利用调制荧光仪在线监测叶绿素荧光

介绍了利用便携式调制荧光仪PAM-2100在线监测叶绿素荧光的技术。该技术不影响植物的自然光合状态,司以在线监测Ft、F′m、Y、rETR、qP、qN或NPQ、PAR和叶温等指标。以凤眼莲为例进行了在线监测,每隔5min监测一次,共进行了225min的监测。结果表明叶绿索荧光参数的变化依赖于PAR的变化。Ft、rETR、qN和NPQ的变化与PAR的变化趋势一致,F′m、Y和qP的变化与PAR的变化相反。通过对风眼莲的在线监测,说明该技术是可靠的,具有简单、快速、灵敏等特点。随着新型调制荧光仪的出现,该技术可能在植物生态学领域得到广泛应用。     

In situ butanol recovery from Clostridium acetobutylicum fermentations by expanded bed adsorption

Although butanol is a promising biofuel, its fermentative production suffers from inhibition caused by end product toxicity. The in situ removal of butanol from cultures via expanded bed adsorption offers an effective strategy for mitigating the effects of product toxicity while eliminating the need to clarify cultures via microfiltration. The hydrophobic polymer resin Dowex Optipore L‐493 was found to be both an effective butanol adsorbent and suitable for use in expanded bed adsorption. Recirculation rates through the adsorption column were strongly correlated with and ultimately controlled rates of butanol uptake from the media which, reaching as high as 41.1 g/L h, easily exceed those of its production in a typical fermentation. Vacuum application with vapor collection was found to be an effective means of adsorbent regeneration, with an average of 81% butanol recovery possible, with butanol concentrations in the cold trap reaching as high as 85.8 g/L. Integration of expanded bed adsorption with a fed‐batch Clostridium acetobutylicum ATCC 824 fermentation and its continuous operation for 38.5 h enabled the net production (i.e., in solution and adsorbed) of butanol and total solvent products at up to 27.2 and 40.7 g/L of culture, respectively, representing 2.2‐ and 2.3‐fold improvements over conventional batch culture. While adsorbent biofouling was found to be minimal, further investigation of biofouling in longer‐term studies will provide useful and further insight regarding the robustness of the process strategy. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 30:68–78, 2014     

In situ monitoring of polymerase extension rate and adaptive feedback control of PCR by using fluorescence measurements

Real time PCR detection systems based on fluorescence detection from intercalating dyes (such as SYBR Green I) typically take only single point measurements during every cycle to quantify the amplification. In this process key information about enzymatic kinetics is lost. In this work we measure SYBR Green I fluorescence intensity every 0.5 s within a cycle during PCR in polypropylene tubes. We observe that the intensity during the extension cycle increases while the template is being extended. Results obtained for different lengths are used to estimate an in vitro polymerase activity rate of Thermus aquaticus and Thermus brockianus. An important practical consequence of this result is that the extension time of each PCR cycle can be individually optimized while the reaction is in progress. We demonstrate this idea of adaptive feedback control and show that the total number of cycles and total time required to reach maximum fluorescence is reduced as compared to conventional PCR.     

In situ background estimation in quantitative fluorescence imaging

Fluorescence imaging of bulk-stained tissue is a popular technique for monitoring the activities in a large population of cells. However, a precise quantification of such experiments is often compromised by an ambiguity of background estimation. Although, in single-cell-staining experiments, background can be measured from a neighboring nonstained region, such a region often does not exist in bulk-stained tissue. Here we describe a novel method that overcomes this problem. In contrast to previous methods, we determined the background of a given region of interest (ROI) using the information contained in the temporal dynamics of its individual pixels. Since no information outside the ROI is needed, the method can be used regardless of the staining profile in the surrounding tissue. Moreover, we extend the method to deal with background inhomogeneities within a single ROI, a problem not yet solved by any of the currently available tools. We performed computer simulations to demonstrate the accuracy of our method and give example applications in ratiometric calcium imaging of bulk-stained olfactory bulb slices. Converting the fluorescence signals into [Ca2+] gives resting values consistent with earlier single-cell staining results, and odorant-induced [Ca2+] transients can be quantitatively compared in different cells. Using these examples we show that inaccurate background subtraction introduces large errors (easily in the range of 100%) in the assessment of both resting [Ca2+] and [Ca2+] dynamics. The proposed method allows us to avoid such errors.     

In situ fluorescence labeling of sheep lung microvascular endothelium

Summary Endothelial cells are intimately involved in a variety of biological processes such as inflammatory disorders, wound healing, and tumor invasion. The finding of endothelial heterogeneity in various tissues has led to major efforts to isolate and culture microvascular endothelial cells in human and animal tissue. In this report we have used phosphatidyl ethanolamine (PE)-labeled liposomes to fluorescently label the sheep lung microvasculature in situ. Using normotensive perfusion pressure, the PE-labeled liposomes did not extravasate into extravascular lung tissue. Mechanical and enzymatic digestion of the lung tissue demonstrated that the PE-labeled liposomes provided a stable label of the vascular lining cells during ex vivo processing. After digestion, the overwhelming majority of the fluorescent label appeared in cellular aggregates. Approximately 80% of these cells demonstrated an in vitro phenotype consistent with microvascular endothelium. A novel monoclonal antibody selective for sheep endothelial cells was developed to confirm the presence of lung endothelium in the fluorescently labeled cellular aggregates. We conclude that in situ fluorescence labeling of vascular lining cells provides an anatomic marker for relevant vascular lining cells and an opportunity to study these cells in vitro.     

Enabling PAT in insect cell bioprocesses: In situ monitoring of recombinant adeno-associated virus production by fluorescence spectroscopy

The process analytical technology (PAT) initiative shifted the bioprocess development mindset towards real-time monitoring and control tools to measure relevant process variables online, and acting accordingly when undesirable deviations occur. Online monitoring is especially important in lytic production systems in which released proteases and changes in cell physiology are likely to affect product quality attributes, as is the case of the insect cell-baculovirus expression vector system (IC-BEVS), a well-established system for production of viral vectors and vaccines. Here, we applied fluorescence spectroscopy as a real-time monitoring tool for recombinant adeno-associated virus (rAAV) production in the IC-BEVS. Fluorescence spectroscopy is simple, yet sensitive and informative. To overcome the strong fluorescence background of the culture medium and improve predictive ability, we combined artificial neural network models with a genetic algorithm-based approach to optimize spectra preprocessing. We obtained predictive models for rAAV titer, cell viability and cell concentration with normalized root mean squared errors of 7%, 4%, and 7%, respectively, for leave-one-batch-out cross-validation. Our approach shows fluorescence spectroscopy allows real-time determination of the best time of harvest to maintain rAAV infectivity, an important quality attribute, and detection of deviations from the golden batch profile. This methodology can be applied to other biopharmaceuticals produced in the IC-BEVS, supporting the use of fluorescence spectroscopy as a versatile PAT tool.     

In situ fermentation monitoring with recombinant firefly luciferase

A novel method is described for the on-line determination of viable cell number. It has been tested in fermentations of Escherichia coli. The cells are transfected with the gene for firefly luciferase and fed low levels of luciferin in the medium. The reaction requires ATP, so the nonviable cells cannot produce light. Thus, light production is linear with viable cell density from innoculation through most of exponential growth. The light emitted by these cells is then conducted from the reaction vessel to the light detection equipment by an optical fiber. With the equipment described below, as few as a 10(6) cells/mL, or an OD(600) of 0.004, are easily detectable and concentrations greater than 10(10) cells/mL are well within range. The data are collected by a computer, so adaptation to on-line control applications is straightforward. During lag phase, this method is much more accurate then optical density measurements. At the end of exponential growth, rapid changes in light production mark carbon source depletion and the onset of cell lysis. A simple model accounts for the luciferin used during the fermentation and corrects the light detected to the proper cell density. (c) 1993 John Wiley & Sons, Inc.     

In situ sensor techniques in modern bioprocess monitoring

New reactor concepts as multi-parallel screening systems or disposable bioreactor systems for decentralized and reproducible production increase the need for new and easy applicable sensor technologies to access data for process control. These sophisticated reactor systems require sensors to work with the lowest sampling volumes or, even better, to measure directly in situ, but in situ sensors are directly incorporated into a reactor or fermenter within the sterility barrier and have therefore to stand the sterilization procedures. Consequently, these in situ sensor technologies should enable the measurement of multi-analytes simultaneously online and in real-time at a low price for the robust sensing element. Current research therefore focuses on the implementation of noninvasive spectroscopic and optical technologies, and tries to employ them through fiber optics attached to disposable sensing connectors. Spectroscopic methods reach from ultraviolet to infrared and further comprising fluorescence and Raman spectroscopy. Also, optic techniques like microscopy are adapted for the direct use in bioreactor systems (Ulber et al. in Anal Bioanal Chem 376:342–348, 2003) as well as various electrochemical methods (Joo and Brown in Chem Rev 108:638–651, 2008). This review shows the variety of modern in situ sensing principles in bioprocess monitoring with emphasis on spectroscopic and optical techniques and the progress in the adaption to latest reactor concepts.     

In situ observation of coral recruitment using fluorescence census techniques

A novel method for the observation of recruitment of fluorescent taxa was developed combining fluorescence census techniques with conventional microscopic examination. The new technique was used to observe coral recruitment on natural limestone plates over a period of 4 months on Meras reef, North Sulawesi, Indonesia. During this period, fluorescence photos were taken of each plate on a weekly basis. This allowed for the detailed observation of fluorescent coral recruitment in-situ. After specific time periods, the plates were sampled and the detected recruits were categorized to family level using their skeletal structure. The fluorescence census technique detected 97.6% of all coral recruits bigger than one millimetre in diameter. The diameter measurements of the recruit's skeletons were used to estimate growth rates at family level. Using the photo series method, the time of settlement of fluorescent recruits on the plates was determined. The results showed roughly linear diameter growth of recruits in the first two months. Acroporidae-polyps were 1.14 mm in diameter when they settled and grew at rate of approximately 0.18 mm in diameter per week. Pocilloporidae-polyps settled at approximately 1.14 mm and showed a growth rate of approximately 0.23 mm in diameter per week. Recruits of three families were observed in detail over time on the artificial plates using this method. While recruitment was fairly continuous, an increase in pocilloporid recruits was observed during the month of September. Similar increases were observed for acroporid recruits during the middle of November and the second quarter of December. This method identifies the limits of detection for the fluorescence census technique used and represents a useful method for the temporal fine scale observation of recruitment in situ.     

In situ monitoring of cell death using Raman microspectroscopy

We investigated the use of Raman microspectroscopy to monitor the molecular changes in human lung carcinoma epithelial cells (A549) when cell death was induced by a toxic chemical. We treated A549 cells with 100 microM Triton X-100 and carried out Raman microspectroscopy measurements in parallel with cell viability and DNA integrity assays at time points of 0, 24, 48, and 72 hours. We found that the important biochemical changes taking place during cell death, such as the degradation of proteins, DNA breakdown, and the formation of lipid vesicles, can be detected with Raman microspectroscopy. A decrease in the intensity of the O-P-O stretching Raman peak corresponding to the DNA molecule phosphate-sugar backbone at 788 cm(-1) indicated DNA disintegration, an observation which was confirmed by DNA integrity analysis. We also found a decrease in the intensity of the Raman peaks corresponding to proteins (1005 cm(-1), 1342 cm(-1)) and an increase in the concentration of lipids (1660 cm(-1), 1303 cm(-1)). These changes are the effects of the complex molecular mechanisms during the induction of cell death, such as protein cleavage due to the activation of caspases, followed by DNA fragmentation.     

Plant cell culture monitoring using an in situ multiwavelength fluorescence probe

A multiwavelength fluorescence probe is proposed for in situ monitoring of Eschscholtzia californica and Catharanthus roseus plant cell cultures. The potential of the probe as a tool for real-time estimation of biomass and production in secondary metabolites has been studied. The probe excitation range is 270-550 nm and the emission range is 310-590 nm, with a step of 20 nm for both excitation and emission filters. Many endogenous fluorophores such as NAD(P)H, riboflavins (riboflavin and derivatives such as FMN, FAD), tryptamine and tryptophan, and fluorescent secondary metabolites were analyzed simultaneously. NAD(P)H fluorescence signal (350/450 nm) showed to be an adequate signal for estimating cells activity. Riboflavins fluorescence signal (450/530 nm) followed C. roseus cell concentration both for the growth phase and after elicitation with jasmonic acid. Fluorescence from the alkaloids interfered with NAD(P)H signal during the production phase. For C. roseus, tryptophan, tryptamine, ajmalicine and serpentine were monitored by the probe. For E. californica, fluorescence from alkaloids overlapped with riboflavins preventing from using the probe to follow cell growth but global alkaloids production could be followed using the probe.     

In situ measurement of bioluminescence and fluorescence in an integrated microbioreactor

Reporter strains of bacteria that emit light or a fluorescent marker in response to specific conditions in their environment are having a significant impact in many areas of biology, including toxicity assays for environmental pollutants, chemical detection, and gene expression profiling. We have demonstrated methods for in situ measurements of bioluminescence and fluorescence from bacterial cultures grown in 50 microL instrumented microbioreactors. Results from microbioreactors were compared to results obtained from conventional 500 mL batch bioreactors and shake flasks. Experiments were conducted with reporter strains of Escherichia coli in which luxCDABE or gfp was fused to a promoter that was either expressed constitutively, or that responded to oxygen limitation. With these reporter strains, we have demonstrated the ability to obtain information on growth conditions within the microbioreactor. We have also shown that the large aspect ratio of the microbioreactor provides a unique advantage over measurements in larger bioreactors by reducing the inner filter effect in on-line measurements and eliminating the need for error-prone off-line dilutions. In addition, continuous on-line monitoring of genes in real-time, when expanded to include entire reporter libraries, could potentially provide a true dynamic picture of cellular gene expression from which the kinetics of gene expression can be untangled and elucidated.     

Single-step multicolor fluorescence In situ hybridization using semiconductor quantum dot-DNA conjugates

We report a rapid method for the direct multicolor imaging of multiple subnuclear genetic sequences using novel quantum dot-based fluorescence in situ hybridization (FISH) probes (QD-FISH). Short DNA oligonucleotides were attached on QDs and used in a single hybridization/detection step of target sites in situ. QD-FISH probes penetrate both intact interphase nuclei and metaphase chromosomes and showed good targeting of dense chromatin domains with minimal steric hindrances. We further demonstrated that QD's broad absorption spectra allowed different colored probes specific for distinct subnuclear genetic sequences to be simultaneously excited with a single excitation wavelength and imaged free of chromatic aberrations in a single exposure. Thus, these results demonstrate that QD-FISH probes are very effective in multicolor FISH applications. This work also documents new possibilities of using QD-FISH probes detection down to the single molecule level.     

In situ dark field microscopy for on-line monitoring of yeast cultures

A new-type in situ probe has been developed to acquire dark field images of yeast in bioreactors. It has been derived from an in situ bright field microscope that is able to measure cell density in bioreactors during fermentation processes. The illumination part of the probe has been replaced with a dark field device, in which an aspheric condenser is used, so that high contrast dark field images can be obtained. The technique of second imaging is implemented to improve the sharpness of the images by means of a relay lens. This new in situ probe is expected to enable the evaluation of the cell viability without staining owing to modern image processing.     

In situ 2D fluorometry and chemometric monitoring of mammalian cell cultures

The main objective of the present study was to investigate the use of in situ 2D fluorometry for monitoring key bioprocess variables in mammalian cell cultures, namely the concentration of viable cells and the concentration of recombinant proteins. All studies were conducted using a recombinant Baby Hamster Kidney (BHK) cell line expressing a fusion glycoprotein IgG1-IL2 cultured in batch and fed-batch modes. It was observed that the intensity of fluorescence signals in the excitation/emission wavelength range of amino acids, vitamins and NAD(P)H changed along culture time, although the dynamics of single fluorophors could not be correlated with the dynamics of the target state variables. Therefore, multivariate chemometric modeling was adopted as a calibration methodology. 2D fluorometry produced large volumes of redundant spectral data, which were first filtered by principal components analysis (PCA). Then, a partial least squares (PLS) regression was applied to correlate the reduced fluorescence maps with the target state variables. Two validation strategies were used to evaluate the predictive capacity of the developed PLS models. Accurate estimations of viable cells density (r(2) = 0.95; 99.2% of variance captured in the training set; r(2) = 0.91; 97.7% of variance captured in the validation set) and of glycoprotein concentration (r(2) = 0.99 and 99.7% of variance captured in the training set; r(2) = 0.99 and 99.3% of variance captured in the validation set) were obtained over a wide range of reactor operation conditions. The results presented herein confirm that 2D fluorometry constitutes a reliable methodology for on-line monitoring of viable cells and recombinant protein concentrations in mammalian cell cultures.     

In situ monitoring of intracellular glucose and glutamine in CHO cell culture

The development of processes to produce biopharmaceuticals industrially is still largely empirical and relies on optimizing both medium formulation and cell line in a product-specific manner. Current small-scale (well plate-based) process development methods cannot provide sufficient sample volume for analysis, to obtain information on nutrient utilization which can be problematic when processes are scaled to industrial fermenters. We envision a platform where essential metabolites can be monitored non-invasively and in real time in an ultra-low volume assay in order to provide additional information on cellular metabolism in high throughput screens. Towards this end, we have developed a model system of Chinese Hamster Ovary cells stably expressing protein-based biosensors for glucose and glutamine. Herein, we demonstrate that these can accurately reflect changing intracellular metabolite concentrations in vivo during batch and fed-batch culture of CHO cells. The ability to monitor intracellular depletion of essential nutrients in high throughput will allow rapid development of improved bioprocesses.     

In situ monitoring of chlorophyll fluorescence to assess the synergistic effect of low temperature and high irradiance stresses inSpirulina cultures grown outdoors in photobioreactors

A chlorophyll fluorescence technique was applied to anin situ study on the effects of low temperature and high light stresses onSpirulina cultures grown outdoors in controlled tubular photobioreactors at high (1.1 g L^–1) and low (0.44 g L^–1) biomass concentrations. Diurnal changes in PSII photochemistry (F _v/F _m) after 15 min of darkness, or in the light (dF/F _m), and non-photochemical (qN) quenching were measured using a portable, pulse-amplitude-modulated fluorometer. The depression of theF _v/F _m ratio ofSpirulina cultures grown outdoors at 25°C (i.e. 10°C below optimum for growth) and 0.44 g L^–1, reached 30% at the middle of the day. At the same time of the day thedF/F _m ratio showed a reduction of up to 52%. The depression of bothF _v/F _m anddF/F _m was lower in the cultures grown at 1.1 g L^–1. Photoinhibition reduced the daily productivity of the culture grown at 0.44 g L^–1 and 25°C by 33% with respect to that grown at 35°C. Changes in the growth yields of the cultures grown under different temperatures and growth rates correlate well with analogous changes in photon yield (dF/F _m). Simple measurements of photochemical yield (F _v/F _m) can be used to test the physiological status ofSpirulina cultures. The results indicate that the saturating pulse fluorescence technique, when usedin situ, is a powerful tool for assessment of the photosynthetic characteristics of outdoor cultures ofSpirulina.     

Histidine-capped copper nanoclusters for in situ amplified fluorescence monitoring of doxycycline through inner filter effect

There is a significant need to accurately measure doxycycline concentrations in view of the adverse effects of an overdose on human health. A fluorescence (FL) detection method was adopted and copper nanoclusters (CuNCs) were synthesized using chemical reduction technology. Based on FL quenching with doxycycline, the prepared CuNCs were used to explore a fluorescent nanoprobe for doxycycline detection. In an optimal sensing environment, this FL nanosensor was sensitive and selective in doxycycline sensing and displayed a linear relationship in the range 0.5–200 μM with a detection limit of 0.092 μΜ. A characterization test demonstrated that CuNCs offered active functional groups for identifying doxycycline using electrostatic interaction and hydrogen bonds. Static quenching and the inner filter effect (IFE) resulted in weakness in the FL of His@CuNCs with doxycycline with great efficiency. This suggested nanosensor was revealed to be a functional model for simple and rapid detection of doxycycline in real samples with very pleasing accuracy.