Spectral Crosstalk Research Articles

Software compensation improves the analysis of heterogeneous tumor samples stained for multiparameter DNA flow cytometry

Background: High concentrations of propidium iodide (PI), in combination with fluorescein isothiocyanate (FITC) and R-phycoerythrin (RPE) used for multiparameter DNA flow cytometry (FCM), cause spectral cross-talk into the green fluorescence channel (FL1). We have evaluated the use of post-acquisition software compensation (N-Color Compensation) in order to correct this spectral cross-talk caused by PI. Method: Cell mixtures were prepared consisting of keratin 8/18 FITC labeled, keratin 8/18 RPE labeled, and unlabeled MCF-7 breast carcinoma cells. DNA was stained with PI (100 μM). Post-acquisition software compensation was applied to correct the spectral cross-talk of PI fluorescence. Secondly, the distribution of the Ki-67 (FITC) protein during the cell cycle (PI) of SiHa cervical carcinoma cells (no software compensation) was compared to the Ki-67 expression pattern of SiHa cells, simultaneously stained for keratin 8 (RPE), after applying software compensation. Finally, software compensation was used to compare the relative levels of PCNA and p53 expression in two clinical ovarian cancer ascites specimens, stained for PCNA or p53 (FITC), keratin 8/18 (RPE), and DNA (PI), with a known p53 status (positive and negative, respectively). Results: The Ki-67 cell cycle-dependent pattern of a triply stained sample (Ki-67 (FITC), keratin 8 (RPE), and DNA (PI)) is restored after software compensation and the results are comparable to the Ki-67 distribution of a sample stained solely for Ki-67 and DNA. P53 expression could only be resolved after using software compensation in the p53 positive ovarian ascites (OA) sample. Conclusions: We conclude that software compensation is a robust and reliable post-acquisition method for the correction of RPE/PI spectral cross-talk, permitting better identification of weakly expressed proteins in heterogeneous clinical tumor samples stained for multiple cellular antigens and DNA using PI.

Electrical and optical characteristics of two color mid wave HgCdTe infrared detectors

Two-color mid wave triple-layer heterojunction HgCdTe detectors were studied using temperature-dependent current–voltage (I–V) measurements, temperature-dependent spectral response measurements, and temperature-dependent noise measurements. The reverse biased dark current shows diffusion-limited behavior for T>125 K. The same data show evidence for generation-recombination-type behavior for the longer wavelength junction at temperatures between 100 and 125 K. For temperatures less than 100 K, the measurements are background limited by photon flux, even though these measurements are performed at nominal zero background. The upper junction shows soft reverse breakdown voltages on the order of about 250 mV, while the bottom junction shows no breakdown for V<500 mV. At 80 K, the R0A product is in excess of 1×106 Ω cm2. In forward bias, the current–voltage characteristics of the lower junction are diffusion limited for all temperatures, while at lower temperatures, the upper junction showed generation-recombination behavior. Optical measurements found a cutoff wavelength of about 4 μm for the lower junction and about 4.5 μm for the upper junction. The spectral crosstalk was less than 3%. At 80 K, the frequency-dependent noise of the shorter wavelength junction showed no dependence on bias, while for the longer wavelength junction, the noise at lower frequencies increased with bias. There is no difference in the noise characteristics when either the photon flux or the temperature is increased.

Improved single laser measurement of two cellular antigens and DNA-ploidy by the combined use of propidium iodide and TO-PRO-3 iodide.

Recently, Frey (Cytometry 17:310-318, 1994) demonstrated that TO-PRO-3 iodide (TP3) can be excited indirectly by a 488 nm laser line through energy transfer by propidium iodide (PI). In the present study, we investigated whether PI-TP3 energy transfer can help to overcome spectral cross talk problems associated with the combined use of fluorescein isothiocyanate (FITC), R-phycoerythrin (PE), and PI. Mixtures of keratin 8/18 FITC-labeled, keratin 8/18-PE-labeled, and unlabeled MCF-7 breast carcinoma cells were prepared and stained for DNA with PI (100 microM). The effect of adding a range of TP3 concentrations (0.001 to 16 microM) to these mixtures was evaluated. The combined use of PI and TP3 was further evaluated using mixtures of unlabeled and p53 FITC-labeled COV362.cl4 ovarian cancer cells and mixtures of unlabeled and p53 FITC-labeled COV362.cl4 cells and peripheral blood lymphocytes (PBL), additionally stained for keratin 8/18 (PE). Finally, a human ovarian ascites tumor specimen was triple-stained for keratin 8/18 (PE), vimentin (FITC) and DNA or keratin 8/18 (PE), PCNA (FITC) and DNA. Addition of TP3 allowed complete correction for spectral cross talk of PE/PI into the green fluorescence detector (FL1). Only minimal (FL1 - %FL2) compensation was required at a TP3 concentration of 2.0 microM in the presence of PI (100 microM). The PI spectral cross talk into the orange fluorescence detector (FL2) was reduced by about 50% using the same photomultiplier (PMT) settings. Although addition of TP3 reduced the signal-to-background ratio by about 30%, the advantage gained through full compensation for spectral cross talk resulted in an improved discrimination of p53-positive and -negative subpopulations in a mixture of human PBL and COV362.cl4 cells. Furthermore, vimentin-negative and PCNA-negative cells were better resolved in a human DNA-aneuploid ovarian ascites tumor after staining the DNA with PI/TP3, rather than with PI alone. We conclude that the addition of TP3 to PI improves the combined measurement by single-laser flow cytometry of DNA-ploidy and antigen expression in heterogenous clinical samples.

Bandgap-Engineering of HgCdTe for Two-Color Ir Detector Arrays BY Movpe

AbstractRecent results on MOVPE growth of multilayer two-color HgCdTe detectors, for simultaneous and independent detection of medium wavelength (MW, 3–5 μm) and long wavelength (LW, 8–12 μm) bands, are reported. The structures are grown in situ on lattice matched (100) CdZnTe in the double-heterojunction p-n-N-P configuration. A barrier layer is placed between the LW and MW absorber layers to prevent diffusion of MW photocarriers into the LW junction and thereby eliminate spectral crosstalk. X-ray double crystal rocking curve widths are ∼ 45 arc-secs, indicating good epitaxial quality. SIMS depth profile measurements of these 28 μm thick structures show well-defined alloy compositions, and arsenic and iodine doping. SIMS data on a series of thirteen films show that good run-to-run repeatability is obtained on thicknesses, compositions, and dopant levels with values close to the device design targets. Depth profile of etch pits through the thickness of the films show etch pit densities in the range of 8×105-5×106 cm−2.

Photonic highway switches based on ring resonators used as frequency-selective components

A new concept for a highway switch, which can be used to connect different optical wavelength division multiplexing data highways for data exchange, is proposed and the system relevant properties are outlined. For the required add-drop filter elements we used ring resonators. Typical characteristics of channel bandwidth, channel spacing, free spectral range, amplification, and cross-talk behavior of a highway switch with double-cavity ring resonators are basically examined and to some extent compared with solutions that were obtained with standard single-ring resonators. A signal flow chart transformation for evaluating filter transfer functions is presented.

Independently accessed back-to-back HgCdTe photodiodes: A new dual-band infrared detector

We report the first data for a new two-color HgCdTe infrared detector for use in large dual-band infrared focal plane arrays (IRFPAs). Referred to as the independently accessed back-to-back photodiode structure, this novel dual-band HgCdTe detector provides independent electrical access to each of two spatially collocated back-to-back HgCdTe photodiodes so that true simultaneous and independent detection of medium wavelength (MW, 3–5 μm) and long wavelength (LW, 8–12 μm) infrared radiation can be accomplished. This new dual-band detector is directly compatible with standard backside-illuminated bump-interconnected hybrid HgCdTe IRFPA technology. It is capable of high fill factor, and allows high quantum efficiency and BLIP sensitivity to be realized in both the MW and LW photodiodes. We report data that demonstrate experimentally the key features of this new dual-band detector. These arrays have a unit cell size of 100 x 100 μm2, and were fabricated from a four-layer p-n-N-P HgCdTe film grown in situ by metalorganic chemical vapor deposition on a CdZnTe substrate. At 80K, the MW detector cutoff wavelength is 4.5 μm and the LW detector cutoff wavelength is 8.0 μm. Spectral crosstalk is less than 3%. Data confirm that the MW and LW photodiodes are electrically and radiometrically independent.

Design and performance of a direct-reading, multichannel spectrometer for the determination of chlorinated purgeable organic compounds by flame infrared-emission spectrometry

The feasibility of determining chlorinated, purgeable organic compounds (POCs) in aqueous samples by flame infrared-emission (FIRE) spectrometry was evaluated with a specially designed, multichannel, dispersive spectrometer having sufficient resolving power to prevent interference from large amounts of non-halogenated POCs that could also be present in the sample. The polychromator was based on a Czerny—Turner optical mounting with a 0.15-m focal length, a nominal focal ratio of f/3, and a reciprocal linear dispersion of 0.15 μm/mm in the first order. The HCl and CO 2 infrared emission bands (3.77 and 4.42 μm, respectively) were monitored in two analytical channels, while a third reference channel, used for background subtraction, monitored the H 2O background emission at 2.8 μm. Instrumental performance was evaluated with dichloromethane, trichloromethane, trichloroethylene, 1,1,2,2-tetrachloroethane and monochlorobenzene as test compounds, and parameters such as the linear dynamic range, reproducibility, detection limit and signal-to-noise ratio, and extent of spectral crosstalk between channels were determined. The feasibility of performing a quantitative analysis of a two-component mixture of dichloromethane and trichloromethane at trace levels was demonstrated.

Observations of natural fluorescence with an underwater radiometer

Measurements of subsurface irradiance spectra in the ocean consistently indicate intensities of long-wavelength visible light (λ≧589 nm) greater than could be derived from the penetration of sunlight. This can be attributed to natural fluorescence and/or to spectral crosstalk due to light leakage through the blocking filters of each discrete detector. A comparison of observed profiles with modelled contributions from these two possible sources indicates that both factors are contributing. At 671 and 694 nm wavelength, the excess signal appears to be predominantly fluorescence while that at 589 nm is mostly crosstalk. Both effects appear to be important at 625 nm although the amount of excess light is small compared to the other wavelengths.

Two-Level Block Encoding for Digital Transmission

Communication channels with no transmission at dc require data encoding to eliminate dc from the signal. Many coding schemes have been developed for this purpose. The scheme discussed here uses code blocks with equal numbers of plus and minus pulses, according to a previously published algorithm. This code is compared to a three-level bipolar coding scheme in terms of spectral characteristics, efficiency, crosstalk noise, power requirements, and hardware requirements. Circuitry is described for coders, decoders, and repeaters. Extensions to other code types are discussed briefly.