In-line Fluorescence Research Articles

In-line monitoring of Bordetella pertussis cultivation using fluorescence spectroscopy.

Fluorescence spectroscopy is a non-invasive and highly sensitive method for bioprocess monitoring. The use of fluorescence spectroscopy is not very well established in the industry for in-line monitoring. In the present work, a 2-D fluorometer with two excitation lights (365 and 405 nm) and emission spectra in the range of 350-850 nm were used for in-line monitoring of two strains of Bordetella pertussis cultivation operated in batch and fed batch. A Partial Least Squares (PLS) based regression model was used for the estimation of cell biomass, amino acids (glutamate and proline) and antigen (Pertactin)produced. It was observed that accurate predictions were achieved when models were calibrated separately for each cell strain and nutrient media formulation. Also, prediction accuracy was improved when dissolved oxygen, agitation and culture volume are added as additional features in the regression model. The proposed approach of combining in-line fluorescence and other online measurements is shown to have good potential for in-line monitoring of bioprocesses.

Monitoring oligomeric forms of amyloid beta in the early stages of aggregation using hybrid X-ray footprinting mass spectrometry with inline fluorescence.

The aggregation-prone Amyloid Beta (Aβ) peptide has been the target of the majority of efforts to develop Alzheimer's Disease therapeutics, most of which have failed or have shown mixed results. It is no longer widely believed that buildup of amyloid fibrils is a causal factor in cognitive decline associated with the disease, and a growing body of evidence now suggests that low molecular weight oligomeric forms of Aβ may be the culprits in disease progression. However, while numerous Aβ fibril structures have been characterized, primarily by NMR and cryo-EM, obtaining structural information on oligomeric forms of Aβ that presumably precede and/or seed fibril formation has proved challenging. These transient forms are heterogeneous, and depend heavily on experimental conditions such as buffer, temperature, concentration, and degree of quiescence during measurement. Here, we present a new approach to delineating structural features of early-stage low molecular weight Aβ oligomers using a solvent accessibility assay in conjunction with inline fluorescence measurements. In this method, inline fluorescence is collected nearly simultaneously from samples during collection of X-ray footprinting mass spectrometry (XFMS) data. In the XFMS method, X-ray irradiation dissociates solvent water to produce hydroxyl radicals, which covalently modify solvent accessible side chains. Liquid chromatography-mass spectrometry is subsequently used to analyze the covalent modifications produced, and the data provide a “water map” at the single residue level, which is used to determine protein structure. This new hybrid approach circumvents issues in batch-to-batch variability of Aβ aggregation which has made this peptide so difficult to study, and provides a method to combine residue-specific structural information nearly simultaneously with global protein structure readouts from either intrinsic protein fluorescence or fluorophores such as Thioflavin T.

In-line Fluorescence Spectroscopy for Quantification of Low Amount of Active Pharmaceutical Ingredient

The pharmaceutical industry is currently implementing new manufacturing principles and modernizing the related processing solutions. A key element in this development is implementation of process analytical technologies (PAT) for measuring product quality in a real-time mode, ideally for a continuously operating processing line. Near-infrared (NIR) spectroscopy is widely used for this purpose, but has limited use for low concentration formulations, due to its inherent detection limit. Light-induced fluorescence (LIF) spectroscopy is a PAT tool that can be used to quantify low concentrations of active pharmaceutical ingredient, and recent development of instrumentation has made it available for in-line applications. In this study, the content of tryptophan in a dynamic powder flow could be measured as low as 0.10 w/w % with LIF spectroscopy with good accuracy of RMSEP = 0.008 w/w %. Both partial least squares regression and support vector machines (SVM) were investigated, but we found SVM to be the better option due to non-linearities between the calibration test and the in-line measurements. With the use of SVM, LIF spectroscopy is a promising candidate for low concentration applications where NIR is not suitable.

Exploring synthetic biology for the development of a sensor cell line for automated bioprocess control

Unfavorable process conditions lead to adverse cultivation states, limited cell growth and thus hamper biotherapeutic protein production. Oxygen deficiency or hyperosmolality are among the most critical process conditions and therefore require continuous monitoring. We established a novel sensor CHO cell line with the ability to automatically sense and report unwanted process conditions by the expression of destabilized fluorescent proteins. To this end, an inducible real-time system to detect hypoxia by hypoxia response elements (HREs) of vascular endothelial growth factor (VEGF) origin reporting limitations by the expression of destabilized green fluorescent protein (GFP) was created. Additionally, we established a technique for observing hyperosmolality by exploiting osmotic response elements (OREs) for the expression of unstable blue fluorescent protein (BFP, FKBP-BFP), enabling the simultaneous automated supervision of two bioprocess parameters by using a dual sensor CHO cell line transfected with a multiplexable monitoring system. We finally also provided a fully automated in-line fluorescence microscopy-based setup to observe CHO cells and their response to varying culture conditions. In summary, we created the first CHO cell line, reporting unfavorable process parameters to the operator, and provided a novel and promising sensor technology accelerating the implementation of the process analytical technology (PAT) initiative by innovative solutions.

Fluorescence enhanced BA-LIFT for single cell detection and isolation

Laser bioprinting is a term that refers to a group of laser-based techniques for printing living cells with high precision and good viability. Most of these techniques are based on modifications of the standard laser induced forward transfer technique (LIFT). When it comes to printing living material, direct laser irradiation should be avoided, therefore an indirect LIFT technique comprising an energy absorption layer should be used. This work presents a blister actuated-LIFT (BA-LIFT) technique which uses a commercial polyimide tape as a uniform energy absorption layer. To increase the potential of the technique for cell selection and printing accuracy, we take advantage of the high optical transmission of the polyimide layer to implement an in-line fluorescence and conventional imaging vision system coaxial with the laser path. With this system and using the appropriate staining methodology it is possible to track and identify different cell types, selecting those to be transferred and tracking cell survival both in the donor and acceptor substrates. We studied the BA-LIFT printability map for sodium alginate and methylcellulose hydrogels in the fluence range from 6.1 to 2.0 J cm−2 together with the cell viability assessment measured by our fluorescence system. The study has revealed that less concentrated, therefore less viscose hydrogel shows better results with lower fluences, whereas hydrogels with higher concentrations present better results at higher fluences. Also, at low fluences 98 % of cell viability was obtained, besides both primary cells and cell lines keep their integrity, proliferating and functional activity. The technique was tested by tracking and targeting mouse hematopoietic progenitor stem cells transferred to assess colony forming units; moreover, natural killer cells were isolated and its activation in a stimulation media was tracked with the fluorescence system.

Calibration of systems for quantitative fluorescence analysis of thin layers.

Imaging fluorescence analysis is a powerful tool for the characterization of thin functional layers. Due to the development of new components such as cost-efficient and long life diode lasers and LEDs as well as sensitive cameras, the number of industrial in situ sensors based on fluorescence analysis technology increased rapidly in recent years. Of crucial importance for all these new sensors are efficient and robust methods for calibration. Although there are many examples for the calibration of laboratory setups for single specialized applications, there is no standardized method for the traceable device independent calibration of imaging fluorescence systems. This paper presents the evaluation of five different methods for the calibration of systems for quantitative fluorescence analysis. Each method is applied for the calibration of an imaging fluorescence laser scanner. In addition to characterizing the precision of the methods, the work analyzes the usability of the methods for different applications. The results show for the first time that a calibrated IR point sensor can be used for the auto calibration of high resolution imaging inline fluorescence sensors. In addition, we present a novel method for the transfer of calibration data between analysis systems with different optical setups by using a solid material fluorescence standard.

Recent Advances in X-Ray Hydroxyl Radical Footprinting at the Advanced Light Source Synchrotron

Synchrotron hydroxyl radical footprinting is a relatively new structural method used to investigate structural features and conformational changes of nucleic acids and proteins in the solution state. It was originally developed at the National Synchrotron Light Source at Brookhaven National Laboratory in the late nineties, and more recently, has been established at the Advanced Light Source at Lawrence Berkeley National Laboratory. The instrumentation for this method is an active area of development, and includes methods to increase dose to the samples while implementing high-throughput sample delivery methods. Improving instrumentation to irradiate biological samples in real time using a sample droplet generator and inline fluorescence monitoring to rapidly determine dose response curves for samples will significantly increase the range of biological problems that can be investigated using synchrotron hydroxyl radical footprinting.

Isotachophoretic Fluorescence in Situ Hybridization of Intact Bacterial Cells.

A counter-pressure-assisted capillary isotachophoresis method in combination with a sieving matrix and ionic spacer was used to perform in-line fluorescence in situ hybridization (FISH) of bacterial cells. A high concentration of sieving matrix (1.8% w/v HEC) was introduced at one end of the capillary, and the bacterial cells were suspended in the spacer electrolyte for injection. Using a 2 min injection with 18 psi counter-pressure, 50% of the cells injected into the capillary were hybridized with the fluorescently labeled oligonucleotide, and the excess unhybridized probe was separated from the hybridized cell-probe complexes in a two-stage ITP method. With an LOD (6.0 × 104 cells/mL) comparable with the CE analysis of a sample processed using an off-line FISH protocol, the total analysis time was reduced from 2.5 h to 30 min. Provided the appropriate probe is selected, this approach can be used for specific detection of bacterial cells in aqueous samples.

Revealing a Size-Resolved Fluorescence-Based Metric for Tracking Oxidative Treatment of Total N-Nitrosamine Precursors in Waters from Wastewater Treatment Plants

N-Nitrosamines make up a nonhalogenated group of disinfection byproducts that form during chloramination, particularly in drinking waters impacted by wastewater treatment plant (WWTP) effluents. Here, we reveal a size-resolved fluorescence-based precursor surrogate for tracking total N-nitrosamine formation potential (TONOFP) through oxidative treatment. Samples were collected at seven WWTPs, at the finished effluent and prior to final disinfection (i.e., secondary effluent). Asymmetric flow field-flow fractionation (AF4) with inline fluorescence detection at excitation and emission wavelengths of 280 and 340 nm, respectively (i.e., I280/340), showed protein-rich natural organic matter peaks between 10–445 and 445–1500 kDa. TONOFP was measured by chemiluminescence on samples dosed with 250 mg L–1 Cl2 monochloramine at pH 7 for 7 days. Metrics from AF4 I280/340 (i.e., area under the curve and peak height) and I257/277 from whole water excitation–emission matrices were leveraged in multivariate models to de...

Surface induced three-peak elution behavior of a monoclonal antibody during cation exchange chromatography

A monoclonal antibody exhibits a two- or three-peak elution behavior when loaded on the CEX resin POROS XS and eluted with a salt gradient. Two peaks are observed without a hold step while a third more strongly retained peak becomes noticeable with a hold time as low as 10min. As the hold time is increased further, the first peak gradually disappears, the second peak initially increases and then decreases, and the third peak continuously increases. Dynamic light scattering shows that the third peak contains significant levels of aggregates formed in the column. Circular dichroism, HX-MS analyses of the eluted fraction, in-line fluorescence detection, and bound-state HX-MS analysis indicate that the aggregates derive from an unfolded intermediate that is slowly formed while the protein is bound to the resin. Aggregate formation does not occur on a different CEX resin, Nuvia HR-S, with similar particle size but with a more homogenous structure or when the sodium acetate load buffer is replaced with arginine acetate. The two early eluting peaks observed for POROS XS regardless of hold time are shown to comprise exclusively monomeric species. A set of biophysical measurements as well as mechanistic modeling support the hypothesis that these two peaks form as a result of the presence of weak and strong binding sites on the resin having, respectively, fast and slow binding kinetics.

Fluorophore Absorption Size Exclusion Chromatography (FA-SEC): An Alternative Method for High-Throughput Detergent Screening of Membrane Proteins.

Membrane proteins play key roles in many fundamental functions in cells including ATP synthesis, ion and molecule transporter, cell signalling and enzymatic reactions, accounting for ~30% genes of whole genomes. However, the hydrophobic nature of membrane proteins frequently hampers the progress of structure determination. Detergent screening is the critical step in obtaining stable detergent-solubilized membrane proteins and well-diffracting protein crystals. Fluorescence Detection Size Exclusion Chromatography (FSEC) has been developed to monitor the extraction efficiency and monodispersity of membrane proteins in detergent micelles. By tracing the FSEC profiles of GFP-fused membrane proteins, this method significantly enhances the throughput of detergent screening. However, current methods to acquire FSEC profiles require either an in-line fluorescence detector with the SEC equipment or an off-line spectrofluorometer microplate reader. Here, we introduce an alternative method detecting the absorption of GFP (FA-SEC) at 485 nm, thus making this methodology possible on conventional SEC equipment through the in-line absorbance spectrometer. The results demonstrate that absorption is in great correlation with fluorescence of GFP. The comparably weaker absorption signal can be improved by using a longer path-length flow cell. The FA-SEC profiles were congruent with the ones plotted by FSEC, suggesting FA-SEC could be a comparable and economical setup for detergent screening of membrane proteins.

Identification of novel isomeric pectic oligosaccharides using hydrophilic interaction chromatography coupled to traveling-wave ion mobility mass spectrometry

Separation and characterization of complex mixtures of pectic oligosaccharides still remains challenging and often requires the use of multiple analytical techniques, especially when isomeric structures are present. In this work, it is demonstrated that the coupling of hydrophilic interaction chromatography (HILIC) to traveling-wave ion mobility mass spectrometry (TWIMMS) enabled the simultaneous separation and characterization of complex mixtures of various isomeric pectic oligosaccharides. Labeling of oligosaccharides with 3-aminoquinoline (3-AQ) improved MS-ionization efficiency of the oligosaccharides and reduced the complexity of the product ion mass spectra, without losing resolution of the HILIC separation. In addition, labeling enabled quantification of oligosaccharides on molar basis using in-line fluorescence detection. Isomeric structures were distinguished using TWIMMS. The 3-AQ-HILIC–TWIMMS method was used to characterize a series of isomeric sugar beet rhamnogalacturonan I derived oligosaccharides carrying a glucuronic acid substituent. Thereby, some novel structural features were identified for the first time: glucuronic acid was attached to O-3 or to O-2 of galacturonic acid residues and a single galacturonic acid residue within an oligomer could contain both an acetyl group and a glucuronic acid substituent.

Nanomolar to sub-picomolar affinity measurements of antibody–antigen interactions and protein multimerizations: Fluorescence-assisted high-performance liquid chromatography

Although several techniques exist for the measurement of high-affinity interactions, it is still challenging to determine dissociation constants around or even below 1pM. During the analysis of several human-derived monoclonal antibodies to adalimumab, we found a clone with a very high affinity that could not be measured using conventional surface plasmon resonance assays. We developed a straightforward and robust method to measure affinities in the nanomolar to sub-picomolar range. The assay is based on separation of bound and free fluorescently labeled antigen using size exclusion chromatography and quantification by in-line fluorescence detection. We describe optimal conditions and procedures that result in a very sensitive assay that can be used to reliably determine ultra-high affinities. Using the method described in this article, a dissociation constant of 0.78pM could be determined for the anti-adalimumab antibody.

Post-column derivatization capillary electrochromatography for detection of biogenic amines in tuna-meat

A system to perform post-column derivatization capillary electrochromatography (CEC) was developed for the first time. The system mainly included a 4-μm (O.D.) silica packed column (200 mm effective length × 0.1 mm inner diameter I.D.) with micro-magnetic particles (MMPs) frits, a T-junction connector, an in-line fluorescence detector and a high-voltage power supply. The system was evaluated by using histamine (HA) as a standard biogenic amine for this study. A 5 μM HA solution was loaded at the anodic site of the capillary column by applying 3 kV for 5 s. Then, HA was electrophoretically eluted with a 20 mM phosphate buffer (pH 7) by applying 3 kV, and was derivatized with 3 mM o-phthalaldehyde (OPA)/ N-acetylcysteine (NAC) in 100 mM borate (pH 10), which was continuously delivered through the reagent-loading capillary tube by gravity into the T-junction connector. HA derivative was finally detected with the in-line fluorescence detector ( λ Ex = 340 nm, λ Em = 450 nm) at 9.7 min after sample loading. To test the utility of this system, it was next employed for its ability to detect the presence of HA and other kinds of biogenic amines, including cadaverine (Cad), spermidine (Spm) and tyramine (Tyr) in tuna-meat, once the validity of the method had been confirmed.

The Mechanism of Assembly and Cofactor Insertion into Rhodobacter capsulatus Xanthine Dehydrogenase

Rhodobacter capsulatus xanthine dehydrogenase (XDH) is a molybdo-flavoprotein that is highly homologous to the homodimeric mammalian xanthine oxidoreductase. However, the bacterial enzyme has an (alphabeta)(2) heterotetrameric structure, and the cofactors were identified to be located on two different polypeptides. We have analyzed the mechanism of cofactor insertion and subunit assembly of R. capsulatus XDH, using engineered subunits with appropriate substitutions in the interfaces. In an (alphabeta) heterodimeric XDH containing the XdhA and XdhB subunits, the molybdenum cofactor (Moco) was shown to be absent, indicating that dimerization of the (alphabeta) subunits has to precede Moco insertion. In an (alphabeta)(2) XDH heterotetramer variant, including only one active Moco-center, the active (alphabeta) site of the chimeric enzyme was shown to be fully active, revealing that the two subunits act independent without cooperativity. Amino acid substitutions at two cysteine residues coordinating FeSI of the two [2Fe-2S] clusters of the enzyme demonstrate that an incomplete assembly of FeSI impairs the formation of the XDH (alphabeta)(2) heterotetramer and, thus, insertion of Moco into the enzyme. The results reveal that the insertion of the different redox centers into R. capsulatus XDH takes place sequentially. Dimerization of two (alphabeta) dimers is necessary for insertion of sulfurated Moco into apo-XDH, the last step of XDH maturation.

Transfer of the Molybdenum Cofactor Synthesized by Rhodobacter capsulatus MoeA to XdhC and MobA

The molybdenum cofactor (Moco) exists in different variants in the cell and can be directly inserted into molybdoenzymes utilizing the molybdopterin (MPT) form of Moco. In bacteria such as Rhodobacter capsulatus and Escherichia coli, MPT is further modified by attachment of a GMP nucleotide, forming MPT guanine dinucleotide (MGD). In this work, we analyzed the distribution and targeting of different forms of Moco to their respective user enzymes by proteins that bind Moco and are involved in its further modification. The R. capsulatus proteins MogA, MoeA, MobA, and XdhC were purified, and their specific interactions were analyzed. Interactions between the protein pairs MogA-MoeA, MoeA-XdhC, MoeA-MobA, and XdhC-MobA were identified by surface plasmon resonance measurements. In addition, the transfer of Moco produced by the MogA-MoeA complex to XdhC was investigated. A direct competition of MobA and XdhC for Moco binding was determined. In vitro analyses showed that XdhC bound to MobA, prevented the binding of Moco to MobA, and thereby inhibited MGD biosynthesis. The data were confirmed by in vivo studies in R. capsulatus cells showing that overproduction of XdhC resulted in a 50% decrease in the activity of bis-MGD-containing Me(2)SO reductase. We propose that, in bacteria, the distribution of Moco in the cell and targeting to the respective user enzymes are accomplished by specific proteins involved in Moco binding and modification.

Selective Substrate Supply in the Regulation of Yeast de Novo Sphingolipid Synthesis

The heat stress response of Saccharomyces cerevisiae is characterized by transient cell cycle arrest, altered gene expression, degradation of nutrient permeases, trehalose accumulation, and translation initiation of heat shock proteins. Importantly heat stress also induces de novo sphingolipid synthesis upon which many of these subprograms of the heat stress response depend. Despite extensive data addressing the roles for sphingolipids in heat stress, the mechanism(s) by which heat induces sphingolipid synthesis remains unknown. This study was undertaken to determine the events and/or factors required for heat stress-induced sphingolipid synthesis. Data presented indicate that heat does not directly alter the in vitro activity of serine palmitoyltransferase (SPT), the enzyme responsible for initiating de novo sphingolipid synthesis. Moreover deletion of the small peptide Tsc3p, which is thought to maximize SPT activity, specifically reduced production of C(20) sphingolipid species by over 70% but did not significantly decrease overall sphingoid base production. In contrast, the fatty-acid synthase inhibitor cerulenin nearly completely blocked sphingoid base production after heat, indicating a requirement for endogenous fatty acids for heat-mediated sphingoid base synthesis. Consistent with this, genetic studies show that fatty acid import does not contribute to heat-induced de novo synthesis under normal conditions. Interestingly the absence of medium serine also ameliorated heat-induced sphingoid base production, indicating a requirement for exogenous serine for the response, and consistent with this finding, disruption of synthesis of endogenous serine did not affect heat-induced sphingolipid synthesis. Serine uptake assays indicated that heat increased serine uptake from medium by 100% during the first 10 min of heat stress. Moreover treatments that increase serine uptake in the absence of heat including acute medium acidification and glucose treatment also enhanced de novo sphingoid base synthesis equivalent to that induced by heat stress. These data agree with findings from mammalian systems that availability of substrates is a key determinant of flux through sphingolipid synthesis. Moreover data presented here indicate that SPT activity can be driven by several factors that increase serine uptake in the absence of heat. These findings may provide insights into the many systems in which de novo synthesis is increased in the absence of elevated in vitro SPT activity.

High sensitivity separation and detection of heparan sulfate disaccharides

Eight Δ-disaccharide standards from heparan sulfate/heparin were derivatized with the fluorophore 4,4-difluoro-5,7- dimethyl-4-bora-3a,4a-diaza- s-indacene-3-propionic acid, hydrazide (BODIPY) via formation of a Schiff's base and separated using HPAEC on a Propac PA1 column with a linear salt gradient and isocratic 150 mM NaOH. Detection was with an in-line fluorescence detector. The standard deviation ( σ n−1 ) in retention times were 0.7–2% over nine runs. The limit of detection, was 100 fmol (100 × 10 −15 mol) of BODIPY labeled Δ-disaccharides, representing considerably improved detection compared to other fluorophore labeled derivatives and, unlike these, required no further purification steps. Separation and improved detection of BODIPY-Δ-disaccharide conjugates will assist the structural analysis of HS and the development of improved sequencing methodologies.

In-line recording of PAM fluorescence of phytoplankton cultures as a new tool for studying effects of fluctuating nutrient supply on photosynthesis

Turbidostat cultures of Dunaliella salina (Chlorophyceae) and Thalassiosira weissflogii (Bacillariophyceae) were grown at fluctuating concentrations of nitrate, phosphate and silicate. In-line measurements of PAM fluorescence were used to monitor the effects of fluctuating nutrient supply on the photochemical efficiency of photosystem II reaction centres of light-adapted cells (ΔF/F′m). Besides the maximal photochemical efficiency of photosystem II reaction centres of dark-adapted cells (F v/F m), chlorophyll a, particulate organic carbon, nitrogen and phosphorus, and the cell number were measured frequently during the experiments. Following nutrient-replete growth, the cells were supplied with medium from which either nitrate, phosphate or silicate was omitted. When significant effects of nutrient starvation were indicated by the fluorescence parameters, a pulse of the deficient nutrient was added to the cultures. Our experimental set-up for in-line fluorescence measurements provided sensitive and reproducible detection of the various fluorescence signals, revealing strong influences of nutrient supply on the photochemical efficiency of photosystem II. In general the fluorescence values changed substantially within 1–30 min after re-addition of the deficient nutrient. Addition of phosphate and silicate induced an immediate characteristic decrease in fluorescence, whereas nitrate addition was characterized by a strong, delayed increase in fluorescence. Complete recovery to pre-starvation fluorescence values took about 48 h in all experiments. The physiological background of nutrient uptake is used to explain the observed tight couplings between fluorescence responses and nutrient re-addition. Our study clearly demonstrates that in-line fluorescence measurements provide a new tool for the investigation of phytoplankton reactions to fluctuating nutrients and offer the possibility to detect nutrient starvation in the field.

In-line recording of PAM fluorescence of phytoplankton cultures as a new tool for studying effects of fluctuating nutrient supply on photosynthesis

Turbidostat cultures of Dunaliella salina (Chlorophyceae) and Thalassiosira weissflogii (Bacillariophyceae) were grown at fluctuating concentrations of nitrate, phosphate and silicate. In-line measurements of PAM fluorescence were used to monitor the effects of fluctuating nutrient supply on the photochemical efficiency of photosystem II reaction centres of light-adapted cells (ΔF/F′m). Besides the maximal photochemical efficiency of photosystem II reaction centres of dark-adapted cells (F v/F m), chlorophyll a, particulate organic carbon, nitrogen and phosphorus, and the cell number were measured frequently during the experiments. Following nutrient-replete growth, the cells were supplied with medium from which either nitrate, phosphate or silicate was omitted. When significant effects of nutrient starvation were indicated by the fluorescence parameters, a pulse of the deficient nutrient was added to the cultures. Our experimental set-up for in-line fluorescence measurements provided sensitive and reproducible detection of the various fluorescence signals, revealing strong influences of nutrient supply on the photochemical efficiency of photosystem II. In general the fluorescence values changed substantially within 1–30 min after re-addition of the deficient nutrient. Addition of phosphate and silicate induced an immediate characteristic decrease in fluorescence, whereas nitrate addition was characterized by a strong, delayed increase in fluorescence. Complete recovery to pre-starvation fluorescence values took about 48 h in all experiments. The physiological background of nutrient uptake is used to explain the observed tight couplings between fluorescence responses and nutrient re-addition. Our study clearly demonstrates that in-line fluorescence measurements provide a new tool for the investigation of phytoplankton reactions to fluctuating nutrients and offer the possibility to detect nutrient starvation in the field.