Imaging Intracellular Zinc by Using a Glyoxal Bis(4-methyl-4-phenyl-3-thiosemicarbazone) Ligand

The ligand glyoxal bis(4-methyl-4-phenyl-3-thiosemicarbazone) (GTSCH2) is shown to be a selective fluorescence turn-on sensor for zinc ions (Zn2+). This sensor is easy to synthesize, exhibits excellent sensitivity and selectivity towards Zn2+ over other physiologically relevant cations, and has sub-nanomolar binding affinity. It displays maximum fluorescence response to Zn2+ when the metal/ligand ratio is 1:1 and displays stable fluorescence over a broad pH range. The potential of GTSCH2 to image Zn2+ inside the cell was demonstrated in MCF-7 cells (human breast cancer cell line) by using flow cytometry and confocal fluorescence microscopy. Cell viability studies reveal that the probe is biocompatible and suitable for cellular applications.

MRT Letter: Light Sheet Based Imaging Flow Cytometry on a Microfluidic Platform

We propose a light sheet based imaging flow cytometry technique for simultaneous counting and imaging of cells on a microfluidic platform. Light sheet covers the entire microfluidic channel and thus omits the necessity of flow focusing and point scanning based technology. Another advantage lies in the orthogonal detection geometry that totally cuts-off the incident light, thereby substantially reducing the background in the detection. Compared to the existing state-of-art techniques the proposed technique shows marked improvement. Using fluorescently-coated Saccharomyces cerevisiae cells we have recorded cell counting with throughput as high as 2,090 cells/min in the low flow rate regime and were able to image the individual cells on-the-go. Overall, the proposed system is cost-effective and simple in channel geometry with the advantage of efficient counting in operational regime of low laminar flow. This technique may advance the emerging field of microfluidic based cytometry for applications in nanomedicine and point of care diagnostics. Microsc. Res. Tech. 76:1101-1107, 2013. (c) 2013 Wiley Periodicals, Inc.

Image reconstruction enables high resolution imaging at large penetration depths in fluorescence microscopy

Imaging thick specimen at a large penetration depth is a challenge in biophysics and material science. Refractive index mismatch results in spherical aberration that is responsible for streaking artifacts, while Poissonian nature of photon emission and scattering introduces noise in the acquired three-dimensional image. To overcome these unwanted artifacts, we introduced a two-fold approach: first, point-spread function modeling with correction for spherical aberration and second, employing maximum-likelihood reconstruction technique to eliminate noise. Experimental results on fluorescent nano-beads and fluorescently coated yeast cells (encaged in Agarose gel) shows substantial minimization of artifacts. The noise is substantially suppressed, whereas the side-lobes (generated by streaking effect) drops by 48.6% as compared to raw data at a depth of 150 mu m. Proposed imaging technique can be integrated to sophisticated fluorescence imaging techniques for rendering high resolution beyond 150 mu m mark. (C) 2013 AIP Publishing LLC.

Monte Carlo simulation of light transport in tissue for optimizing light delivery in photoacoustic imaging of the sentinel lymph node

Noninvasive or minimally invasive identification of sentinel lymph node (SLN) is essential to reduce the surgical effects of SLN biopsy. Photoacoustic (PA) imaging of SLN in animal models has shown its promise for clinical use in the future. Here, we present a Monte Carlo simulation for light transport in the SLN for various light delivery configurations with a clinical ultrasound probe. Our simulation assumes a realistic tissue layer model and also can handle the transmission/reflectance at SLN-tissue boundary due to the mismatch of refractive index. Various light incidence angles show that for deeply situated SLNs the maximum absorption of light in the SLN is for normal incidence. We also show that if a part of the diffused reflected photons is reflected back into the skin using a reflector, the absorption of light in the SLN can be increased significantly to enhance the PA signal. (C) 2013 Society of Photo-Optical Instrumentation Engineers (SPIE)

Studies on thin film based flexible gold electrode arrays for resistivity imaging in electrical impedance tomography

Practical phantoms are essential to assess the electrical impedance tomography (EIT) systems for their validation, calibration and comparison purposes. Metal surface electrodes are generally used in practical phantoms which reduce the SNR of the boundary data due to their design and development errors. Novel flexible and biocompatible gold electrode arrays of high geometric precision are proposed to improve the boundary data quality in EIT. The flexible gold electrode arrays are developed on flexible FR4 sheets using thin film technology and practical gold electrode phantoms are developed with different configurations. Injecting a constant current to the phantom boundary the surface potentials are measured by a LabVIEW based data acquisition system and the resistivity images are reconstructed in EIDORS. Boundary data profile and the resistivity images obtained from the gold electrode phantoms are compared with identical phantoms developed with stainless steel electrodes. Surface profilometry, microscopy and the impedance spectroscopy show that the gold electrode arrays are smooth, geometrically precised and less resistive. Results show that the boundary data accuracy and image quality are improved with gold electrode arrays. Results show that the diametric resistivity plot (DRP), contrast to noise ratio (CNR), percentage of contrast recovery (PCR) and coefficient of contrast (COC) of reconstructed images are improved in gold electrode phantoms. (C) 2013 Elsevier Ltd. All rights reserved.

Carbohydrate-appended photocytotoxic (imidazophenanthroline)-oxovanadium(IV) complexes for cellular targeting and imaging

Oxovanadium(IV) complexes VO(aip)(L)](ClO4)(2) (L = phtpy, 1; stpy, 2) and VO(pyip)(L)](ClO4)(2) (L = phtpy, 3; stpy, 4), where aip is 2-(9-anthryl)-1H-imidazo4,5-f]1,10] phenanthroline, pyip is 2-(1-pyrenyl)-1Himidazo4,5-f]1,10] phenanthroline, phtpy is (4'-phenyl)-2,2': 6',2 `'-terpyridine and stpy is (2,2': 6', 2 `'-terpyridin-4'-oxy) ethyl-beta-D-glucopyranoside, were prepared, characterized and their DNA binding and photocleavage activity, cellular uptake and photocytotoxicity in visible light were studied. The complexes are avid binders to calf thymus DNA (K-b similar to 10(5) mol(-1)). They efficiently cleave pUC19 DNA in red light of 705 nm via the formation of HO center dot species. The glucose appended complexes 2 and 4 showed higher photocytotoxicity in HeLa and Hep G2 cells over the normal HEK 293T cells. No such preference was observed for the phtpy complexes 1 and 3. No significant difference in IC50 values was observed for the HEK 293T cells. Cell cycle analysis showed that the glucose appended complexes 2 and 4 are more photocytotoxic in cancer cells than in normal cells. Fluorescence microscopy was done to study the cellular localization of complex 4 having a pendant pyrenyl group.

Imaging cellular signalling: many `moving tales' in MAP kinase odyssey

Cellular signalling events are at the core of every adaptive response. Signalling events link environmental changes to physiological responses, consequently allowing cellular and organismal sustenance and survival. Classical approaches to study cellular signalling have relied on a variety of cell disruptive techniques which yield limited kinetic information, while the underlying events are much more complex. In this article, we discuss how modern live cell imaging microscopy has found increasing utilization in revealing spatio temporal dynamics of various signalling pathways. Utilizing the well studied mitogen-activated protein kinase (MAPK) signalling cascade as a template, the design, construction and utilization of `mobile' (translocation proficient) biosensors, suitable for studying MAPK signalling in living cells are described in detail. Experimental setup and results obtained from these biosensors, based on different proteins involved in the MAPK signalling cascade, have been described along with the setup of a microscope optimal for live cell imaging applications. Utilizing the ability to activate or deactivate signalling pathways using defined activators and specific pharmacological inhibitors, we also show how these sensors can yield unique spatial and temporal kinetic information of signalling in living cells.

Co-registration of speech production datasets from electromagnetic articulography and real-time magnetic resonance imaging

This paper describes a spatio-temporal registration approach for speech articulation data obtained from electromagnetic articulography (EMA) and real-time Magnetic Resonance Imaging (rtMRI). This is motivated by the potential for combining the complementary advantages of both types of data. The registration method is validated on EMA and rtMRI datasets obtained at different times, but using the same stimuli. The aligned corpus offers the advantages of high temporal resolution (from EMA) and a complete mid-sagittal view (from rtMRI). The co-registration also yields optimum placement of EMA sensors as articulatory landmarks on the magnetic resonance images, thus providing richer spatio-temporal information about articulatory dynamics. (C) 2014 Acoustical Society of America

High Speed Imaging of Generation and Collapse of Multielectron Bubbles in Liquid Helium

We present experimental results on the generation and collapse of multielectron bubbles in liquid helium. By applying voltage pulses to a tungsten tip above the surface of the liquid, millimetre sized deformations were formed. Using high speed photography, we have imaged the disintegration of these deformations into bubbles of sizes ranging from ten to few hundred microns. At temperatures less than 2 K, the bubbles split into smaller bubbles and then disappeared in a time scale of few milliseconds. Smaller bubbles were formed at temperatures around 3 K, but were visible for more than hundreds of milliseconds. Although we have not been able to measure their charge directly, some of these bubbles responded to electric fields, implying these were indeed multielectron bubbles. With the existing theoretical picture, it is not possible to understand the strong dependence of the lifetime of multielectron bubbles on temperature.

A Vision-Based Micro-Newton Static Force Sensor Using a Displacement-Amplifying Compliant Mechanism (DaCM)

A micro-newton static force sensor is presented here as a packaged product. The sensor, which is based on the mechanics of deformable objects, consists of a compliant mechanism that amplifies the displacement caused by the force that is to be measured. The output displacement, captured using a digital microscope and analyzed using image processing techniques, is used to calculate the force using precalibrated force-displacement curve. Images are scanned in real time at a frequency of 15 frames per second and sampled at around half the scanning frequency. The sensor was built, packaged, calibrated, and tested. It has simulated and measured stiffness values of 2.60N/m and 2.57N/m, respectively. The smallest force it can reliably measure in the presence of noise is about 2 mu N over a range of 1.4mN. The off-the-shelf digital microscope aside, all of its other components are purely mechanical; they are inexpensive and can be easily made using simple machines. Another highlight of the sensor is that its movable and delicate components are easily replaceable. The sensor can be used in aqueous environment as it does not use electric, magnetic, thermal, or any other fields. Currently, it can only measure static forces or forces that vary at less than 1Hz because its response time and bandwidth are limited by the speed of imaging with a camera. With a universal serial bus (USB) connection of its digital microscope, custom-developed graphical user interface (GUI), and related software, the sensor is fully developed as a readily usable product.

Model-Resolution-Based Basis Pursuit Deconvolution Improves Diffuse Optical Tomographic Imaging

The image reconstruction problem encountered in diffuse optical tomographic imaging is ill-posed in nature, necessitating the usage of regularization to result in stable solutions. This regularization also results in loss of resolution in the reconstructed images. A frame work, that is attributed by model-resolution, to improve the reconstructed image characteristics using the basis pursuit deconvolution method is proposed here. The proposed method performs this deconvolution as an additional step in the image reconstruction scheme. It is shown, both in numerical and experimental gelatin phantom cases, that the proposed method yields better recovery of the target shapes compared to traditional method, without the loss of quantitativeness of the results.

Iron(III) benzhydroxamates of dipicolylamines for photocytotoxicity in red light and cellular imaging

Benzhydroxamate (BHA) iron(III) complexes Fe(BHA)(L)ClICI (I, 2)], where L is (phenyl)dipicolylamine (phdpa in I) and (pyrenyl)dipicolylamine (pydpa in 2), were prepared and their photocytotoxicity in visible (400-700 nm) and red (600-720 nm) light was studied. Complex 1 was structurally characterized by X-ray crystallography. The complexes have high-spin iron(III) centers. Complex 2, with a pyrenyl fluorophore, was used for cellular imaging, showing both mitochondrial and nuclear localization in the fluorescence microscopic study. The complex exhibited photocytotoxicity in red light in HeLa cancer cells, giving IC50 value of 24.4(+/- 0.4) pM, but remained essentially non-toxic in the dark. The involvement of reactive oxygen species and an apoptotic nature of cell death were observed from the cellular studies. (C) 2014 Elsevier Ltd. All rights reserved.

Three Dimensional Fluorescence Imaging Using Multiple Light-Sheet Microscopy

We developed a multiple light-sheet microscopy (MLSM) system capable of 3D fluorescence imaging. Employing spatial filter in the excitation arm of a SPIM system, we successfully generated multiple light-sheets. This improves upon the existing SPIM system and is capable of 3D volume imaging by simultaneously illuminating multiple planes in the sample. Theta detection geometry is employed for data acquisition from multiple specimen layers. This detection scheme inherits many advantages including, background reduction, cross-talk free fluorescence detection and high-resolution at long working distance. Using this technique, we generated 5 equi-intense light-sheets of thickness approximately 7: 5 mm with an inter-sheet separation of 15 mm. Moreover, the light-sheets generated by MLSM is found to be 2 times thinner than the state-of-art SPIM system. Imaging of fluorescently coated yeast cells of size 4 +/- 1 mm (encaged in Agarose gel-matrix) is achieved. Proposed imaging technique may accelerate the field of fluorescence microscopy, cell biology and biophotonics.

Studies on microscopic structure of diesel sprays under atmospheric and high gas pressures

In the present work, the spray structure of diesel from a 200-mu m, single-hole solenoid injector is studied using microscopic imaging at injection pressures of 700, 1000 and 1400 bar for various gas pressures. A long-distance microscope with a high resolution camera is used for spray visualization with a direct imaging technique. This study shows that even at very high injection pressures, the spray structure in an ambient environment of atmospheric pressure reveals presence of entangled ligaments and non-spherical droplets during the injection period. With increase in the injection pressure, the ligaments tend to get smaller and spread radially. The spray structure studies are also conducted at high gas pressures in a specially designed high pressure chamber with optical access. The near nozzle spray structure at the end of the injection shows that the liquid jet breakup is improved with increase in gas density. The droplet size measurement is possible only late in the injection duration when the breakup appears to be complete and mostly spherical droplets are observed. Hence, droplet size measurements are performed after 1.3 ms from start of the injection pulse. Spatial and temporal variation in Sauter Mean `Diameter (SMD) is observed and reported for the case corresponding to an injection pressure of 700 bar. Overall, this study has highlighted the importance of verifying the extentof atomization and droplet shape even in dense sprays before using conventional dropsizing methods such as PDPA.