Optical sectioning plays an important role for in-vivo or 3D optical microscopy. We have developed reflection-mode dynamic speckle-field interferometric microscopy (DSIM) to image targets with high spatial resolution, as well as high depth sectioning. In DSIM, single-shot, wide-field quantitative phase image can be obtained by using broadband, time-varying speckle-field in Linnik interferometer combined with off-axis holography. With broadband laser (center wavelength 900nm, bandwidth ~ 100nm), and high numerical aperture objective lens (1.0NA, water immersion), we could demonstrate up to ~600 nm depth-sectioning. However, there was no concrete theoretical model to describe the system. In this study, we developed a theoretical model to investigate the depth-sectioning effect of DSIM based on the theory of optical diffraction tomography with 1st order Born approximation. We systematically studied the dependence of the depth-sectioning, and image contrast in accordance with the bandwidth, and numerical aperture, respectively, and it shows a good agreement with experiment. We also studied the effect of the aberration, and dispersion induced by the target specimen for better understanding of the system dealing with thick targets. In addition, our model can be easily extended for general optical coherence microscopy, and will help for better designing of such optical systems that requires high depth resolution.
Changes in the cellular homeostasis in response to a stimuli, disease or therapeutic intervention are multifaceted in nature, and cannot be grasped by routinely employed targeted imaging that focuses on a small set of suspected molecules or genes. Novel approaches relying on global analysis of cellular features, from morphology to the composite biomolecular status (notably chemical composition and molecular conformation), is a pre-requisite for accurate monitoring of cellular processes. In the present study label-free profiling of normal skin fibroblasts (Hs895.Sk) exposed to sub-lethal doses of ultra-violet radiation has been performed using quantitative phase imaging and Raman spectroscopy. Spectral differences in the Raman fingerprint region indicates differences in the protein and nucleic acid composition. These differences were successfully utilized to develop an automated classification model based on principal component analysis. Distinct changes in the cellular morphology were observed and validated through quantitative phase imaging. Significant dose dependent differences in different biophysical parameters such as dry mass and matter density were observed. Combination of these two techniques, one suited for detection of subtle morphological/biophysical alterations while the other appropriate for capturing molecular perturbations, could pave the way to address issues of label-free monitoring of cellular responses in response to an external stimulus. These findings can provide an accurate understanding of different markers associated with radiation damage and would assist in providing a quantitative tool to our future studies on designing alternate diagnostic tools.
To exploit photonics technologies for in vivo studies in life science and biomedicine, it is necessary to efficiently deliver light energy to the target objects embedded deep within complex biological tissues. However, light waves diffuse randomly inside complex media due to multiple scattering, and only a small fraction reaches the target object. Here we present a method to counteract the random diffusion and to focus ‘snake-like’ multiple-scattered waves to the embedded target. To realize this, we experimentally identified time-gated reflection eigenchannels that have extraordinarily large reflectance at a specific flight time where most of the multiple-scattered waves have interacted with the target object. By injecting light to these eigenchannels, we achieved more than 10-fold enhancement in light energy delivery compared to ordinary wave diffusion cases. This method works up to depths of approximately 2 times the transport mean free path at which target objects are completely invisible by ballistic optical imaging. This work will lay a foundation for enhancing the working depth of imaging, sensing, and light stimulation.
KEYWORDS: Prisms, Imaging systems, Interferometry, Holography, 3D displays, Time of flight cameras, Cameras, Time metrology, Image resolution, Digital holography
With the advancement of 3D display technology, 3D imaging of macroscopic objects has drawn much attention as they provide the contents to display. The most widely used imaging methods include a depth camera, which measures time of flight for the depth discrimination, and various structured illumination techniques. However, these existing methods have poor depth resolution, which makes imaging complicated structures a difficult task. In order to resolve this issue, we propose an imaging system based upon low-coherence interferometry and off-axis digital holographic imaging. By using light source with coherence length of 200 micro, we achieved the depth resolution of 100 micro. In order to map the macroscopic objects with this high axial resolution, we installed a pair of prisms in the reference beam path for the long-range scanning of the optical path length. Specifically, one prism was fixed in position, and the other prism was mounted on a translation stage and translated in parallel to the first prism. Due to the multiple internal reflections between the two prisms, the overall path length was elongated by a factor of 50. In this way, we could cover a depth range more than 1 meter. In addition, we employed multiple speckle illuminations and incoherent averaging of the acquired holographic images for reducing the specular reflections from the target surface. Using this newly developed system, we performed imaging targets with multiple different layers and demonstrated imaging targets hidden behind the scattering layers. The method was also applied to imaging targets located around the corner.
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