A kV-MV approach to CBCT metal artifact reduction using multi-layer MV-CBCT.

Objective. To demonstrate that complete cone beam CT (CBCT) scans from both MV-energy and kV-energy LINAC sources can reduce metal artifacts in radiotherapy guidance, while maintaining standard-of-care x-ray doses levels.Approach. MV-CBCT and kV-CBCT scans are acquired at half normal dose. The impact of lowered dose on MV-CBCT data quality is mitigated by the use of a 4-layer MV-imager prototype and reduced LINAC energy settings (2.5 MV) to improve photon capture. Additionally, the MV-CBCT is used to determine the 3D position and pose of metal implants, which in turn is used to guide model-based poly-energetic correction and interleaving of the kV-CBCT and MV-CBCT data. Certain edge-preserving regularization steps incorporated into the model-based correction algorithm further reduce MV data noise.Main results. The method was tested in digital phantoms and a real pelvis phantom with large 2.5″ spherical inserts, emulating hip replacements of different materials. The proposed method demonstrated an appealing compromise between the high contrast of kV-CBCT and low artifact content of MV-CBCT. Contrast-to-noise improved 3-fold compared to MV-CBCT with a clinical 1-layer architecture at matched dose (37 mGy) and edge blur levels. Visual delineation of the bladder and prostate improved noteably over kV- or MV-CBCT alone.Significance. The proposed method demonstrates that a full MV-CBCT scan can be combined with kV-CBCT to reduce metal artifacts without resorting to complicated beam collimation strategies to limit the MV-CBCT dose contribution. Additionally, significant improvements in CNR can be achieved as compared to metal artifact reduction through current clinical MV-CBCT practices.

Monte Carlo model of a prototype flat-panel detector for multi-energy applications in radiotherapy.

BACKGROUND: The incorporation of multi-energy capabilities into radiotherapy flat-panel detectors offers advantages including enhanced soft tissue visualization by reduction of signal from overlapping anatomy such as bone in 2D image projections; creation of virtual monoenergetic images for 3D contrast enhancement, metal artefact reduction and direct acquisition of relative electron density. A novel dual-layer on-board imager offering dual energy processing capabilities is being designed. As opposed to other dual-energy implementation techniques which require separate acquisition with two different x-ray spectra, the dual-layer detector design enables simultaneous acquisition of high and low energy images with a single exposure. A computational framework is required to optimize the design parameters and evaluate detector performance for specific clinical applications. PURPOSE: In this study, we report on the development of a Monte Carlo (MC) model of the imager including model validation. METHODS: The stack-up of the dual-layer imager (DLI) was implemented in GEANT4 Application for Tomographic Emission (GATE). The DLI model has an active area of 43×43 cm2 , with top and bottom Cesium Iodide (CsI) scintillators of 600 and 800 µm thickness, respectively. Measurement of spatial resolution and imaging of dedicated multi-material dual-energy (DE) phantoms were used to validate the model. The modulation transfer function (MTF) of the detector was calculated for a 120 kVp x-ray spectrum using a 0.5 mm thick tantalum edge rotated by 2.5o . For imaging validation, the DE phantom was imaged using a 140 kVp x-ray spectrum. For both validation simulations, corresponding measurements were done using an initial prototype of the imager. Agreement between simulations and measurement was assessed using normalized root mean square error (NRMSE) and 1D profile difference for the MTF and phantom images respectively. Further comparison between measurement and simulation was made using virtual monoenergetic images (VMIs) generated from basis material images derived using precomputed look-up tables. RESULTS: The MTF of the bottom layer of the dual-layer model shows values decreasing more quickly with spatial frequency, compared to the top layer, due to the thicker bottom scintillator thickness and scatter from the top layer. A comparison with measurement shows NRMSE of 0.013 and 0.015 as well as identical MTF50 of 0.8 mm1 and 1.0 mm1 for the top and bottom layer respectively. For the DE imaging of the DE-phantom, although a maximum deviation of 3.3% is observed for the 10 mm aluminum and Teflon inserts at the top layer, the agreement for all other inserts is less than 2.2% of the measured value at both layers. Material decomposition of simulated scatter-free DE images gives an average accuracy in PMMA and aluminum composition of 4.9% and 10.3% for 11-30 mm PMMA and 1-10 mm aluminum objects respectively. A comparison of decomposed values using scatter containing measured and simulated DE images shows good agreement within statistical uncertainty. CONCLUSION: Validation using both MTF and phantom imaging shows good agreement between simulation and measurements. With the present configuration of the digital prototype, the model can generate material decomposed images and virtual monoenergetic images.

Frequency-dependent optimal weighting approach for megavoltage multilayer imagers.

Multi-layer imaging (MLI) devices improve the detective quantum efficiency (DQE) while maintaining the spatial resolution of conventional mega-voltage (MV) x-ray detectors for applications in radiotherapy. To date, only MLIs with identical detector layers have been explored. However, it may be possible to instead use different scintillation materials in each layer to improve the final image quality. To this end, we developed and validated a method for optimally combining the individual images from each layer of MLI devices that are built with heterogeneous layers. Two configurations were modeled within the GATE Monte Carlo package by stacking different layers of a terbium doped gadolinium oxysulfide Gd2O2S:Tb (GOS) phosphor and a LKH-5 glass scintillator. Detector response was characterized in terms of the modulation transfer function (MTF), normalized noise power spectrum (NNPS) and DQE. Spatial frequency-dependent weighting factors were then analytically derived for each layer such that the total DQE of the summed combination image would be maximized across all spatial modes. The final image is obtained as the weighted sum of the sub-images from each layer. Optimal weighting factors that maximize the DQE were found to be the quotient of MTF and NNPS of each layer in the heterogeneous MLI detector. Results validated the improvement of the DQE across the entire frequency domain. For the LKH-5 slab configuration, DQE(0) increases between 2%-3% (absolute), while the corresponding improvement for the LKH-5 pixelated configuration was 7%. The performance of the weighting method was quantitatively evaluated with respect to spatial resolution, contrast-to-noise ratio (CNR) and signal-to-noise ratio (SNR) of simulated planar images of phantoms at 2.5 and 6 MV. The line pair phantom acquisition exhibited a twofold increase in CNR and SNR, however MTF was degraded at spatial frequencies greater than 0.2 lp mm-1. For the Las Vegas phantom, the weighting improved the CNR by around 30% depending on the contrast region while the SNR values are higher by a factor of 2.5. These results indicate that the imaging performance of MLI systems can be enhanced using the proposed frequency-dependent weighting scheme. The CNR and SNR of the weighted combined image are improved across all spatial scales independent of the detector combination or photon beam energy.

Abbreviated on-treatment CBCT using roughness penalized mono-energization of kV-MV data and a multi-layer MV imager.

Simultaneous acquisition of cone beam CT (CBCT) projections using both the kV and MV imagers of an image guided radiotherapy system reduces set-up scan times-a benefit to lung cancer radiation oncology patients-but increases noise in the 3D reconstruction. In this article, we present a kV-MV scan time reduction technique that uses two noise-reducing measures to achieve superior performance. The first is a high-DQE multi-layer MV imager prototype. The second is a beam hardening correction algorithm which combines poly-energetic modeling with edge-preserving, regularized smoothing of the projections. Performance was tested in real acquisitions of the Catphan 604 and a thorax phantom. Percent noise was quantified from voxel values in a soft tissue volume of interest (VOI) while edge blur was quantified from a VOI straddling a boundary between air and soft material. Comparisons in noise/resolution performance trade-off were made between our proposed approach, a dose-equivalent kV-only scan, and a kV-MV reconstruction technique previously published by Yinet al(2005Med. Phys.329). The proposed technique demonstrated lower noise as a function of spatial resolution than the baseline kV-MV method, notably a 50% noise reduction at typical edge blur levels. Our proposed method also exhibited fainter non-uniformity artifacts and in some cases superior contrast. Overall, we find that the combination of a multi-layer MV imager, acquiring at a LINAC source energy of 2.5 MV, and a denoised beam hardening correction algorithm enables noise, resolution, and dose performance comparable to standard kV-imager only set-up CBCT, but with nearly half the gantry rotation time.

GPU-accelerated Monte Carlo simulation of MV-CBCT.

Monte Carlo simulation (MCS) is one of the most accurate computation methods for dose calculation and image formation in radiation therapy. However, the high computational complexity and long execution time of MCS limits its broad use. In this paper, we present a novel strategy to accelerate MCS using a graphic processing unit (GPU), and we demonstrate the application in mega-voltage (MV) cone-beam computed tomography (CBCT) simulation. A new framework that generates a series of MV projections from a single simulation run is designed specifically for MV-CBCT acquisition. A Geant4-based GPU code for photon simulation is incorporated into the framework for the simulation of photon transport through a phantom volume. The FastEPID method, which accelerates the simulation of MV images, is modified and integrated into the framework. The proposed GPU-based simulation strategy was tested for its accuracy and efficiency in a Catphan 604 phantom and an anthropomorphic pelvis phantom with beam energies at 2.5 MV, 6 MV, and 6 MV FFF. In all cases, the proposed GPU-based simulation demonstrated great simulation accuracy and excellent agreement with measurement and CPU-based simulation in terms of reconstructed image qualities. The MV-CBCT simulation was accelerated by factors of roughly 900-2300 using an NVIDIA Tesla V100 GPU card against a 2.5 GHz AMD Opteron™ Processor 6380.

A rapid, accurate image simulation strategy for mega-voltage cone-beam computed tomography.

Intensive computation time is required to simulate images of electronic portal imaging device (EPID) using Monte Carlo (MC) technique, limiting the development of applications associated with EPID, such as mega-voltage cone-beam computed tomography (MV-CBCT). In this study, a fast, accurate simulation strategy for MV-CBCT utilizing the FastEPID technique has been developed and validated. During FastEPID simulation, photon detection was determined by pre-calculated photon energy deposition efficiency (η) and particle transport within the EPID was replaced with a pre-calculated optical photon spread function. This method is capable of reducing the time required for EPID image simulation by a factor of 90-140, without compromising image quality. MV-CBCT images reconstructed from the FastEPID simulated projections have been validated against measurement in terms of mean Hounsfield unit (HU), noise, and cupping artifact. These images were obtained with both a Catphan 604 phantom and an anthropomorphic pelvis phantom, under treatment beam energies of 2.5 MV, 6 MV, and 6 MV flattening filter free. The agreement between measurement and simulation was excellent in all cases. This novel strategy was capable of reducing the run time of a full scan simulation of MV-CBCT performed on a CPU cluster to a matter of hours, rather than weeks or months required by a conventional approach. Multiple applications associated with MV-CBCT (e.g. imager design optimization) are anticipated to gain from the implementation of this novel simulation strategy.

Low-dose megavoltage cone-beam computed tomography using a novel multi-layer imager (MLI).

PURPOSE: The feasibility of low-dose megavoltage cone-beam acquisition (MVCBCT) using a novel, high detective quantum efficiency (DQE) multi-layer imager (MLI) was investigated. The aim of this work was to reconstruct MVCBCT images using the MLI at different total dose levels, and assess Hounsfield Unit (HU) accuracy, noise and contrast-to-noise ratio (CNR) for low-dose megavoltage cone-beam acquisition. METHODS: The MLI has four stacked layers; each layer contains a combination of copper filter/converter, gadolinium oxysulfide (GOS) scintillator and a-Si detector array. In total, 720 projections of a CATPHAN® phantom were acquired over 360° at 2.5, 6, and 6 MV flattening filter free (FFF) beam energies on a Varian TrueBeam LINAC. The dose per projection was 0.01, 0.0167, and 0.05 MU for 2.5, 6, and 6 MV FFF, respectively. MVCBCT images were reconstructed with varying numbers of projections to provide a range of doses for evaluation. Hounsfield Unit uniformity, accuracy, noise and CNR were estimated. Improvements were quantified relative to the standard AS1200 single-layer imager. RESULTS: Average HU uniformity for the MLI reconstructions was within a range of 95%-99% for all of the energies studied. Relative electron density estimation from HU values was within 0.4% ± 1.8% from nominal values. The CNR for MVCBCT based on MLI projections was 2-4× greater than from AS1200 projections. The 2.5 MV beam acquisition with the MLI exhibited the lowest noise and the best balance between CNR and dose for low-dose reconstructions. CONCLUSIONS: Megavoltage cone-beam acquisition imaging with a novel MLI prototype mounted on a clinical linear accelerator demonstrated substantial improvement over the standard AS1200 EPID. Further optimization of MVCBCT reconstruction, particularly for 2.5 MV acquisitions, will improve image metrics. Overall, the MLI improves CNR at substantially lower doses than currently required by conventional detectors. This new high DQE detector could provide high-quality MVCBCT at clinically acceptable doses.

A novel method for fast image simulation of flat panel detectors.

We have developed a novel method for fast image simulation of flat panel detectors, based on the photon energy deposition efficiency and the optical spread function (OSF). The proposed method, FastEPID, determines the photon detection using photon energy deposition and replaces particle transport within the detector with precalculated OSFs. The FastEPID results are validated against experimental measurement and conventional Monte Carlo simulation in terms of modulation transfer function (MTF), signal-to-noise ratio (SNR), contrast-to-noise ratio (CNR), contrast, and relative difference of pixel value, obtained with a slanted slit image, Las Vegas phantom, and anthropomorphic pelvis phantom. Excellent agreement is observed between simulation and measurement in all cases. Without degrading image quality, the FastEPID method is capable of reducing simulation time up to a factor of 150. Multiple applications, such as imager design optimization for planar and volumetric imaging, are expected to benefit from the implementation of the FastEPID method.

Characterizing a novel scintillating glass for application to megavoltage cone-beam computed tomography.

PURPOSE: The purpose of this study was to evaluate the performance of a prototype electric portal imaging device (EPID) with a high detective quantum efficiency (DQE) scintillator, LKH-5. Specifically, image quality in context of both planar and megavoltage (MV) cone-beam computed tomography (CBCT) is analyzed. METHODS: Planar image quality in terms of modulation transfer function (MTF), noise power spectrum (NPS), and DQE are measured and compared to an existing EPID (AS-1200) using the 6 MV beamline for a Varian TrueBeam linac. Imager performance is contextualized for three-dimensional (3D), MV-CBCT performance by measuring imager lag and analyzing the expected degradation of the DQE as a function of dose. Finally, comparisons between reconstructed images of the Catphan phantom in terms of qualitative quality and signal-difference-to-noise ratio (SDNR) are made for 6 MV images using both conventional and LKH-5 EPIDs as well as for the kilovoltage (kV) on-board imager (OBI). RESULTS: Analysis of the NPS reveals linearity at all measured doses using the prototype LKH-5 detector. While the first zero of the MTF is much lower for the LKH-5 detector than the conventional EPID (0.6 cycles/mm vs 1.6 cycles/mm), the normalized NPS (NNPS) multiplied by total quanta (qNNPS) of the LKH-5 detector is roughly a factor of seven to eight times lower, yielding a DQE(0) of approximately 8%. First, second, and third frame lag were measured at approximately 23%, 5%, and 1%, respectively, although no noticeable image artifacts were apparent in reconstructed volumes. Analysis of low-dose performance reveals that DQE(0) remains at 80% of its maximum value at a dose as low as 7.5 × 10-6  MU. For a 400 projection technique, this represents a total scan dose of 0.0030 MU, suggesting that if imaging doses are increased to a value typical of kV-CBCT scans (~2.7 cGy), the LKH-5 detector will retain quantum noise limited performance. Finally, comparing Catphan scans, the prototype detector exhibits much lower image noise than the conventional EPID, resulting in improved small object representation. Furthermore, SDNR of H2 O and polystyrene cylinders improved from -1.95 and 2.94 to -15 and 18.7, respectively. CONCLUSIONS: Imaging performance of the prototype LKH-5 detector was measured and analyzed for both planar and 3D contexts. Improving noise transfer of the detector results in concurrent improvement of DQE(0). For 3D imaging, temporal characteristics were adequate for artifact-free performance and at relevant doses, the detector retained quantum noise limited performance. Although quantitative MTF measurements suggest poorer resolution, small object representation of the prototype imager is qualitatively improved over the conventional detector due to the measured reduction in noise.

Feasibility of closed-MLC tracking using high sensitivity and multi-layer electronic portal imagers.

In radiation therapy, improvements in treatment conformality are often limited by movement of target tissue. To better treat the target, tumor tracking strategies involving beam's-eye-view (BEV) have been explored. However, localization surrogates like implanted fiducial markers may sometimes leave the field-of-view (FOV), as defined by the linear accelerator (LINAC) multi-leaf collimator (MLC). Radiation leakage through the MLC has been measured previously at approximately 1%-2%. High sensitivity prototype detectors imagers may improve the ability to visualize objects outside of the MLC FOV during treatment. The present study presents a proof-of-concept for tracking fiducial markers outside the MLC FOV by employing high sensitivity detectors using a high-efficiency, prototype scintillating glass called LKH-5 and also investigates the impact of multi-layer imager (MLI) architecture. It was found that by improving the detector efficiency, using either of these methods results in a reduction of dose required for fiducial marker visibility. Further, image correction by a rectangular median filter will improve fiducial marker representation in the MLC blocked images. Quantified by measuring the peak-to-sidelobe ratio (PSR) of the normalized cross correlation (NCC) between a template of the fiducial marker with the blocked MLC acquisition, visibility has been found at a threshold of roughly 5 for all configurations with a 3 × 3 cm2 ROI. For typical gadolinium oxysulfide (GOS) detectors in single and simulated 4-layer configurations, the minimum dose required for visualization was 20 and 10 MU, respectively. For LKH-5 detectors in single and simulated 4-layer configurations, this minimum dose was reduced to 4 and 2 MU, respectively. With a 6 MV flattening filter free (FFF) beam dose rate of 1400 MU min-1, the maximum detector frame rate while maintaining fiducial visibility is approximately 12 fps for a 4-layer LKH-5 configuration.

Physics considerations in MV-CBCT multi-layer imager design.

Megavoltage (MV) cone-beam computed tomography (CBCT) using an electronic portal imaging (EPID) offers advantageous features, including 3D mapping, treatment beam registration, high-z artifact suppression, and direct radiation dose calculation. Adoption has been slowed by image quality limitations and concerns about imaging dose. Developments in imager design, including pixelated scintillators, structured phosphors, inexpensive scintillation materials, and multi-layer imager (MLI) architecture have been explored to improve EPID image quality and reduce imaging dose. The present study employs a hybrid Monte Carlo and linear systems model to determine the effect of detector design elements, such as multi-layer architecture and scintillation materials. We follow metrics of image quality including modulation transfer function (MTF) and noise power spectrum (NPS) from projection images to 3D reconstructions to in-plane slices and apply a task based figure-of-merit, the ideal observer signal-to-noise ratio (d') to determine the effect of detector design on object detectability. Generally, detectability was limited by detector noise performance. Deploying an MLI imager with a single scintillation material for all layers yields improvement in noise performance and d' linear with the number of layers. In general, improving x-ray absorption using thicker scintillators results in improved DQE(0). However, if light yield is low, performance will be affected by electronic noise at relatively high doses, resulting in rapid image quality degradation. Maximizing image quality in a heterogenous MLI detector (i.e. multiple different scintillation materials) is most affected by limiting total noise. However, while a second-order effect, maximizing total spatial resolution of the MLI detector is a balance between the intensity contribution of each layer against its individual MTF. So, while a thinner scintillator may yield a maximal individual-layer MTF, its quantum efficiency will be relatively low in comparison to a thicker scintillator and thus, intensity contribution may be insufficient to noticeably improve the total detector MTF.

Multi-layer imager design for mega-voltage spectral imaging.

The architecture of multi-layer imagers (MLIs) can be exploited to provide megavoltage spectral imaging (MVSPI) for specific imaging tasks. In the current work, we investigated bone suppression and gold fiducial contrast enhancement as two clinical tasks which could be improved with spectral imaging. A method based on analytical calculations that enables rapid investigation of MLI component materials and thicknesses was developed and validated against Monte Carlo computations. The figure of merit for task-specific imaging performance was the contrast-to-noise ratio (CNR) of the gold fiducial when the CNR of bone was equal to zero after a weighted subtraction of the signals obtained from each MLI layer. Results demonstrated a sharp increase in the CNR of gold when the build-up component or scintillation materials and thicknesses were modified. The potential for low-cost, prompt implementation of specific modifications (e.g. composition of the build-up component) could accelerate clinical translation of MVSPI.

Leveraging multi-layer imager detector design to improve low-dose performance for megavoltage cone-beam computed tomography.

While megavoltage cone-beam computed tomography (CBCT) using an electronic portal imaging device (EPID) provides many advantages over kilovoltage (kV) CBCT, clinical adoption is limited by its high doses. Multi-layer imager (MLI) EPIDs increase DQE(0) while maintaining high resolution. However, even well-designed, high-performance MLIs suffer from increased electronic noise from each readout, degrading low-dose image quality. To improve low-dose performance, shift-and-bin addition (ShiBA) imaging is proposed, leveraging the unique architecture of the MLI. ShiBA combines hardware readout-binning and super-resolution concepts, reducing electronic noise while maintaining native image sampling. The imaging performance of full-resolution (FR); standard, aligned binned (BIN); and ShiBA images in terms of noise power spectrum (NPS), electronic NPS, modulation transfer function (MTF), and the ideal observer signal-to-noise ratio (SNR)-the detectability index (d')-are compared. The FR 4-layer readout of the prototype MLI exhibits an electronic NPS magnitude 6-times higher than a state-of-the-art single layer (SLI) EPID. Although the MLI is built on the same readout platform as the SLI, with each layer exhibiting equivalent electronic noise, the multi-stage readout of the MLI results in electronic noise 50% higher than simple summation. Electronic noise is mitigated in both BIN and ShiBA imaging, reducing its total by ~12 times. ShiBA further reduces the NPS, effectively upsampling the image, resulting in a multiplication by a sinc2 function. Normalized NPS show that neither ShiBA nor BIN otherwise affects image noise. The LSF shows that ShiBA removes the pixilation artifact of BIN images and mitigates the effect of detector shift, but does not quantifiably improve the MTF. ShiBA provides a pre-sampled representation of the images, mitigating phase dependence. Hardware binning strategies lower the quantum noise floor, with 2 × 2 implementation reducing the dose at which DQE(0) degrades by 10% from 0.01 MU to 0.004 MU, representing 20% improvement in d'.

Spectral imaging using clinical megavoltage beams and a novel multi-layer imager.

We assess the feasibility of clinical megavoltage (MV) spectral imaging for material and bone separation with a novel multi-layer imager (MLI) prototype. The MLI provides higher detective quantum efficiency and lower noise than conventional electronic portal imagers. Simulated experiments were performed using a validated Monte Carlo model of the MLI to estimate energy absorption and energy separation between the MLI components. Material separation was evaluated experimentally using solid water and aluminum (Al), copper (Cu) and gold (Au) for 2.5 MV, 6 MV and 6 MV flattening filter free (FFF) clinical photon beams. An anthropomorphic phantom with implanted gold fiducials was utilized to further demonstrate bone/gold separation. Weighted subtraction imaging was employed for material and bone separation. The weighting factor (w) was iteratively estimated, with the optimal w value determined by minimization of the relative signal difference ([Formula: see text]) and signal-difference-to-noise ratio (SDNR) between material (or bone) and the background. Energy separation between layers of the MLI was mainly the result of beam hardening between components with an average energy separation between 34 and 47 keV depending on the x-ray beam energy. The minimum average energy of the detected spectrum in the phosphor layer was 123 keV in the top layer of the MLI with the 2.5 MV beam. The w values that minimized [Formula: see text] and SDNR for Al, Cu and Au were 0.89, 0.76 and 0.64 for 2.5 MV; for 6 MV FFF, w was 0.98, 0.93 and 0.77 respectively. Bone suppression in the anthropomorphic phantom resulted in improved visibility of the gold fiducials with the 2.5 MV beam. Optimization of the MLI design is required to achieve optimal separation at clinical MV beam energies.

Development and laboratory-scale testing of a fully automated online flow cytometer for drinking water analysis.

Accurate and sensitive online detection tools would benefit both fundamental research and practical applications in aquatic microbiology. Here, we describe the development and testing of an online flow cytometer (FCM), with a specific use foreseen in the field of drinking water microbiology. The system incorporated fully automated sampling and fluorescent labeling of bacterial nucleic acids with analysis at 5-min intervals for periods in excess of 24 h. The laboratory scale testing showed sensitive detection (< 5% error) of bacteria over a broad concentration range (1 × 10(3) -1 × 10(6) cells mL(-1) ) and particularly the ability to track both gradual changes and dramatic events in water samples. The system was tested with bacterial pure cultures as well as indigenous microbial communities from natural water samples. Moreover, we demonstrated the possibility of using either a single fluorescent dye (e.g., SYBR Green I) or a combination of two dyes (SYBR Green I and Propidium Iodide), thus broadening the application possibilities of the system. The online FCM approach described herein has considerable potential for routine and continuous monitoring of drinking water, optimization of specific drinking water processes such as biofiltration or disinfection, as well as aquatic microbiology research in general.

Real-time on-line flow cytometry for bioprocess monitoring.

As the understanding of variation is the key to a good process and product quality one should pay attention to dynamics on the single-cell level. The basic idea of this approach was to qualify and quantify variations on the single-cell level during bioreactor cultivations by monitoring the expression of an eGFP tagged target protein (human membrane protein) using fully automated real-time, flow injection flow cytometry (FI-FCM). The FI-FCM system consists of a sampling- and defoaming- as well as of a dilution-section. It allows a very short monitoring interval (5 min) and is able to dilute the reactor sample by a factor ranging up to more than 10,000. In bioreactor cultivations of recombinant Pichia pastoris expressing the eGFP tagged target protein, high correlations (R(2)≥ 0.97) between the FI-FCM fluorescent signal and other, however, population-averaged fluorescence signals (off-line fluorescence, in situ fluorescence probe) were obtained. FI-FCM is the only method able to distinguish between few cells with high fluorescence and many cells with low fluorescence intensity and proved that cells differ significantly from each other within the population during bioreactor cultivations. Single-cell fluorescence was distributed over a broad range within the cell population. These distributions strongly suggest that (a) the AOX-I promoter is leaky and (b) a fraction of the population is able to express more protein of interest within shorter time and (c) a fraction of the population does not express the fusion protein at all. These findings can help in the selection of high producing, stable strains. To show the platform-independency of the system, it has successfully been tested during bioreactor cultivations of three different strains (P. pastoris, Saccharomyces cerevisiae, Escherichia coli). Along with its applications in PAT, the FI-FCM could be used as a platform-independent (prokaryotes and eukaryotes) method in various other applications; for example in the closed-loop-control of bioprocesses using different kinds of fluorescent reporters, (waste- and drinking-) water analysis, clone selection in combination with FACS or even for surgery applications.

Cobalt-catalyzed hydrohydrazination of dienes and enynes: access to allylic and propargylic hydrazides.

[reaction: see text] The cobalt-catalyzed hydrohydrazination reaction of dienes and enynes is presented. Allylic and propargylic hydrazines were obtained in synthetically useful yields (allylic amines, 60-90%; propargylic amines, 47-83%) and good chemo- and regioselectivity.