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.

Spatial Filter Based Bessel-Like Beam for Improved Penetration Depth Imaging in Fluorescence Microscopy

Monitoring and visualizing specimens at a large penetration depth is a challenge. At depths of hundreds of microns, several physical effects (such as, scattering, PSF distortion and noise) deteriorate the image quality and prohibit a detailed study of key biological phenomena. In this study, we use a Bessel-like beam in-conjugation with an orthogonal detection system to achieve depth imaging. A Bessel-like penetrating diffractionless beam is generated by engineering the back-aperture of the excitation objective. The proposed excitation scheme allows continuous scanning by simply translating the detection PSF. This type of imaging system is beneficial for obtaining depth information from any desired specimen layer, including nano-particle tracking in thick tissue. As demonstrated by imaging the fluorescent polymer-tagged-CaCO3 particles and yeast cells in a tissue-like gel-matrix, the system offers a penetration depth that extends up to 650 mu m. This achievement will advance the field of fluorescence imaging and deep nano-particle tracking.

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.

Terpyridine Oxovanadium(IV) Complexes of Phenanthroline Bases for Cellular Imaging and Photocytotoxicity in HeLa Cells

Oxovanadium(IV) complexes VO(N-N-N)(N-N)](NO3)(2) (1-4) of (4'-phenyl)-2,2': 6',2 `'-terpyridine (ph-tpy in 1 and 2) or (4'-pyrenyl)-2,2':6',2 `'-terpyridine (py-tpy in 3 and 4) having N-N as 1,10-phenanthroline (phen in 1 and 3) or dipyrido3,2-a:2',3'-c]phenazine (dppz in 2 and 4) are prepared and characterized. The crystal structure of 1 has VO2+ group in VN5O coordination geometry. The terpyridine ligand coordinates in a meridional binding mode. The phen ligand displays a chelating mode of binding with an N-donor site trans to the vanadyl oxo group. The complexes show a d-d band in the range of 710-770 nm in aqueous DMF (4:1 v/v). The complexes exhibit an irreversible V-IV/V-III redox response near -1.0 V vs. SCE in aqueous DMF/0.1 M KCl. The complexes bind to CT DNA giving K-b values within 3.5 x 10(5) to 1.2 x 10(6) M-1. The complexes show poor chemical nuclease activity in dark. Complexes 2-4 show photonuclease activity in UV-A light of 365 nm forming O-1(2) and (OH)-O-center dot. Complex 4 shows DNA photocleavage activity at near-IR light of 785 nm forming (OH)-O-center dot radicals. Complexes 2 and 4 show significant photocytotoxicity in HeLa cancer cells. Uptake of the complexes in HeLa cells, studied by fluorescence imaging, show predominantly cytosolic localization inside the cells.

Impact of metal binding on the antitumor activity and cellular imaging of a metal chelator cationic imidazopyridine derivative

A new water soluble cationic imidazopyridine species, viz. (1E)-1-((pyridin-2-yl)methyleneamino)-3-(3(pyridin-2-yl) imidazo1,5-a]pyridin-2(3H)-yl)propan-2-ol (1), as a metal chelator is prepared as its PF6 salt and characterized. Compound 1 shows fluorescence at 438 nm on excitation at 342 nm in Tris-HCl buffer giving a fluorescence quantum yield (phi) of 0.105 and a life-time of 5.4 ns. Compound 1, as an avid DNA minor groove binder, shows pUC19 DNA cleavage activity in UV-A light of 365 nm forming singlet oxygen species in a type-II pathway. The photonuclease potential of 1 gets enhanced in the presence of Fe2+, Cu2+ or Zn2+. Compound 1 itself displays anticancer activity in HeLa, HepG2 and Jurkat cells with an enhancement on addition of the metal ions. Photodynamic effect of 1 at 365 nm also gets enhanced in the presence of Fe2+ and Zn2+. Fluorescence-based cell cycle analysis shows a significant dead cell population in the sub-G1 phase of the cell cycle suggesting apoptosis via ROS generation. A significant change in the nuclear morphology is observed from Hoechst 33258 and an acridine orange/ethidium bromide (AO/EB) dual nuclear staining suggesting apoptosis in cells when treated with 1 alone or in the presence of the metal ions. Apoptosis is found to be caspase-dependent. Fluorescence imaging to monitor the distribution of 1 in cells shows that 1 in the presence of metal ions accumulates predominantly in the cytoplasm. Enhanced uptake of 1 into the cells within 12 h is observed in the presence of Fe2+ and Zn2+.

Fast image reconstruction for fluorescence microscopy

Real-time image reconstruction is essential for improving the temporal resolution of fluorescence microscopy. A number of unavoidable processes such as, optical aberration, noise and scattering degrade image quality, thereby making image reconstruction an ill-posed problem. Maximum likelihood is an attractive technique for data reconstruction especially when the problem is ill-posed. Iterative nature of the maximum likelihood technique eludes real-time imaging. Here we propose and demonstrate a compute unified device architecture (CUDA) based fast computing engine for real-time 3D fluorescence imaging. A maximum performance boost of 210x is reported. Easy availability of powerful computing engines is a boon and may accelerate to realize real-time 3D fluorescence imaging. Copyright 2012 Author(s). This article is distributed under a Creative Commons Attribution 3.0 Unported License. http://dx.doi.org/10.1063/1.4754604]

Multidimensional data reconstruction for two color fluorescence microscopy

We propose an iterative data reconstruction technique specifically designed for multi-dimensional multi-color fluorescence imaging. Markov random field is employed (for modeling the multi-color image field) in conjunction with the classical maximum likelihood method. It is noted that, ill-posed nature of the inverse problem associated with multi-color fluorescence imaging forces iterative data reconstruction. Reconstruction of three-dimensional (3D) two-color images (obtained from nanobeads and cultured cell samples) show significant reduction in the background noise (improved signal-to-noise ratio) with an impressive overall improvement in the spatial resolution (approximate to 250 nm) of the imaging system. Proposed data reconstruction technique may find immediate application in 3D in vivo and in vitro multi-color fluorescence imaging of biological specimens. (C) 2012 American Institute of Physics. http://dx.doi.org/10.1063/1.4769058]

Efficient generation of diffraction-limited multi-sheet pattern for biological imaging

We demonstrate a new technique to generate multiple light-sheets for fluorescence microscopy. This is possible by illuminating the cylindrical lens using multiple copies of Gaussian beams. A diffraction grating placed just before the cylindrical lens splits the incident Gaussian beam into multiple beams traveling at different angles. Subsequently, this gives rise to diffraction-limited light-sheets after the Gaussian beams pass through the combined cylindrical lens-objective sub-system. Direct measurement of field at and around the focus of objective lens shows multi-sheet pattern with an average thickness of 7.5 mu m and inter-sheet separation of 380 mu m. Employing an independent orthogonal detection sub-system, we successfully imaged fluorescently-coated yeast cells (approximate to 4 mu m) encaged in agarose gel-matrix. Such a diffraction-limited sheet-pattern equipped with dedicated detection system may find immediate applications in the field of optical microscopy and fluorescence imaging. (C) 2015 Optical Society of America

Real-time maximum a-posteriori image reconstruction for fluorescence microscopy

Rapid reconstruction of multidimensional image is crucial for enabling real-time 3D fluorescence imaging. This becomes a key factor for imaging rapidly occurring events in the cellular environment. To facilitate real-time imaging, we have developed a graphics processing unit (GPU) based real-time maximum a-posteriori (MAP) image reconstruction system. The parallel processing capability of GPU device that consists of a large number of tiny processing cores and the adaptability of image reconstruction algorithm to parallel processing (that employ multiple independent computing modules called threads) results in high temporal resolution. Moreover, the proposed quadratic potential based MAP algorithm effectively deconvolves the images as well as suppresses the noise. The multi-node multi-threaded GPU and the Compute Unified Device Architecture (CUDA) efficiently execute the iterative image reconstruction algorithm that is similar to 200-fold faster (for large dataset) when compared to existing CPU based systems. (C) 2015 Author(s). All article content, except where otherwise noted, is licensed under a Creative Commons Attribution 3.0 Unported License.

Enhancement Mechanism of Fluorescence Intensity in Presence of Plasmonic Nanoparticles

The emission intensity of fluorophore molecule may change in presence of strong plasmon field induced by nanoparticles. The enhancement intensity is optimized through selective clustering or functionalization of nanoparticles in closed vicinity of fluorophore. Our study is aimed at understanding the enhancement mechanism of fluorescence intensity in presence of gold nanoparticles to utilize it in molecular sensing and in situ imaging in the microfluidic lab-on-chip device. Related phenomena are studied in situ in a microfluidic channel via fluorescence imaging. Detailed analysis is carried out to understand the possible mechanism of enhancement of fluorescence due to nanoparticles. In the present experimental study we show that SYTO9 fluorescence intensity increased in presence of Au nanoparticles of similar to 20 nm diameter. The fluorescence intensity is 20 time more compared to that in absence of Au nanoparticles. The enhancement of fluorescence intensity is attributed to the plasmonic resonance of Au nanoparticle at around the fluorescence emission wavelength. Underlying fundamental mechanism via dipole interaction model is explored for quantitative correlation of plasmonic enhancement properties.

Highly fluorescent GFPm2+-based genome integration-proficient promoter probe vector to study Mycobacterium tuberculosis promoters in infected macrophages

Study of activity of cloned promoters in slow-growing Mycobacterium tuberculosis during long-term growth conditions in vitro or inside macrophages, requires a genome-integration proficient promoter probe vector, which can be stably maintained even without antibiotics, carrying a substrate-independent, easily scorable and highly sensitive reporter gene. In order to meet this requirement, we constructed pAKMN2, which contains mycobacterial codon-optimized gfpm2+ gene, coding for GFPm2+ of highest fluorescence reported till date, mycobacteriophage L5 attP-int sequence for genome integration, and a multiple cloning site. pAKMN2 showed stable integration and expression of GFPm2+ from M. tuberculosis and M. smegmatis genome. Expression of GFPm2+, driven by the cloned minimal promoters of M. tuberculosis cell division gene, ftsZ (MtftsZ), could be detected in the M. tuberculosis/pAKMN2-promoter integrants, growing at exponential phase in defined medium in vitro and inside macrophages. Stable expression from genome-integrated format even without antibiotic, and high sensitivity of detection by flow cytometry and fluorescence imaging, in spite of single copy integration, make pAKMN2 useful for the study of cloned promoters of any mycobacterial species under long-term in vitro growth or stress conditions, or inside macrophages.

Distribution of transverse chain fluctuations in harmonically confined semiflexible polymers

Two different experimental studies of polymer dynamics based on single-molecule fluorescence imaging have recently found evidence of heterogeneities in the widths of the putative tubes that surround filaments of F-actin during their motion in concentrated solution. In one J. Glaser, D. Chakraborty, K. Kroy, I. Lauter, M. Degawa, N. Kirchesner, B. Hoffmann, R. Merkel, and M. Giesen, Phys. Rev. Lett. 105, 037801 (2010)], the observations were explained in terms of the statistics of a worm-like chain confined to a potential determined self-consistently by a binary collision approximation, and in the other B. Wang, J. Guan, S. M. Anthony, S. C. Bae, K. S. Schweizer, and S. Granick, Phys. Rev. Lett. 104, 118301 (2010)], they were explained in terms of the scaling properties of a random fluid of thin rods. In this paper, we show, using an exact path integral calculation, that the distribution of the length-averaged transverse fluctuations of a harmonically confined weakly bendable rod (one possible realization of a semiflexible chain in a tube), is in good qualitative agreement with the experimental data, although it is qualitatively different in analytic structure from the earlier theoretical predictions. We also show that similar path integral techniques can be used to obtain an exact expression for the time correlation function of fluctuations in the tube cross section. (C) 2012 American Institute of Physics. http://dx.doi.org/10.1063/1.4712306]

Photocytotoxic ferrocene-appended (L-tyrosine)copper(II) complexes of phenanthroline bases

Copper(II) complexes of ferrocene(Fc)-conjugated reduced Schiff base of L-tyrosine (Fc-TyrH), viz., Cu(Fc-Tyr)(L)](ClO4), where L is 1,10-phenanthroline (phen, 1), dipyrido3,2-d:2',3'-f]quinoxaline (dpq, 2), dipyrido3,2-a:2',3'-c]phenazine (dppz, 3) and 2-(naphthalen-1-yl)-1H-imidazo4,5-f]1,10]phenanthroline (nip, 4), were prepared and tested for their photocytotoxicity in cancer cells. Cu(Fc-Phe)(phen)](-ClO4) (5) of L-phenylalanine and Cu(Ph-Tyr)(L)(ClO4)] of the reduced Schiff base Ph-TyrH derived from benzaldehyde and L-tyrosine having phen (6) and dppz (7), and Cu(Ph-Phe)(phen)(ClO4)] (8) using L-phenylalanine were prepared and used as controls. Complexes 5 and 6 were structurally characterized by X-ray crystallography. A copper(II)-based d-d band near 600 nm and a ferrocenyl band at similar to 450 nm were observed in DMF-Tris-HCI buffer (1:4 v/v) in respective complexes. The complexes are photocleavers of pUC19 DNA in visible light forming (OH)-O-center dot radicals. They are cytotoxic in HeLa (human cervical cancer) and MCF-7 (human breast cancer) cells showing an enhancement of cytotoxicity in visible light. Fluorescence imaging shows nuclear localization of the complexes.

Charge Injection through Nanocomposite Electrode in Microfluidic Channel for Electrical Lysis of Biological Cells

Several concepts have been developed in the recent years for nanomaterial based integrated MEMS platform in order to accelerate the process of biological sample preparation followed by selective screening and identification of target molecules. In this context, there exist several challenges which need to be addressed in the process of electrical lysis of biological cells. These are due to (i) low resource settings while achieving maximal lysis (ii) high throughput of target molecules to be detected (iii) automated extraction and purification of relevant molecules such as DNA and protein from extremely small volume of sample (iv) requirement of fast, accurate and yet scalable methods (v) multifunctionality toward process monitoring and (vi) downward compatibility with already existing diagnostic protocols. This paper reports on the optimization of electrical lysis process based on various different nanocomposite coated electrodes placed in a microfluidic channel. The nanocomposites are synthesized using different nanomaterials like Zinc nanorod dispersion in polymer. The efficiency of electrical lysis with various different electrode coatings has been experimentally verified in terms of DNA concentration, amplification and protein yield. The influence of the coating thickness on the injection current densities has been analyzed. We further correlate experimentally the current density vs. voltage relationship with the extent of bacterial cell lysis. A coupled multiphysics based simulation model is used to predict the cell trajectories and lysis efficiencies under various electrode boundary conditions as estimated from experimental results. Detailed in-situ fluorescence imaging and spectroscopy studies are performed to validate various hypotheses.

MECHANISM OF CELL LYSIS IN MICROFLUIDIC CHANNEL WITH INTEGRATED NANOCOMPOSITE ELECTRODES

This paper reports on the characterization of an integrated micro-fluidic platform for controlled electrical lysis of biological cells and subsequent extraction of intracellular biomolecules. The proposed methodology is capable of high throughput electrical cell lysis facilitated by nano-composite coated electrodes. The nano-composites are synthesized using Carbon Nanotube and ZnO nanorod dispersion in polymer. Bacterial cells are used to demonstrate the lysis performance of these nanocomposite electrodes. Investigation of electrical lysis in the microchannel is carried out under different parameters, one with continuous DC application and the other under DC biased AC electric field. Lysis in DC field is dependent on optimal field strength and governed by the cell type. By introducing the AC electrical field, the electrokinetics is controlled to prevent cell clogging in the micro-channel and ensure uniform cell dispersion and lysis. Lysis mechanism is analyzed with time-resolved fluorescence imaging which reveal the time scale of electrical lysis and explain the dynamic behavior of GFP-expressing E. coli cells under the electric field induced by nanocomposite electrodes. The DNA and protein samples extracted after lysis are compared with those obtained from a conventional chemical lysis method by using a UV-Visible spectroscopy and fluorimetry. The paper also focuses on the mechanistic understanding of the nano-composite coating material and the film thickness on the leakage charge densities which lead to differential lysis efficiency.