Cooled Charge-coupled Device Research Articles

A high-speed multispectral spinning-disk confocal microscope system for fluorescent speckle microscopy of living cells

Fluorescent speckle microscopy (FSM) uses a small fraction of fluorescently labeled subunits to give macromolecular assemblies such as the cytoskeleton fluorescence image properties that allow quantitative analysis of movement and subunit turnover. We describe a multispectral microscope system to analyze the dynamics of multiple cellular structures labeled with spectrally distinct fluorophores relative to one another over time in living cells. This required a high-resolution, highly sensitive, low-noise, and stable imaging system to visualize the small number of fluorophores making up each fluorescent speckle, a means by which to switch between excitation wavelengths rapidly, and a computer-based system to integrate image acquisition and illumination functions and to allow a convenient interface for viewing multispectral time-lapse data. To reduce out-of-focus fluorescence that degrades speckle contrast, we incorporated the optical sectioning capabilities of a dual-spinning-disk confocal scanner. The real-time, full-field scanning allows the use of a low-noise, fast, high-dynamic-range, and quantum-efficient cooled charge-coupled device (CCD) as a detector as opposed to the more noisy photomultiplier tubes used in laser-scanning confocal systems. For illumination, our system uses a 2.5-W Kr/Ar laser with 100–300 mW of power at several convenient wavelengths for excitation of few fluorophores in dim FSM specimens and a four-channel polychromatic acousto-optical modulator fiberoptically coupled to the confocal to allow switching between illumination wavelengths and intensity control in a few microseconds. We present recent applications of this system for imaging the cytoskeleton in migrating tissue cells and neurons.

Noninvasive imaging of cationic lipid-mediated delivery of optical and PET reporter genes in living mice.

Gene therapy involves the safe and effective delivery of one or more genes of interest to target cells in vivo. The advantages of using nonviral delivery systems include ease of preparation, low toxicity, and weak immunogenicity. Nonviral delivery methods, when combined with a noninvasive, clinically applicable imaging assay, will greatly aid in the optimization of gene therapy approaches for cancer. We demonstrate cationic lipid-mediated noninvasive monitoring of reporter gene expression of firefly (Photinus pyralis) luciferase (fl) and a mutant herpes simplex virus type I thymidine kinase (HSV1-sr39tk, tk) in living mice using a cooled charge coupled device (CCD) camera and positron emission tomography (PET), respectively. We observe a high level of fl and tk reporter gene expression predominantly in the lungs after a single injection of the extruded DOTAP:cholesterol DNA liposome complexes by way of the tail vein, seen to be time- and dose-dependent. We observe a good correlation between the in vivo bioluminescent signal and the ex vivo firefly luciferase enzyme (FL) activity in different organs. We further demonstrate the feasibility of noninvasively imaging both optical and PET reporter gene expression in the same animal using the CCD camera and microPET, respectively.

Sub-Doppler Spectral Resolution and Improved Sensitivity in a Cesium Resonance Fluorescence Imaging Monochromator

Improvements in spectral resolution and sensitivity of a cesium based resonance fluorescence imaging monochromator (RFIM) are described. Alignment of the two lasers used in the excitation scheme in a co-propagating geometry have resulted in a detector spectral bandpass (270 MHz) that is narrower than the Doppler broadened profile (380 MHz) of cesium vapor at room temperature. Improvements in the experimental design, namely the addition of an image intensifier and cooled charge-coupled device (CCD) camera, have resulted in increased sensitivity; we report a limit of detection of 108 photons per pixel limited by image intensifier noise. Images of the cesium D2 line emission (852.12 nm) from a cesium hollow cathode lamp were compared to an argon emission line (4Sº1/2 → 4P3/2) at 852.14 nm from a silver/argon hollow cathode lamp as a demonstration of the spectral resolution. The ability of the RFIM to discriminate between photons exciting the F = 4 vs. F = 3 ground state hyperfine level (a frequency difference of 9.2 GHz) of the cesium 62S1/2 state was also investigated to further demonstrate the spectral selectivity. The potential use of the described RFIM for chemical imaging applications is discussed.

Molecular engineering of a two-step transcription amplification (TSTA) system for transgene delivery in prostate cancer.

Gene therapy is founded on the concept that tissue-specific promoters can express heterologous genes for molecular imaging or therapeutic applications. The engineering of cell-specific enhancers to improve potency and the development of two-step transcriptional activation (TSTA) approaches have significantly improved the efficacy of transgene expression. Here we combine these technologies to create a robust, titratable, androgen-responsive system targeted to prostate cancer cells. Our "chimeric" TSTA system uses a duplicated variant of the prostate-specific antigen (PSA) gene enhancer to express GAL4 derivatives fused to one, two, or four VP16 activation domains. We targeted the resulting activators to cells with reporter templates bearing one, two, or five GAL4 binding sites upstream of firefly luciferase. We monitored activity via firefly luciferase assays in transfected cell extracts and in live nude mice using a cooled charge-coupled device (CCD) imaging system. In this system, we found that firefly luciferase expression in prostate cancer cells can be varied over an 800-fold range. We also found that a single plasmid bearing the optimized enhancer, GAL4-VP16 derivative, and reporter expressed firefly luciferase at 20-fold higher levels than the cytomegalovirus enhancer. We discuss the implications of this strategy and its application to molecular imaging and therapy.

Performance of a tracking device based on the GEM scintillation

A tracking device based on gas electron multiplier scintillation using a high-resolution cooled charge-coupled device (CCD) camera was developed. The CCD is sensitive to the 400-1000 nm band, and standard glass photographic lenses are used. The active gas is an Ar-CF/sub 4/ mixture. The prototype detector is described and images of single alpha tracks are presented. The light yield and detector performance for several other gaseous mixtures, such as /sup 4/He-CF/sub 4/, are also investigated.

A model that uses the induction phase of lux gene-dependent bioluminescence in Pseudomonas fluorescens HK44 to quantify cell density in translucent porous media

A cooled charge-coupled device (CCD) camera was used to follow the kinetics of induction of lux gene-dependent bioluminescence in Pseudomonas fluorescens HK44 held either in aqueous suspensions minus sand, saturated or unsaturated translucent sand (0.348 and 0.07 cm 3 H 2O/cm 3 of sand, respectively), and at cell densities ranging between 1×10 6 and 8.5×10 8 cells/ml. Before O 2 availability became a limiting factor, the rate of light emission ( L) increased with the square of time ( t) and linearly with increasing cell density ( c). A nonlinear model was developed that contains a “rate of increase in light emission” constant, B′, which is determined directly from the slope of a plot of √ L/ c against t. The model predicted the behavior of lux induction in HK44 under a variety of conditions. Similar B′ values were determined [49.0–57.6×10 −10 light units/(cell min 2)] for cell suspensions held in aqueous medium minus sand, in saturated or unsaturated 40/50 grade sand (0.36 mm grain diameter) and in two other textural classes of translucent sand. Although both the growth phase, and the presence of glucose during lux induction affected the first detectable time (FDT) of bioluminescence by HK44 in sand, the kinetics of induction of light emission were similar among treatments (stationary phase cells plus glucose, B′=61.6±3.2, log phase cells plus glucose, B′=63.2±7.2). The potential exists to use a combination of a CCD camera system, an inducible lux gene containing bioluminescent bacterium, and a light transmission chamber to nonintrusively visualize and quantify in real time the interactions between bacterial growth and unsaturated flow of water and solutes in porous media.

Noninvasive optical imaging of firefly luciferase reporter gene expression in skeletal muscles of living mice.

The ability to monitor reporter gene expression noninvasively offers significant advantages over current techniques such as postmortem tissue staining or enzyme activity assays. Here we demonstrate a novel method of repetitively tracking in vivo gene expression of firefly luciferase (FL) in skeletal muscles of mice using a cooled charged coupled device (CCD) camera. We first show that the cooled CCD camera provides consistent and reproducible results within +/-8% standard deviation from mean values, and a detection sensitivity (range tested: 1 x 10(4) - 1 x 10(9) plaque form-ing units (pfu)) of 1 x 10(6) pfu of E1-deleted adenovirus expressing FL driven by a cytomegalovirus promoter (Ad-CMV-FL). The duration and magnitude of adenoviral mediated (1 x 10(9) pfu) FL gene expression were then followed over time. FL gene expression in immunocompetent Swiss Webster mice peaks within the first 48 hours, falls by 98% after 20 days, and persists for >150 days. In contrast, FL activity in nude mice remains elevated for >110 days. Finally, transduced Swiss Webster and nude mice were sacrificed to show that the in vivo CCD signals correlate well with in vitro luciferase enzyme assays (r(2)=0.91 and 0.96, respectively). Our findings demonstrate the ability of the cooled CCD camera to sensitively and noninvasively track the location, magnitude, and persistence of FL gene expression. Monitoring of gene therapy studies in small animals may be aided considerably with further extensions of this technique.

Imaging transgene expression in live animals.

Monitoring the expression of therapeutic genes in targeted tissues in disease models is important to assessing the effectiveness of systems of gene therapy delivery. We applied a new light-detection cooled charged-coupled device (CCCD) camera for continuous in vivo assessment of commonly used gene therapy delivery systems (such as ex vivo manipulated cells, viral vectors, and naked DNA), without the need to kill animals. We examined a variety of criteria related to real-time monitoring of luciferase (luc) gene expression in tissues including bone, muscle, salivary glands, dermis, liver, peritoneum, testis, teeth, prostate, and bladder in living mice and rats. These criteria included determination of the efficiency of infection/transfection of various viral and nonviral delivery systems, promoter specificity, and visualization of luciferase activity, and of the ability of luciferin to reach various organs. The exposure time for detection of luc activity by the CCCD camera is relatively short (approximately 2 minutes) compared with the intensified CCD camera photon-counting method (approximately 15 minutes). Here we transduce a variety of vectors (such as viruses, transfected cells, and naked DNA) by various delivery methods, including electroporation, systemic injection of viruses, and tail-vein, high-velocity-high-volume administration of DNA plasmids. The location, intensity, and duration of luc expression in different organs were determined. The distribution of luciferin is most probably not a barrier for the detection of in vivo luciferase activity. We showed that the CCCD photon detection system is a simple, reproducible, and applicable method that enables the continuous monitoring of a gene delivery system in living animals.

Selective distribution of porphyrins in skin thick basal cell carcinoma after topical application of methyl 5-aminolevulinate

Topical photodynamic therapy (PDT) of superficial basal cell carcinoma (BCC) with 5-aminolevulinic acid (ALA) has achieved promising clinical results. However, the efficacy of this therapy for thick BCC is dramatically decreased by a limited diffusion of hydrophilic ALA into the tumor. Lipophilic esters of ALA may enhance their penetration into the lesion. In this randomized, open clinical study, microscopic fluorescence photometry incorporating a light-sensitive thermo-electrically cooled charge-coupled device (CCD) camera was employed to investigate the penetration of methyl 5-aminolevulinate-induced porphyrin fluorescence in thick BCC lesions. Both the distribution pattern and the amount of porphyrins in 32 lesions of 16 patients were studied after topical application of 16, 80 or 160 mg/g of methyl 5-aminolevulinate for 3 or 18 h. A highly selective and homogeneous distribution of methyl 5-aminolevulinate-induced porphyrin fluorescence was seen in all lesions studied, with much less fluorescence in the adjacent normal skin tissues. In lesions of up to 2 mm thickness the application of 160 mg/g methyl 5-aminolevulinate for 3 h showed the highest ratio of porphyrin fluorescence depth to tumor depth (0.98±0.04), thus providing a biologic rationale for a clinical PDT trial with this regimen.

RERG is a novel ras-related, estrogen-regulated and growth-inhibitory gene in breast cancer.

Using microarray analysis, we identified a unique ras superfamily gene, termed RERG (ras-related and estrogen-regulated growth inhibitor), whose expression was decreased or lost in a significant percentage of primary human breast tumors that show a poor clinical prognosis. Importantly, high RERG expression correlated with expression of a set of genes that define a breast tumor subtype that is estrogen receptor-positive and associated with a slow rate of tumor cell proliferation and a favorable prognosis for these cancer patients. RERG mRNA expression was induced rapidly in MCF-7 cells stimulated by beta-estradiol and repressed by tamoxifen treatment. Like Ras, RERG protein exhibited intrinsic GDP/GTP binding and GTP hydrolysis activity. Unlike Ras proteins, RERG lacks a known recognition signal for COOH-terminal prenylation and was localized primarily in the cytoplasm. Expression of RERG protein in MCF-7 breast carcinoma cells resulted in a significant inhibition of both anchorage-dependent and anchorage-independent growth in vitro and inhibited tumor formation in nude mice. These features of RERG are strikingly different from most Ras superfamily GTP-binding pro-teins and suggest that the loss of RERG expression may contribute to breast tumorigenesis.

Analysis of Biological Structures by Electron Energy Loss Spectroscopy and Imaging

As techniques for electron energy-loss spectroscopy (EELS) reach a higher degree of optimization, detection limits for analyzing biological structures are approaching those predicted by theory. in favorable specimens, single atom detection is predicted for elemental maps acquired by means of the scanning transmission electron microscope (STEM) equipped with a field emission source, paralleldetection EELS and a spectrum-imaging system. to obtain such results, the electron detector should have a detective quantum efficiency close to unity and a well behaved point-spread function; such design features are now available with a cooled charge-couple device (CCD) array. The energy-filtering transmission electron microscope (EFTEM) provides a complementary approach to mapping elements occurring at higher concentrations but distributed over larger regions of the specimen. Use of an optimized CCD detector in the EFTEM now enables accurate quantitation in addition to high analytical sensitivity, albeit not at the single atom level.

Determination of Ultratrace-Level Fluorescent Tracer Concentrations in Environmental Samples Using a Combination of HPLC Separation and Laser-Excited Fluorescence Multiwavelength Emission Detection: Application to Testing of Geothermal Well Brines

Lowering the limits of detection for fluorescent tracers in environmental samples can reduce the cost and environmental impact of tracer testing and allow a wider variety of tracer dyes to be used. Detecting dyes in environmental water samples at the sub-part-pertrillion level raises significant challenges in the sensitivity and selectivity of measurement. In the present work, we address this issue by developing a high-sensitivity multiwavelength fluorescence detector for high-performance liquid chromatography (HPLC)-separated samples. The fluorescence flow-cell utilizes fiber-optic coupling of laser excitation and the collected emission, which is dispersed in a short spectrograph and detected with a cooled charge-coupled device (CCD). The HPLC separation step not only resolves the target tracer from fluorescent impurities in the sample but also transfers the dye molecules into a solution of reproducible composition which provides a constant Raman scattering background against which the tracer fluorescence spectrum may be detected. The combination of emission-wavelength and elution-time measurement provides a multidimensional data set that improves selectivity for detecting a tracer. For analysis of the data, partial least-squares modeling is tested, along with two methods that do not require prior knowledge of interfering species: rank annihilation and self-modeling curve resolution. The method is developed and tested on fluorescein standards, and a detection limit for fluorescein of 40 ppq (4.0 × 10−14 g/mL) is predicted. This capability was tested on geothermal well samples, in which ∼40 ppq fluorescein could be detected in the presence of 15-fold greater fluorescence from an unknown interferent.

Miniaturization of a Hepatitis C Virus RNA Polymerase Assay Using a −102°C Cooled CCD Camera-Based Imaging System

Innovations in detection technologies have allowed us to develop a novel assay in 1536-well plate format and assess the advantages of screen miniaturization compared with conventional high-throughput compound screening in 96- or 384-well plates. An HCV RNA polymerase assay has been miniaturized in 1536-well plates by using a new detection technology known as LEADseeker homogeneous imaging system. It uses a −102°C cooled charge-coupled device (CCD) camera and newly designed scintillation proximity microparticles. The miniaturized assay used europium-doped streptavidin-coated yttrium oxide (YOx) or polystyrene (PS) microspheres to capture biotin-labeled [3H]RNA product transcripts. Beads in proximity to the radioisotope convert the emitted β− particles into photons having wavelengths in the red region of the visible spectrum, optimal for detection by the CCD camera. Because the camera collects light from all wells of the plate simultaneously, 1536-well plates are imaged as rapidly as 384-well plates, on the order of 10 min per plate. The assay has a signal to background of approximately 20-fold, satisfactory for high-throughput robotics screening. The enzyme kinetics and potency of a known inhibitor were similar to those obtained from the conventional assay using scintillation proximity assay (SPA) beads and a scintillation plate counter. Furthermore, the newly developed microbeads (emitting at 610 to 620 nm) are less prone to quenching effects caused by yellow-colored compounds, than conventional SPA beads or scintillation fluid (emitting at 400 to 480 nm region). Thus, the LEADseeker imaging system is a useful new tool for miniaturization of assays for high-throughput screening.

Digital fluorescence microscopy of cell cytoplasts with and without the centrosome

This chapter describes the experimental protocols used for the preparation of samples for high-resolution digital fluorescence microscopy. For high-resolution digital fluorescence microscopy of microtubules (MTs), Cy3-tagged tubulin subunits are injected in living cells. Injected cells are treated with cytochalasin and nocodazole to disrupt actin filaments and MTs, respectively, and then enucleated by centrifugation. After washing out the drugs, images of fluorescently labeled microtubules in cytoplasts are captured with a cooled charge-coupled device (CCD) camera. In cytoplasts lacking the centrosome, decreased MT stability produces an elevated level of free tubulin and imaging of MTs is complicated by unusually high background fluorescence. Therefore, every effort must be made to improve the signal-to-noise ratio. A sensitive cooled CCD camera is used for capturing images. MTs are labeled with a very bright fluorophore to maximize the signal. Porcine brain tubulin subunits are conjugated with Cy3, which is significantly brighter than many other available fluorophores. The chapter describes the protocol used for the preparation of Cy3-tagged porcine brain tubulin subunits.

Chromosomes are target sites for photodynamic therapy as demonstrated by subcellular laser microirradiation

The present investigation has been undertaken to examine the possibility that the cell nucleus, and specifically the genetic material, is a target site for photodynamic therapy. PTK 2 and Hep-2 cells are pretreated with a medium containing 15 μg/ml (0.09 mM) 5-aminolevulinic acid (ALA). Individual fluorescence images are recorded for each selected cell using a cooled charge-coupled device (CCD). A laser microbeam system generating 630 nm is used for subcellular-region irradiation of specific targets: chromosomes, the mitotic spindle, the perispindle region and the peripheral cytoplasm. Nuclei of interphase cells are also irradiated. Data comparing the sensitivities of the different subcellular microirradiation sites in ALA-treated mitotic cells demonstrate that under the irradiation conditions used, the chromosome is the most sensitive subcellular target followed by the perispindle region, the peripheral cytoplasm and spindle, and, lastly, the interphase nucleus.

Digital Video Microscopy: A Practical Visual Analysis Technique for the Conservator

Video microscopy has been an important application in science, medicine, and industry. It has also been used in many conservation laboratories as part of the daily routine of examining and treating objects. These microscopy systems are normally composed of a cooled charged coupled device (CCD) video camera and a high-resolution color monitor used together with a microscope. Further capabilities are gained by digitizing such systems with the addition of a video capture card and computer software. This enhancement makes it possible to approximate the side-by-side visual comparison capabilities of very expensive comparison microscopes used in the forensic sciences. Such capabilities can be a valuable tool in today's conservation laboratory. It is possible to use side-by-side visual comparison studies in the course of identifying materials, examining various interactions between materials during pretreatment spot-testing, or monitoring the actual treatment phase. This article provides a brief overview of digital video microscopy and examines some of its applications that may be suitable for routine conservation use.

Digital Video Microscopy: A Practical Visual Analysis Technique for the Conservator

Video microscopy has been an important application in science, medicine, and industry. It has also been used in many conservation laboratories as part of the daily routine of examining and treating objects. These microscopy systems are normally composed of a cooled charged coupled device (CCD) video camera and a high-resolution color monitor used together with a microscope. Further capabilities are gained by digitizing such systems with the addition of a video capture card and computer software. This enhancement makes it possible to approximate the side-by-side visual comparison capabilities of very expensive comparison microscopes used in the forensic sciences. Such capabilities can be a valuable tool in today's conservation laboratory. It is possible to use side-by-side visual comparison studies in the course of identifying materials, examining various interactions between materials during pretreatment spot-testing, or monitoring the actual treatment phase. This article provides a brief overview of digital video microscopy and examines some of its applications that may be suitable for routine conservation use.

Single-molecule fluorescence observed with mercury lamp illumination.

We demonstrate that it is possible to observe single fluorescent molecules using a standard fluorescence microscope with mercury lamp excitation and an inexpensive cooled charge-coupled device (CCD) camera. With this equipment, we have been able to observe single molecules of tetramethyl-rhodamine, rhodamine 6G, fluorescein isothiocyanate and green fluorescent protein. Immobilized molecules were observed both in air and in aqueous solution.

Raman optical activity instrument for studies of biopolymer structure and dynamics

The latest version of a multi-channel Raman optical activity (ROA) instrument implementing incident circular polarization (ICP) modulation within a in a backscattering geometry and optimized for measurements on biopolymers in aqueous solution is described. It is based on a fast single-stage, imaging (stigmatic) monochromator equipped with a high optical density holographic notch filter, a holographic transmission grating and a thinned back-illuminated thermoelectrically cooled charge-coupled device (CCD) detector with a high quantum efficiency. A large-aperture longitudinal electro-optic modulator (Pockels cell) is employed to switch between orthogonal circular polarization states in the incident laser radiation. A thick Lyot depolarizer is used for depolarization of the backscattered Raman radiation. This backscattering ICP CCD ROA design is currently realized with two prototype instruments dedicated to studies of the structure and dynamics of biopolymers in aqueous solution. Backscattered ICP ROA spectra of the following samples are presented as typical examples of the excellent performance characteristics: poly(L-glutamic acid) in α-helical and disordered conformations; bovine α-lactalbumin in native and acid molten globule states; human immunoglobulin; calf thymus DNA and both magnesium-bound and magnesium-free phenylalanine-specific transfer RNA; and filamentous bacterial viruses Pf1 and M13. Copyright © 1999 John Wiley & Sons, Ltd.

Vibrational levels and statistical analysis of the X(1Σg+) ground state of CS2

Single rovibronic levels in the perturbed B21(Δu1) state of CS2 molecules in a cold molecular beam were excited by ultraviolet (UV) photons from a frequency doubled single mode continuous wave (cw) dye laser. The dispersed fluorescence spectrum, detected by a liquid nitrogen (LN) cooled charge coupled device (CCD) array behind a monochromator allowed the determination of vibrational term values in the X(1Σg+) state from the (0,00,0) level up to 20 000 cm−1. Based on calculations including Fermi resonances, most of the measured vibrational bands could be assigned to several vibronic ground state level progressions. A statistical analysis of the positions of all measured levels shows for the nearest neighbor distances and the Δ3 statistics a pure Poissonian distribution. This indicates that no strong correlation between neighboring vibrational levels nor long range interactions are present and therefore no chaotic behavior could be found in contrast to the cases of NO2 and SO2.