HyScreen: A Ground-Based Imaging System for High-Resolution Red and Far-Red Solar-Induced Chlorophyll Fluorescence.

Solar-induced chlorophyll fluorescence (SIF) is used as a proxy of photosynthetic efficiency. However, interpreting top-of-canopy (TOC) SIF in relation to photosynthesis remains challenging due to the distortion introduced by the canopy's structural effects (i.e., fluorescence re-absorption, sunlit-shaded leaves, etc.) and sun-canopy-sensor geometry (i.e., direct radiation infilling). Therefore, ground-based, high-spatial-resolution data sets are needed to characterize the described effects and to be able to downscale TOC SIF to the leafs where the photosynthetic processes are taking place. We herein introduce HyScreen, a ground-based push-broom hyperspectral imaging system designed to measure red (F687) and far-red (F760) SIF and vegetation indices from TOC with single-leaf spatial resolution. This paper presents measurement protocols, the data processing chain and a case study of SIF retrieval. Raw data from two imaging sensors were processed to top-of-canopy radiance by dark-current correction, radiometric calibration, and empirical line correction. In the next step, the improved Fraunhofer line descrimination (iFLD) and spectral-fitting method (SFM) were used for SIF retrieval, and vegetation indices were calculated. With the developed protocol and data processing chain, we estimated a signal-to-noise ratio (SNR) between 50 and 200 from reference panels with reflectance from 5% to 95% and noise equivalent radiance (NER) of 0.04 (5%) to 0.18 (95%) mW m-2 sr-1 nm-1. The results from the case study showed that non-vegetation targets had SIF values close to 0 mW m-2 sr-1 nm-1, whereas vegetation targets had a mean F687 of 1.13 and F760 of 1.96 mW m-2 sr-1 nm-1 from the SFM method. HyScreen showed good performance for SIF retrievals at both F687 and F760; nevertheless, we recommend further adaptations to correct for the effects of noise, varying illumination and sensor optics. In conclusion, due to its high spatial resolution, Hyscreen is a promising tool for investigating the relationship between leafs and TOC SIF as well as their relationship with plants' photosynthetic capacity.

A highly sensitive and multiplexed method for focused transcript analysis.

We describe a novel, multiplexed method for focused transcript analysis of tens to hundreds of genes. In this method TRAC (transcript analysis with aid of affinity capture) mRNA targets, a set of amplifiable detection probes of distinct sizes and biotinylated oligo(dT) capture probe are hybridized in solution. The formed sandwich hybrids are collected on magnetic streptavidin-coated microparticles and washed. The hybridized probes are eluted, optionally amplified by a PCR using a universal primer pair and detected with laser-induced fluorescence and capillary electrophoresis. The probes were designed by using a computer program developed for the purpose. The TRAC method was adapted to 96-well format by utilizing an automated magnetic particle processor. Here we demonstrate a simultaneous analysis of 18 Saccharomyces cerevisiae transcripts from two experimental conditions and show a comparison with a qPCR system. The sensitivity of the method is significantly increased by the PCR amplification of the hybridized and eluted probes. Our data demonstrate a bias-free use of at least 16 cycles of PCR amplification to increase probe signal, allowing transcript analysis from 2.5 ng of the total mRNA sample. The method is fast and simple and avoids cDNA conversion. These qualifications make it a potential, new means for routine analysis and a complementing method for microarrays and high density chips.

Rapid and multiplexed transcript analysis of microbial cultures using capillary electophoresis-detectable oligonucleotide probe pools.

A rapid assay for multiplex transcript analysis based on solution hybridization with pools of oligonucleotide probes was developed. In this assay called TRAC (transcript analysis with aid of affinity capture) the mRNAs to be studied are hybridized with gene-specific detection probe pools and biotinylated oligo(dT) and captured on streptavidin-coated magnetic particles. Unbound sample material and nonspecifically bound detection probes are removed and the target-specific probes are eluted and detected by capillary electrophoresis. Simultaneous treatment of 96 samples was automated using a magnetic bead particle processor. The assay enabled detection of in vitro transcribed RNA at the level of 30 amol (20 pg) and over a 300-fold linear range. Besides extracted RNA, crude cell lysates were directly used as samples. The assay was used for transcriptional analysis of selected mRNAs in the filamentous fungus Trichoderma reesei in two experimental conditions. TRAC analysis was highly reproducible, providing expression results that were consistent with conventional Northern blot analysis. The whole procedure starting from sample collecting can be carried out in 2 h, making this assay suitable for high-throughput analysis of a limited set of mRNAs e.g. in gene expression monitoring of production organism in microbial bioprocesses.

Light transmission through a high index dielectric hole in a metal film surrounded by surface corrugations.

We analyze transmission of a normally incident plane wave through a 100nm diameter hole in a silver film that is filled with a high index dielectric and is surrounded by 300nm wide surface grooves. Specifically, we study the dependency of the transmission efficiency on the number of grooves, groove depth, and the horizontal distance between the groove and the central hole. We observe that the investigated structure exhibits over five orders of magnitude larger transmission efficiency versus a single hole without the dielectric filling.

Multiplexed quantification of bacterial 16S rRNA by solution hybridization with oligonucleotide probes and affinity capture.

Multiplexed and quantitative analysis of nucleic acid sequences in complex mixtures is essential in various applications of microbiological research. We have developed a method based on solution hybridization between biotinylated nucleic acid targets and multiple fluorophore-labeled oligonucleotide probes of distinct sizes. The biotin-nucleic acid-probe complexes are captured on magnetic streptavidin-coated microparticles and washed. The hybridized probes are eluted and their identity and quantity are determined by capillary electrophoresis. The signal intensities of the recorded probes correspond to the amount of target nucleic acid in the mixture, and the size indicates the target. Based on this principle and 16S rRNA-specific oligonucleotide probes, we set up an application for the relative quantification of different groups of clostridia and related organisms in a mixed bacterial population. The lower detection limit is 0.05 ng of total RNA and the linear range of measurement is 10(2). The method allowed accurate and highly repeatable quantification of the proportion of clostridia in human feces. Further, we discuss other applications of the method such as quantitative transcriptional analysis of eukaryotic microorganisms, which can be performed without conversion of mRNA to cDNA.

Light transmission through a high index dielectric-filled sub-wavelength hole in a metal film.

We investigate transmission of a normally incident, linearly polarized plane wave through a circular sub-wavelength hole in a metal film filled by a high index dielectric medium. We demonstrate for the first time that the trans-mission efficiency of such holes exhibits a Fabry-Pérot like behaviour versus thickness of the metal film, similar to that exhibited by sub-wavelength slits in metal films illuminated by TM-polarized plane waves. We show that by reducing the imaginary part of the propagation constant of the hybrid HE11 mode and by fortifying the Fabry-Pérot resonance, the high index dielectric filling can greatly enhance light transmission through a circular sub-wavelength hole.

Assigning probes into a small number of pools separable by electrophoresis.

MOTIVATION: Measuring transcriptional expression levels (transcriptional profiling) has become one of the most important methods in functional genomics. Still, new measuring methods are needed to obtain more reliable, quantitative data about transcription on a genomic scale. In this paper we concentrate on certain computational optimization problems arising in the design of one such novel method. From a computational point of view the key feature of the new method is that the hybridized probes are distinguished from each other based on their different size. Therefore the probes have to be assigned into pools such that the probes in the same pool have unique sizes different enough from each other. Identification of expressed RNA is given by probe pool and probe size while quantification is given by the label of the probe, e.g. fluorescence intensity. RESULTS: We show how to computationally find the probes and assign them into pools for a whole genome such that (i) each gene has a specific probe suitable for amplification and hybridization, and (ii) the expression level measurement can be done in a minimal number of pools separable by electrophoresis in order to minimize the total experiment cost of the measurement. Our main result is a polynomial-time approximation algorithm for assigning the probes into pools. We demonstrate the feasibility of the procedure by selecting probes for the yeast genome and assigning them into less than 100 pools. The probe sequences and their assignment into pools are available for academic research on request from the authors.

Numerical study of near-field writing on a phase-change optical disk.

Absorption in the phase-change layer of an optical disk located in the near field of a Fabry-Perot laser diode is studied with a combination of finite-difference time domain (FDTD) analysis and a phenomenological laser model that predicts the operational characteristics of a laser diode. Some numerical simulations are performed and results are presented. In addition, the combined FDTD/laser-simulation model is described briefly.