Sentinel node mapping using hysteroscopic injection of indocyanine green and laparoscopic near-infrared fluorescence imaging in endometrial cancer staging.

Herein is presented a technique for minimally invasive sentinel node mapping. The patient had apparently early stage endometrial cancer. Sentinel node mapping was performed using a hysteroscopic injection of indocyanine green followed by laparoscopic sentinel node detection via near-infrared fluorescence. This technique ensures delineation of lymphatic drainage from the tumor area, thus achieving accurate detection of sentinel nodes.

New bodipy dyads for two-photons fluorescence imaging in cells

Fluorescent probes are essential tools for studying biological systems. The last decade has witnessed particular interest in the development of two-photon excitable probes, due to their advantageous features in tissue imaging compared to the corresponding one-photon probes [1]. Recently, we have designed and synthetized an aminonaphthalimide–BODIPY derivative as energy transfer cassettes and were found to show very fast and efficient BODIPY fluorescence sensitization [2]. This was observed upon one- and two-photon excitation, which extends the application range of the investigated bichromophoric dyads in terms of accessible excitation wavelengths. In order to increase the two-photon absorption of the system aminonaphthalimide fluorophore was replace with a Prodan analog (BODIPY dyad 1), which presents found a variety of applications as probes and labels in biology [3]. The two-photon absorption cross-section  of the dyads is significantly incremented by the presence of the 6-acetyl-2-naphthylamine donor group. The emission maximum of a BODIPY fluorophore can significantly be red-shifted in comparison to their precursors by conjugation with aromatic aldehydes. [4] We use a synthetic strategy to obtain BODIPY dyad 2 that incorporates an imidazole ring. This molecule can be used in biological media as a near-neutral pH indicator based on one- and two-photon excitable BODIPY acceptor.

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.

Bright-field and fluorescence chip-scale microscopy for biological imaging

Optical microscopy is an essential tool in biological science and one of the gold standards for medical examinations. Miniaturization of microscopes can be a crucial stepping stone towards realizing compact, cost-effective and portable platforms for biomedical research and healthcare. This thesis reports on implementations of bright-field and fluorescence chip-scale microscopes for a variety of biological imaging applications. The term “chip-scale microscopy” refers to lensless imaging techniques realized in the form of mass-producible semiconductor devices, which transforms the fundamental design of optical microscopes.

Our strategy for chip-scale microscopy involves utilization of low-cost Complementary metal Oxide Semiconductor (CMOS) image sensors, computational image processing and micro-fabricated structural components. First, the sub-pixel resolving optofluidic microscope (SROFM), will be presented, which combines microfluidics and pixel super-resolution image reconstruction to perform high-throughput imaging of fluidic samples, such as blood cells. We discuss design parameters and construction of the device, as well as the resulting images and the resolution of the device, which was 0.66 µm at the highest acuity. The potential applications of SROFM for clinical diagnosis of malaria in the resource-limited settings is discussed.

Next, the implementations of ePetri, a self-imaging Petri dish platform with microscopy resolution, are presented. Here, we simply place the sample of interest on the surface of the image sensor and capture the direct shadow images under the illumination. By taking advantage of the inherent motion of the microorganisms, we achieve high resolution (~1 µm) imaging and long term culture of motile microorganisms over ultra large field-of-view (5.7 mm × 4.4 mm) in a specialized ePetri platform. We apply the pixel super-resolution reconstruction to a set of low-resolution shadow images of the microorganisms as they move across the sensing area of an image sensor chip and render an improved resolution image. We perform longitudinal study of Euglena gracilis cultured in an ePetri platform and image based analysis on the motion and morphology of the cells. The ePetri device for imaging non-motile cells are also demonstrated, by using the sweeping illumination of a light emitting diode (LED) matrix for pixel super-resolution reconstruction of sub-pixel shifted shadow images. Using this prototype device, we demonstrate the detection of waterborne parasites for the effective diagnosis of enteric parasite infection in resource-limited settings.

Then, we demonstrate the adaptation of a smartphone’s camera to function as a compact lensless microscope, which uses ambient illumination as its light source and does not require the incorporation of a dedicated light source. The method is also based on the image reconstruction with sweeping illumination technique, where the sequence of images are captured while the user is manually tilting the device around any ambient light source, such as the sun or a lamp. Image acquisition and reconstruction is performed on the device using a custom-built android application, constructing a stand-alone imaging device for field applications. We discuss the construction of the device using a commercial smartphone and demonstrate the imaging capabilities of our system.

Finally, we report on the implementation of fluorescence chip-scale microscope, based on a silo-filter structure fabricated on the pixel array of a CMOS image sensor. The extruded pixel design with metal walls between neighboring pixels successfully guides fluorescence emission through the thick absorptive filter to the photodiode layer of a pixel. Our silo-filter CMOS image sensor prototype achieves 13-µm resolution for fluorescence imaging over a wide field-of-view (4.8 mm × 4.4 mm). Here, we demonstrate bright-field and fluorescence longitudinal imaging of living cells in a compact, low-cost configuration.

Innovations of wide-field optical-sectioning fluorescence microscopy : toward high-speed volumetric bio-imaging with simplicity

Optical microscopy has become an indispensable tool for biological researches since its invention, mostly owing to its sub-cellular spatial resolutions, non-invasiveness, instrumental simplicity, and the intuitive observations it provides. Nonetheless, obtaining reliable, quantitative spatial information from conventional wide-field optical microscopy is not always intuitive as it appears to be. This is because in the acquired images of optical microscopy the information about out-of-focus regions is spatially blurred and mixed with in-focus information. In other words, conventional wide-field optical microscopy transforms the three-dimensional spatial information, or volumetric information about the objects into a two-dimensional form in each acquired image, and therefore distorts the spatial information about the object. Several fluorescence holography-based methods have demonstrated the ability to obtain three-dimensional information about the objects, but these methods generally rely on decomposing stereoscopic visualizations to extract volumetric information and are unable to resolve complex 3-dimensional structures such as a multi-layer sphere.

The concept of optical-sectioning techniques, on the other hand, is to detect only two-dimensional information about an object at each acquisition. Specifically, each image obtained by optical-sectioning techniques contains mainly the information about an optically thin layer inside the object, as if only a thin histological section is being observed at a time. Using such a methodology, obtaining undistorted volumetric information about the object simply requires taking images of the object at sequential depths.

Among existing methods of obtaining volumetric information, the practicability of optical sectioning has made it the most commonly used and most powerful one in biological science. However, when applied to imaging living biological systems, conventional single-point-scanning optical-sectioning techniques often result in certain degrees of photo-damages because of the high focal intensity at the scanning point. In order to overcome such an issue, several wide-field optical-sectioning techniques have been proposed and demonstrated, although not without introducing new limitations and compromises such as low signal-to-background ratios and reduced axial resolutions. As a result, single-point-scanning optical-sectioning techniques remain the most widely used instrumentations for volumetric imaging of living biological systems to date.

In order to develop wide-field optical-sectioning techniques that has equivalent optical performance as single-point-scanning ones, this thesis first introduces the mechanisms and limitations of existing wide-field optical-sectioning techniques, and then brings in our innovations that aim to overcome these limitations. We demonstrate, theoretically and experimentally, that our proposed wide-field optical-sectioning techniques can achieve diffraction-limited optical sectioning, low out-of-focus excitation and high-frame-rate imaging in living biological systems. In addition to such imaging capabilities, our proposed techniques can be instrumentally simple and economic, and are straightforward for implementation on conventional wide-field microscopes. These advantages together show the potential of our innovations to be widely used for high-speed, volumetric fluorescence imaging of living biological systems.

Use of fluorescence lifetime imaging microscopy (FLIM) as a timer of cell cycle S phase

Incorporation of thymidine analogues in replicating DNA, coupled with antibody and fluorophore staining, allows analysis of cell proliferation, but is currently limited to monolayer cultures, fixed cells and end-point assays. We describe a simple microscopy imaging method for live real-time analysis of cell proliferation, S phase progression over several division cycles, effects of anti-proliferative drugs and other applications. It is based on the prominent (~ 1.7-fold) quenching of fluorescence lifetime of a common cell-permeable nuclear stain, Hoechst 33342 upon the incorporation of 5-bromo-2’-deoxyuridine (BrdU) in genomic DNA and detection by fluorescence lifetime imaging microscopy (FLIM). We show that quantitative and accurate FLIM technique allows high-content, multi-parametric dynamic analyses, far superior to the intensity-based imaging. We demonstrate its uses with monolayer cell cultures, complex 3D tissue models of tumor cell spheroids and intestinal organoids, and in physiological study with metformin treatment.

Synthesis of a multimodal molecular imaging probe based on a hyperbranched polymer architecture

Molecular imaging is utilised in modern medicine to aid in the diagnosis and treatment of disease by allowing its spatiotemporal state to be examined in vivo. This study focuses on the development of novel multimodal molecular imaging agents based on hyperbranched polymers that combine the complementary capabilities of optical fluorescence imaging and positron emission tomography-computed tomography (PET/CT) into one construct. RAFT-mediated polymerisation was used to prepare two hydrophilic hyperbranched polymers that were differentiated by their size and level of branching. The multiple functional end-groups facilitated covalent attachment of both near infrared fluorescent dyes for optical imaging, as well as a copper chelator allowing binding of 64Cu as a PET radio nuclei. In vivo multimodal imaging of mice using PET/CT and planar optical imaging was first used to assess the biodistribution of the polymeric materials and it was shown that the larger and more branched polymer had a significantly longer circulation time. The larger constructs were also shown to exhibit enhanced accumulation in solid tumours in a murine B16 melanoma model. Importantly, it was demonstrated that the PET modality gave rise to high sensitivity immediately after injection of the agent, while the optical modality facilitated extended longitudinal studies, thus highlighting how the complementary capabilities of the molecular imaging agents can be useful for studying various diseases, including cancer.

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.