh corrosive environment inside the human body and to minimize the losses caused by wear. Recently, researches are carried out in developing bioactive surfaces on metallic implants, which supports the growth and proliferation of cells on to these surfaces. Titanium silicon nitride (TiSiN) hard nanocomposites thin films were fabricated on Ti alloys (Ti-6Al–4 V) by pulsed direct current (DC) reactive magnetron sputtering. The films were characterized for its microstructural and electrochemical behavior. The higher charge transfer resistance (Rct) and positive shift in Ecorr value of TiSiN/Ti alloys than the bare Ti-alloys indicates a better corrosion resistance offered by the TiSiN thin films to the underlying substrates. The biological response to TiSiN/Ti alloys and control bare Ti-alloys was measured in vitro using cell-based assays with two main outcomes. Firstly, neither the Ti alloy nor the TiSiN thin film was cytotoxic to cells. Secondly, the TiSiN thin film promoted differentiation of human bone cells above the bare control Ti alloy as measured by alkaline phosphatase and calcium production. TiSiN thin films provide better corrosion resistance and protect the underlying metal from the corrosive environment. The thin film surface is both biocompatible and bioactive as indicated from the cytotoxicity and biomineralization studies.
In the first part of this chapter, we describe how the new concept of digital optics applied to the field of holographic microscopy has made it possible to quantitatively and accurately measure the phase retardation induced on the transmitted wavefront by the observed transparent specimen, allowing thus to develop a reliable and flexible digital holographic quantitative phase microscopy (DH-QPM).In the second part the most relevant DH-QPM applications in the field of cell biology are presented. Particularly, applications taking directly advantage of benefits provided by digital optics particularly off-line autofocusing and extended depth of focus, are outlined. Otherwise, special emphasis is placed on how important biophysical cell parameters including absolute cell volume, dry mass, protein content, transmembrane water movements, cell membrane fluctuations etc. can be derived from the quantitative phase signal (QPS) and used to characterize cell dynamics, analyze specific biological mechanisms and discriminate between physiological and pathophysiological states. In the last part, we present how transmembrane water movement measurements can be used to resolve neuronal network activity.
We present a phase shifting interferometry system for the study of the
early adhesion process for osteoblast-like cells, through an
interference microscope. Optical phase maps from the cells are obtained
experimentally as a function of cell adhesion time. The process is
carried out on surfaces of metallic materials relevant to the
development of bone implants. The surfaces were subjected to various
levels of mechanical polishing and their roughness was measured using
the same experimental technique mentioned before. Morphological changes
of the cell can be measured over their optical phase maps while the cell
adhesion process is accomplished. The experimental technique shows a
suitable feature as to the observed time scale, and also shows a high
stockiness and precision for the determination of the optical phase.
In this review, we summarize how the new concept of digital optics applied to the field of holographic microscopy has allowed the development of a reliable and flexible digital holographic quantitative phase microscopy (DH-QPM) technique at the nanoscale particularly suitable for cell imaging. Particular emphasis is placed on the original biological information provided by the quantitative phase signal. We present the most relevant DH-QPM applications in the field of cell biology, including automated cell counts, recognition, classification, three-dimensional tracking, discrimination between physiological and pathophysiological states, and the study of cell membrane fluctuations at the nanoscale. In the last part, original results show how DH-QPM can address two important issues in the field of neurobiology, namely, multiple-site optical recording of neuronal activity and noninvasive visualization of dendritic spine dynamics resulting from a full digital holographic microscopy tomographic approach. Expected final online publication date for the Annual Review of Biomedical Engineering Volume 15 is July 11, 2013. Please see http://www.annualreviews.org/catalog/pubdates.aspx for revised estimates.
