TY  - JOUR
A1  - Khadem, S. M. J.
A1  - Hille, Carsten
A1  - Löhmannsröben, Hans-Gerd
A1  - Sokolov, Igor M.
T1  - What information is contained in the fluorescence correlation spectroscopy curves, and where
JF  - Physical review : E, Statistical, nonlinear and soft matter physics
Y1  - 2016
U6  - https://doi.org/10.1103/PhysRevE.94.022407
SN  - 2470-0045
SN  - 2470-0053
VL  - 94
PB  - American Physical Society
CY  - College Park
ER  - 
TY  - JOUR
A1  - Klauß, André
A1  - Koenig, Marcelle
A1  - Hille, Carsten
T1  - Upgrade of a Scanning Confocal Microscope to a Single-Beam Path STED Microscope
JF  - PLoS one
N2  - By overcoming the diffraction limit in light microscopy, super-resolution techniques, such as stimulated emission depletion (STED) microscopy, are experiencing an increasing impact on life sciences. High costs and technically demanding setups, however, may still hinder a wider distribution of this innovation in biomedical research laboratories. As far-field microscopy is the most widely employed microscopy modality in the life sciences, upgrading already existing systems seems to be an attractive option for achieving diffraction-unlimited fluorescence microscopy in a cost-effective manner. Here, we demonstrate the successful upgrade of a commercial time-resolved confocal fluorescence microscope to an easy-to-align STED microscope in the single-beam path layout, previously proposed as "easy-STED", achieving lateral resolution <lambda/10 corresponding to a five-fold improvement over a confocal modality. For this purpose, both the excitation and depletion laser beams pass through a commercially available segmented phase plate that creates the STED-doughnut light distribution in the focal plane, while leaving the excitation beam unaltered when implemented into the joint beam path. Diffraction-unlimited imaging of 20 nm-sized fluorescent beads as reference were achieved with the wavelength combination of 635 nm excitation and 766 nm depletion. To evaluate the STED performance in biological systems, we compared the popular phalloidin-coupled fluorescent dyes Atto647N and Abberior STAR635 by labeling F-actin filaments in vitro as well as through immunofluorescence recordings of microtubules in a complex epithelial tissue. Here, we applied a recently proposed deconvolution approach and showed that images obtained from time-gated pulsed STED microscopy may benefit concerning the signal-to-background ratio, from the joint deconvolution of sub-images with different spatial information which were extracted from offline time gating.
Y1  - 2015
U6  - https://doi.org/10.1371/journal.pone.0130717
SN  - 1932-6203
VL  - 10
IS  - 6
PB  - PLoS
CY  - San Fransisco
ER  - 
TY  - JOUR
A1  - Lahn, Mattes
A1  - Dosche, Carsten
A1  - Hille, Carsten
T1  - Two-photon microscopy and fluorescence lifetime imaging reveal stimulus-induced intracellular Na+ and Cl- changes in cockroach salivary acinar cells
JF  - American journal of physiology : Cell physiology
N2  - Lahn M, Dosche C, Hille C. Two-photon microscopy and fluorescence lifetime imaging reveal stimulus-induced intracellular Na+ and Cl- changes in cockroach salivary acinar cells. Am J Physiol Cell Physiol 300: C1323-C1336, 2011. First published February 23, 2011; doi: 10.1152/ajpcell.00320.2010.-The intracellular ion homeostasis in cockroach salivary acinar cells during salivation is not satisfactorily understood. This is mainly due to technical problems regarding strong tissue autofluorescence and ineffective ion concentration quantification. For minimizing these problems, we describe the successful application of two-photon (2P) microscopy partly in combination with fluorescence lifetime imaging microscopy (FLIM) to record intracellular Na+ and Cl- concentrations ([Na+](i), [Cl-](i)) in cockroach salivary acinar cells. Quantitative 2P-FLIM Cl- measurements with the dye N-(ethoxycarbonylmethyl)-6-methoxy-quinolinium bromide indicate that the resting [Cl-](i) is 1.6 times above the Cl- electrochemical equilibrium but is not influenced by pharmacological inhibition of the Na+-K+-2Cl(-) cotransporter (NKCC) and anion exchanger using bumetanide and 4,4'-diisothiocyanatodihydrostilbene-2,2'-disulfonic acid disodium salt. In contrast, rapid Cl- reuptake after extracellular Cl- removal is almost totally NKCC mediated both in the absence and presence of dopamine. However, in physiological saline [Cl-](i) does not change during dopamine stimulation although dopamine stimulates fluid secretion in these glands. On the other hand, dopamine causes a decrease in the sodium-binding benzofuran isophthalate tetra-ammonium salt (SBFI) fluorescence and an increase in the Sodium Green fluorescence after 2P excitation. This opposite behavior of both dyes suggests a dopamine-induced [Na+](i) rise in the acinar cells, which is supported by the determined 2P-action cross sections of SBFI. The [Na+](i) rise is Cl- dependent and inhibited by bumetanide. The Ca2+-ionophore ionomycin also causes a bumetanide-sensitive [Na+](i) rise. We propose that a Ca2+-mediated NKCC activity in acinar peripheral cells attributable to dopamine stimulation serves for basolateral Na+ uptake during saliva secretion and that the concomitantly transported Cl- is recycled back to the bath.
KW  - epithelial ion transport
KW  - Na+-K+-2Cl(-) cotransporter
KW  - MQAE
KW  - SBFI
KW  - 2P cross section
Y1  - 2011
U6  - https://doi.org/10.1152/ajpcell.00320.2010
SN  - 0363-6143
VL  - 300
IS  - 6
SP  - C1323
EP  - C1336
PB  - American Chemical Society
CY  - Bethesda
ER  - 
TY  - JOUR
A1  - Wessig, Pablo
A1  - Behrends, Nicole
A1  - Kumke, Michael Uwe
A1  - Eisold, Ursula
A1  - Meiling, Til
A1  - Hille, Carsten
T1  - Two-photon FRET pairs based on coumarin and DBD dyes
JF  - RSC Advances
N2  - The synthesis and photophysical properties of two new FRET pairs based on coumarin as a donor and DBD dye as an acceptor are described. The introduction of a bromo atom dramatically increases the two-photon excitation (2PE) cross section providing a 2PE-FRET system, which is also suitable for 2PE-FLIM.
Y1  - 2016
U6  - https://doi.org/10.1039/c6ra03983a
SN  - 2046-2069
VL  - 6
SP  - 33510
EP  - 33513
PB  - Royal Society of Chemistry
CY  - Cambridge
ER  - 
TY  - JOUR
A1  - Hille, Carsten
A1  - Lahn, Mattes
A1  - Löhmannsröben, Hans-Gerd
A1  - Dosche, Carsten
T1  - Two-photon fluorescence lifetime imaging of intracellular chloride in cockroach salivary glands
Y1  - 2009
UR  - http://www.rsc.org/Publishing/Journals/pp/index.asp
U6  - https://doi.org/10.1039/B813797H
SN  - 1474-905X
ER  - 
TY  - JOUR
A1  - Sagolla, Kristina
A1  - Löhmannsröben, Hans-Gerd
A1  - Hille, Carsten
T1  - Time-resolved fluorescence microscopy for quantitative Ca2+ imaging in living cells
JF  - Analytical & bioanalytical chemistry
N2  - Calcium (Ca2+) is a ubiquitous intracellular second messenger and involved in a plethora of cellular processes. Thus, quantification of the intracellular Ca2+ concentration ([Ca2+](i)) and of its dynamics is required for a comprehensive understanding of physiological processes and potential dysfunctions. A powerful approach for studying [Ca2+](i) is the use of fluorescent Ca2+ indicators. In addition to the fluorescence intensity as a common recording parameter, the fluorescence lifetime imaging microscopy (FLIM) technique provides access to the fluorescence decay time of the indicator dye. The nanosecond lifetime is mostly independent of variations in dye concentration, allowing more reliable quantification of ion concentrations in biological preparations. In this study, the feasibility of the fluorescent Ca2+ indicator Oregon Green Bapta-1 (OGB-1) for two-photon fluorescence lifetime imaging microscopy (2P-FLIM) was evaluated. In aqueous solution, OGB-1 displayed a Ca2+-dependent biexponential fluorescence decay behaviour, indicating the presence of a Ca2+-free and Ca2+-bound dye form. After sufficient dye loading into living cells, an in situ calibration procedure has also unravelled the Ca2+-free and Ca2+-bound dye forms from a global biexponential fluorescence decay analysis, although the dye's Ca2+ sensitivity is reduced. Nevertheless, quantitative [Ca2+](i) recordings and its stimulus-induced changes in salivary gland cells could be performed successfully. These results suggest that OGB-1 is suitable for 2P-FLIM measurements, which can gain access to cellular physiology.
KW  - Fluorescence lifetime
KW  - TCSPC
KW  - Two-photon excitation
KW  - 2P cross section
KW  - Epithelial ion transport
KW  - OGB-1
Y1  - 2013
U6  - https://doi.org/10.1007/s00216-013-7290-6
SN  - 1618-2642
VL  - 405
IS  - 26
SP  - 8525
EP  - 8537
PB  - Springer
CY  - Heidelberg
ER  - 
TY  - JOUR
A1  - Hille, Carsten
A1  - Berg, Maik
A1  - Bressel, Lena
A1  - Munzke, Dorit
A1  - Primus, Philipp
A1  - Löhmannsröben, Hans-Gerd
A1  - Dosche, Carsten
T1  - Time-domain fluorescence lifetime imaging for intracellular pH sensing in living tissues
N2  - pH sensing in living cells represents one of the most prominent topics in biochemistry and physiology. In this study we performed one-photon and two-photon time-domain fluorescence lifetime imaging with a laser-scanning microscope using the time-correlated single-photon counting technique for imaging intracellular pH levels. The suitability of different commercial fluorescence dyes for lifetime-based pH sensing is discussed on the basis of in vitro as well of in situ measurements. Although the tested dyes are suitable for intensity-based ratiometric measurements, for lifetime- based techniques in the time-domain so far only BCECF seems to meet the requirements of reliable intracellular pH recordings in living cells.
Y1  - 2008
U6  - https://doi.org/10.1007/s00216-008-2147-0
ER  - 
TY  - JOUR
A1  - Marelja, Zvonimir
A1  - Chowdhury, Mita Mullick
A1  - Dosche, Carsten
A1  - Hille, Carsten
A1  - Baumann, Otto
A1  - Löhmannsröben, Hans-Gerd
A1  - Leimkühler, Silke
T1  - The L-cysteine desulfurase NFS1 is localized in the cytosol where it provides the sulfur for molybdenum cofactor biosynthesis in humans
JF  - PLoS one
N2  - In humans, the L-cysteine desulfurase NFS1 plays a crucial role in the mitochondrial iron-sulfur cluster biosynthesis and in the thiomodification of mitochondrial and cytosolic tRNAs. We have previously demonstrated that purified NFS1 is able to transfer sulfur to the C-terminal domain of MOCS3, a cytosolic protein involved in molybdenum cofactor biosynthesis and tRNA thiolation. However, no direct evidence existed so far for the interaction of NFS1 and MOCS3 in the cytosol of human cells. Here, we present direct data to show the interaction of NFS1 and MOCS3 in the cytosol of human cells using Forster resonance energy transfer and a split-EGFP system. The colocalization of NFS1 and MOCS3 in the cytosol was confirmed by immunodetection of fractionated cells and localization studies using confocal fluorescence microscopy. Purified NFS1 was used to reconstitute the lacking molybdoenzyme activity of the Neurospora crassa nit-1 mutant, giving additional evidence that NFS1 is the sulfur donor for Moco biosynthesis in eukaryotes in general.
Y1  - 2013
U6  - https://doi.org/10.1371/journal.pone.0060869
SN  - 1932-6203
VL  - 8
IS  - 4
PB  - PLoS
CY  - San Fransisco
ER  - 
TY  - JOUR
A1  - Günther, Erika
A1  - Klauß, André
A1  - Toro-Nahuelpan, Mauricio
A1  - Schüler, Dirk
A1  - Hille, Carsten
A1  - Faivre, Damien
T1  - The in vivo mechanics of the magnetotactic backbone as revealed by correlative FLIM-FRET and STED microscopy
JF  - Scientific reports
N2  - Protein interaction and protein imaging strongly benefit from the advancements in time-resolved and superresolution fluorescence microscopic techniques. However, the techniques were typically applied separately and ex vivo because of technical challenges and the absence of suitable fluorescent protein pairs. Here, we show correlative in vivo fluorescence lifetime imaging microscopy Forster resonance energy transfer (FLIM-FRET) and stimulated emission depletion (STED) microscopy to unravel protein mechanics and structure in living cells. We use magnetotactic bacteria as a model system where two proteins, MamJ and MamK, are used to assemble magnetic particles called magnetosomes. The filament polymerizes out of MamK and the magnetosomes are connected via the linker MamJ. Our system reveals that bacterial filamentous structures are more fragile than the connection of biomineralized particles to this filament. More importantly, we anticipate the technique to find wide applicability for the study and quantification of biological processes in living cells and at high resolution.
Y1  - 2019
U6  - https://doi.org/10.1038/s41598-019-55804-5
SN  - 2045-2322
VL  - 9
PB  - Nature Publ. Group
CY  - London
ER  - 
TY  - JOUR
A1  - Bruun, Kristina
A1  - Hille, Carsten
T1  - Study on intracellular delivery of liposome encapsulated quantum dots using advanced fluorescence microscopy
JF  - Scientific reports
N2  - Quantum dots increasingly gain popularity for in vivo applications. However, their delivery and accumulation into cells can be challenging and there is still lack of detailed information. Thereby, the application of advanced fluorescence techniques can expand the portfolio of useful parameters for a more comprehensive evaluation. Here, we encapsulated hydrophilic quantum dots into liposomes for studying cellular uptake of these so-called lipodots into living cells. First, we investigated photophysical properties of free quantum dots and lipodots observing changes in the fluorescence decay time and translational diffusion behaviour. In comparison to empty liposomes, lipodots exhibited an altered zeta potential, whereas their hydrodynamic size did not change. Fluorescence lifetime imaging microscopy (FLIM) and fluorescence correlation spectroscopy (FCS), both combined with two-photon excitation (2P), were used to investigate the interaction behaviour of lipodots with an insect epithelial tissue. In contrast to the application of free quantum dots, their successful delivery into the cytosol of salivary gland duct cells could be observed when applying lipodots. Lipodots with different lipid compositions and surface charges did not result in considerable differences in the intracellular labelling pattern, luminescence decay time and diffusion behaviour. However, quantum dot degradation after intracellular accumulation could be assumed from reduced luminescence decay times and blue-shifted luminescence signals. In addition to single diffusing quantum dots, possible intracellular clustering of quantum dots could be assumed from increased diffusion times. Thus, by using a simple and manageable liposome carrier system, 2P-FLIM and 2P-FCS recording protocols could be tested, which are promising for investigating the fate of quantum dots during cellular interaction.
Y1  - 2019
U6  - https://doi.org/10.1038/s41598-019-46732-5
SN  - 2045-2322
VL  - 9
PB  - Nature Publ. Group
CY  - London
ER  - 
TY  - JOUR
A1  - Khadem, S. M. J.
A1  - Hille, Carsten
A1  - Löhmannsröben, Hans-Gerd
A1  - Sokolov, Igor M.
T1  - Spot variation fluorescence correlation spectroscopy by data post-processing
JF  - Scientific reports
N2  - Spot variation fluorescence correlation spectroscopy (SV-FCS) is a variant of the FCS techniques which may give useful information about the structural organisation of the medium in which the diffusion takes place. We show that the same results can be obtained by post-processing the photon count data from ordinary FCS measurements. By using this method, one obtains the fluorescence autocorrelation functions for sizes of confocal volume, which are effectively smaller than that of the initial FCS measurement. The photon counts of the initial experiment are first transformed into smooth intensity trace using kernel smoothing method or to a piecewise-continuous intensity trace using binning and then a non-linear transformation is applied to this trace. The result of this transformation mimics the photon count rate in an experiment performed with a smaller confocal volume. The applicability of the method is established in extensive numerical simulations and directly supported in in-vitro experiments. The procedure is then applied to the diffusion of AlexaFluor647-labeled streptavidin in living cells.
Y1  - 2017
U6  - https://doi.org/10.1038/s41598-017-05672-8
SN  - 2045-2322
VL  - 7
SP  - 1
EP  - 9
PB  - Nature Publ. Group
CY  - London
ER  - 
TY  - JOUR
A1  - Jahn, Karolina
A1  - Buschmann, Volker
A1  - Hille, Carsten
T1  - Simultaneous Fluorescence and Phosphorescence Lifetime Imaging Microscopy in Living Cells
JF  - Scientific reports
N2  - In living cells, there are always a plethora of processes taking place at the same time. Their precise regulation is the basis of cellular functions, since small failures can lead to severe dysfunctions. For a comprehensive understanding of intracellular homeostasis, simultaneous multiparameter detection is a versatile tool for revealing the spatial and temporal interactions of intracellular parameters. Here, a recently developed time-correlated single-photon counting (TCSPC) board was evaluated for simultaneous fluorescence and phosphorescence lifetime imaging microscopy (FLIM/PLIM). Therefore, the metabolic activity in insect salivary glands was investigated by recording ns-decaying intrinsic cellular fluorescence, mainly related to oxidized flavin adenine dinucleotide (FAD) and the mu s-decaying phosphorescence of the oxygen-sensitive ruthenium-complex Kr341. Due to dopamine stimulation, the metabolic activity of salivary glands increased, causing a higher pericellular oxygen consumption and a resulting increase in Kr341 phosphorescence decay time. Furthermore, FAD fluorescence decay time decreased, presumably due to protein binding, thus inducing a quenching of FAD fluorescence decay time. Through application of the metabolic drugs antimycin and FCCP, the recorded signals could be assigned to a mitochondrial origin. The dopamine-induced changes could be observed in sequential FLIM and PLIM recordings, as well as in simultaneous FLIM/PLIM recordings using an intermediate TCSPC timing resolution.
Y1  - 2015
U6  - https://doi.org/10.1038/srep14334
SN  - 2045-2322
VL  - 5
PB  - Nature Publ. Group
CY  - London
ER  - 
TY  - JOUR
A1  - Jahn, Karolina
A1  - Buschmann, Volker
A1  - Hille, Carsten
T1  - Simultaneous Fluorescence and Phosphorescence Lifetime Imaging Microscopy in Living Cells
JF  - Scientific Reports
N2  - In living cells, there are always a plethora of processes taking place at the same time. Their precise regulation is the basis of cellular functions, since small failures can lead to severe dysfunctions. For a comprehensive understanding of intracellular homeostasis, simultaneous multiparameter detection is a versatile tool for revealing the spatial and temporal interactions of intracellular parameters. Here, a recently developed time-correlated single-photon counting (TCSPC) board was evaluated for simultaneous fluorescence and phosphorescence lifetime imaging microscopy (FLIM/PLIM). Therefore, the metabolic activity in insect salivary glands was investigated by recording ns-decaying intrinsic cellular fluorescence, mainly related to oxidized flavin adenine dinucleotide (FAD) and the μs-decaying phosphorescence of the oxygen-sensitive ruthenium-complex Kr341. Due to dopamine stimulation, the metabolic activity of salivary glands increased, causing a higher pericellular oxygen consumption and a resulting increase in Kr341 phosphorescence decay time. Furthermore, FAD fluorescence decay time decreased, presumably due to protein binding, thus inducing a quenching of FAD fluorescence decay time. Through application of the metabolic drugs antimycin and FCCP, the recorded signals could be assigned to a mitochondrial origin. The dopamine-induced changes could be observed in sequential FLIM and PLIM recordings, as well as in simultaneous FLIM/PLIM recordings using an intermediate TCSPC timing resolution.
KW  - Confocal microscopy
KW  - Fluorescence imaging
KW  - Fluorescence spectroscopy
KW  - Fluorescent probes
Y1  - 2015
U6  - https://doi.org/10.1038/srep14334
SN  - 2045-2322
IS  - 5
PB  - Nature Publishing Group
CY  - London
ER  - 
TY  - JOUR
A1  - Roder, Phillip
A1  - Hille, Carsten
T1  - Local tissue manipulation via a force- and pressure-controlled AFM micropipette for analysis of cellular processes
JF  - Scientific reports
N2  - Local manipulation of complex tissues at the single-cell level is challenging and requires excellent sealing between the specimen and the micromanipulation device. Here, biological applications for a recently developed loading technique for a force-and pressure-controlled fluidic force microscope micropipette are described. This technique allows for the exact positioning and precise spatiotemporal control of liquid delivery. The feasibility of a local loading technique for tissue applications was investigated using two fluorescent dyes, with which local loading behaviour could be optically visualised. Thus, homogeneous intracellular distribution of CellTracker Red and accumulation of SYTO 9 Green within nuclei was realised in single cells of a tissue preparation. Subsequently, physiological micromanipulation experiments were performed. Salivary gland tissue was pre-incubated with the Ca2+-sensitive dye OGB-1. An intracellular Ca2+ rise was then initiated at the single-cell level by applying dopamine via micropipette. When pre-incubating tissue with the nitric oxide (NO)-sensitive dye DAF-FM, NO release and intercellular NO diffusion was observed after local application of the NO donor SNP. Finally, local micromanipulation of a well-defined area along irregularly shaped cell surfaces of complex biosystems was shown for the first time for the fluidic force microscope micropipette. Thus, this technique is a promising tool for the investigation of the spatiotemporal effects of locally applied substances in complex tissues.
Y1  - 2018
U6  - https://doi.org/10.1038/s41598-018-24255-9
SN  - 2045-2322
VL  - 8
PB  - Nature Publ. Group
CY  - London
ER  - 
TY  - JOUR
A1  - Techen, Anne
A1  - Hille, Carsten
A1  - Dosche, Carsten
A1  - Kumke, Michael Uwe
T1  - Fluorescence study of drug-carrier interactions in CTAB/PBS buffer model systems
JF  - Journal of colloid and interface science
N2  - The well-known cationic surfactant hexadecyltrimethylammonium bromide (CTAB) was used as a model carrier to study drug-carrier interactions with fluorescence probes (5-hexadecanoylaminofluorescein (HAF) and 2,10-bis-(3-aminopropyloxy)dibenzo[aj]perylene-8,16-dione (NIR 628) by applying ensemble as well as single molecule fluorescence techniques. The impact of the probes on the micelle parameters (critical micelle concentration, average aggregation number, hydrodynamic radius) was investigated under physiological conditions. In the presence of additional electrolytes, such as buffer, the critical micelle concentration decreased by a factor of about 10. In contrast, no influence of the probes on the critical micelle concentration and on average aggregation number was observed. The results show that HAF does not affect the characteristics of CTAB micelles. Analyzing fluorescence correlation spectroscopy data and time-resolved anisotropy decays in terms of the "two-step" in combination with the "wobbling-in-cone" model, it was proven that HAF and NIR 628 are differently associated with the micelles. Based on ensemble and single molecule fluorescence experiments, intra- and intermicellar energy transfer process between the two dyes were probed and characterized.
KW  - Hexadecyltrimethylammonium bromide
KW  - 5-Hexadecanoylaminofluorescein
KW  - 2,10-Bis-(3-aminopropyloxy)dibenzo[aj]perylene-8,16-dione
KW  - Fluorescence correlation spectroscopy
KW  - Fluorescence anisotropy
KW  - Single-molecule FRET
Y1  - 2012
U6  - https://doi.org/10.1016/j.jcis.2012.03.063
SN  - 0021-9797
VL  - 377
SP  - 251
EP  - 261
PB  - Elsevier
CY  - San Diego
ER  - 
TY  - JOUR
A1  - Rein, Julia
A1  - Zimmermann, Bernhard
A1  - Hille, Carsten
A1  - Lang, Ingo
A1  - Walz, Bernd
A1  - Baumann, Otto
T1  - Fluorescence measurements of serotonin-induced V-ATPase-dependent pH changes at the luminal surface in salivary glands of the blowfly Calliphora vicina
N2  - Secretion in blowfly salivary glands is induced by the neurohormone serotonin and powered by a vacuolar-type H+- ATPase (V-ATPase) located in the apical membrane of the secretory cells. We have established a microfluorometric method for analysing pH changes at the luminal surface of the secretory epithelial cells by using the fluorescent dye 5-N- hexadecanoyl-aminofluorescein (HAF). After injection of HAF into the lumen of the tubular salivary gland, the fatty acyl chain of the dye molecule partitions into the outer leaflet of the plasma membrane and its pH-sensitive fluorescent moiety is exposed at the cell surface. Confocal imaging has confirmed that HAF distributes over the entire apical membrane of the secretory cells and remains restricted to this membrane domain. Ratiometric analysis of HAF fluorescence demonstrates that serotonin leads to a reversible dose-dependent acidification at the luminal surface. Inhibition by concanamycin A confirms that the serotonin-induced acidification at the luminal surface is due to H+ transport across the apical membrane via V-ATPase. Measurements with pH-sensitive microelectrodes corroborate a serotonin-induced luminal acidification and demonstrate that luminal pH decreases by about 0.4 pH units at saturating serotonin concentrations. We conclude that ratiometric measurements of HAF fluorescence provide an elegant method for monitoring V-ATPase-dependent H+ transport in the blowfly salivary gland in vivo and for analysing the spatiotemporal pattern of pH changes at the luminal surface
Y1  - 2006
UR  - http://jeb.biologists.org/
U6  - https://doi.org/10.1242/Jeb.02187
SN  - 0022-0949
ER  - 
TY  - JOUR
A1  - Schulze, Sven
A1  - Wehrhold, Michel
A1  - Hille, Carsten
T1  - Femtosecond-Pulsed laser written and etched fiber bragg gratings for fiber-optical biosensing
JF  - Sensors
N2  - We present the development of a label-free, highly sensitive fiber-optical biosensor for online detection and quantification of biomolecules. Here, the advantages of etched fiber Bragg gratings (eFBG) were used, since they induce a narrowband Bragg wavelength peak in the reflection operation mode. The gratings were fabricated point-by-point via a nonlinear absorption process of a highly focused femtosecond-pulsed laser, without the need of prior coating removal or specific fiber doping. The sensitivity of the Bragg wavelength peak to the surrounding refractive index (SRI), as needed for biochemical sensing, was realized by fiber cladding removal using hydrofluoric acid etching. For evaluation of biosensing capabilities, eFBG fibers were biofunctionalized with a single-stranded DNA aptamer specific for binding the C-reactive protein (CRP). Thus, the CRP-sensitive eFBG fiber-optical biosensor showed a very low limit of detection of 0.82 pg/L, with a dynamic range of CRP detection from approximately 0.8 pg/L to 1.2 mu g/L. The biosensor showed a high specificity to CRP even in the presence of interfering substances. These results suggest that the proposed biosensor is capable for quantification of CRP from trace amounts of clinical samples. In addition, the adaption of this eFBG fiber-optical biosensor for detection of other relevant analytes can be easily realized.
KW  - fiber Bragg gratings
KW  - ultra-fast laser inscription
KW  - fiber etching
KW  - nanostructure fabrication
KW  - fiber-optical sensors
KW  - aptamers
KW  - C-reactive protein
KW  - biomarker
Y1  - 2018
U6  - https://doi.org/10.3390/s18092844
SN  - 1424-8220
VL  - 18
IS  - 9
PB  - MDPI
CY  - Basel
ER  - 
TY  - JOUR
A1  - Hille, Carsten
A1  - Walz, Bernd
T1  - Dopamine-induced graded intracellular Ca2+ elevation via the Na+-Ca2+ exchanger operating in the Ca2+-entry mode in cockroach salivary ducts
N2  - Stimulation with the neurotransmitter dopamine causes an amplitude-modulated increase in the intracellular Ca2+ concentration ([Ca2+](i)) in epithelial cells of the ducts of cockroach salivary glands. This is completely attributable to a Ca2+ influx from the extracellular space. Additionally, dopamine induces a massive [Na+](i) elevation via the Na+- K+-2Cl(-) cotransporter (NKCC). We have reasoned that Ca2+-entry is mediated by the Na+-Ca2+ exchanger (NCE) operating in the Ca2+-entry mode. To test this hypothesis, [Ca2+](i) and [Na+](i) were measured by using the fluorescent dyes Fura- 2, Fluo-3, and SBFI. Inhibition of Na+-entry from the extracellular space by removal of extracellular Na+ or inhibition of the NKCC by 10 mu M bumetanide did not influence resting [Ca2+]i but completely abolished the dopamine-induced [Ca2+](i) elevation. Simultaneous recordings of [Ca2+](i) and [Na+](i) revealed that the dopamine-induced [Na+](i) elevation preceded the [Ca2+](i) elevation. During dopamine stimulation, the generation of an outward Na+ concentration gradient by removal of extracellular Na+ boosted the [Ca2+](i) elevation. Furthermore, prolonging the dopamine-induced [Na+](i) rise by blocking the Na+/K+-ATPase reduced the recovery from [Ca2+](i) elevation. These results indicate that dopamine induces a massive NKCC-mediated elevation in [Na+](i), which reverses the NCE activity into the reverse mode causing a graded [Ca2+](i) elevation in the duct cells.
Y1  - 2006
UR  - http://www.sciencedirect.com/science/journal/01434160
U6  - https://doi.org/10.1016/j.ceca.2005.11.006
SN  - 0143-4160
ER  - 
TY  - JOUR
A1  - Sass, Stephan
A1  - Stöcklein, Walter F. M.
A1  - Klevesath, Anja
A1  - Hurpin, Jeanne
A1  - Menger, Marcus
A1  - Hille, Carsten
T1  - Binding affinity data of DNA aptamers for therapeutic anthracyclines from microscale thermophoresis and surface plasmon resonance spectroscopy
JF  - The analyst : the analytical journal of the Royal Society of Chemistry
N2  - Anthracyclines like daunorubicin (DRN) and doxorubicin (DOX) play an undisputed key role in cancer treatment, but their chronic administration can cause severe side effects. For precise anthracycline analytical systems, aptamers are preferable recognition elements. Here, we describe the detailed characterisation of a single-stranded DNA aptamer DRN-10 and its truncated versions for DOX and DRN detection. Binding affinities were determined from surface plasmon resonance (SPR) and microscale thermophoresis (MST) and combined with conformational data from circular dichroism (CD). Both aptamers displayed similar nanomolar binding affinities to DRN and DOX, even though their rate constants differed as shown by SPR recordings. SPR kinetic data unravelled a two-state reaction model including a 1 : 1 binding and a subsequent conformational change of the binding complex. This model was supported by CD spectra. In addition, the dissociation constants determined with MST were always lower than that from SPR, and especially for the truncated aptamer they differed by two orders of magnitude. This most probably reflects the methodological difference, namely labelling for MST vs. immobilisation for SPR. From CD recordings, we suggested a specific G-quadruplex as structural basis for anthracycline binding. We concluded that the aptamer DRN-10 is a promising recognition element for anthracycline detection systems and further selected aptamers can be also characterised with the combined methodological approach presented here.
Y1  - 2019
U6  - https://doi.org/10.1039/c9an01247h
SN  - 0003-2654
SN  - 1364-5528
VL  - 144
IS  - 20
SP  - 6064
EP  - 6073
PB  - Royal Society of Chemistry
CY  - Cambridge
ER  - 
TY  - JOUR
A1  - Klauß, André
A1  - Conrad, Florian
A1  - Hille, Carsten
T1  - Binary phase masks for easy system alignment and basic aberration sensing with spatial light modulators in STED microscopy
JF  - Scientific reports
Y1  - 2017
U6  - https://doi.org/10.1038/s41598-017-15967-5
SN  - 2045-2322
VL  - 7
PB  - Nature Publ. Group
CY  - London
ER  -