@article{RoderHille2015, author = {Roder, Phillip and Hille, Carsten}, title = {A Multifunctional Frontloading Approach for Repeated Recycling of a Pressure-Controlled AFM Micropipette}, series = {PLoS ONE}, volume = {10}, journal = {PLoS ONE}, number = {12}, publisher = {Public Library of Science}, address = {Lawrence, Kan.}, issn = {1932-6203}, doi = {10.1371/journal.pone.0144157}, year = {2015}, abstract = {Fluid force microscopy combines the positional accuracy and force sensitivity of an atomic force microscope (AFM) with nanofluidics via a microchanneled cantilever. However, adequate loading and cleaning procedures for such AFM micropipettes are required for various application situations. Here, a new frontloading procedure is described for an AFM micropipette functioning as a force- and pressure-controlled microscale liquid dispenser. This frontloading procedure seems especially attractive when using target substances featuring high costs or low available amounts. Here, the AFM micropipette could be filled from the tip side with liquid from a previously applied droplet with a volume of only a few μL using a short low-pressure pulse. The liquid-loaded AFM micropipettes could be then applied for experiments in air or liquid environments. AFM micropipette frontloading was evaluated with the well-known organic fluorescent dye rhodamine 6G and the AlexaFluor647-labeled antibody goat anti-rat IgG as an example of a larger biological compound. After micropipette usage, specific cleaning procedures were tested. Furthermore, a storage method is described, at which the AFM micropipettes could be stored for a few hours up to several days without drying out or clogging of the microchannel. In summary, the rapid, versatile and cost-efficient frontloading and cleaning procedure for the repeated usage of a single AFM micropipette is beneficial for various application situations from specific surface modifications through to local manipulation of living cells, and provides a simplified and faster handling for already known experiments with fluid force microscopy.}, language = {en} } @misc{RoderHille2015, author = {Roder, Phillip and Hille, Carsten}, title = {A Multifunctional Frontloading Approach for Repeated Recycling of a Pressure-Controlled AFM Micropipette}, url = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus4-86592}, year = {2015}, abstract = {Fluid force microscopy combines the positional accuracy and force sensitivity of an atomic force microscope (AFM) with nanofluidics via a microchanneled cantilever. However, adequate loading and cleaning procedures for such AFM micropipettes are required for various application situations. Here, a new frontloading procedure is described for an AFM micropipette functioning as a force- and pressure-controlled microscale liquid dispenser. This frontloading procedure seems especially attractive when using target substances featuring high costs or low available amounts. Here, the AFM micropipette could be filled from the tip side with liquid from a previously applied droplet with a volume of only a few μL using a short low-pressure pulse. The liquid-loaded AFM micropipettes could be then applied for experiments in air or liquid environments. AFM micropipette frontloading was evaluated with the well-known organic fluorescent dye rhodamine 6G and the AlexaFluor647-labeled antibody goat anti-rat IgG as an example of a larger biological compound. After micropipette usage, specific cleaning procedures were tested. Furthermore, a storage method is described, at which the AFM micropipettes could be stored for a few hours up to several days without drying out or clogging of the microchannel. In summary, the rapid, versatile and cost-efficient frontloading and cleaning procedure for the repeated usage of a single AFM micropipette is beneficial for various application situations from specific surface modifications through to local manipulation of living cells, and provides a simplified and faster handling for already known experiments with fluid force microscopy.}, language = {en} } @article{RoderHille2015, author = {Roder, Phillip and Hille, Carsten}, title = {A Multifunctional Frontloading Approach for Repeated Recycling of a Pressure-Controlled AFM Micropipette}, series = {PLoS one}, volume = {10}, journal = {PLoS one}, number = {12}, publisher = {PLoS}, address = {San Fransisco}, issn = {1932-6203}, doi = {10.1371/journal.pone.0144157}, pages = {10}, year = {2015}, abstract = {Fluid force microscopy combines the positional accuracy and force sensitivity of an atomic force microscope (AFM) with nanofluidics via a microchanneled cantilever. However, adequate loading and cleaning procedures for such AFM micropipettes are required for various application situations. Here, a new frontloading procedure is described for an AFM micropipette functioning as a force-and pressure-controlled microscale liquid dispenser. This frontloading procedure seems especially attractive when using target substances featuring high costs or low available amounts. Here, the AFM micropipette could be filled from the tip side with liquid from a previously applied droplet with a volume of only a few mu L using a short low-pressure pulse. The liquid-loaded AFM micropipettes could be then applied for experiments in air or liquid environments. AFM micropipette frontloading was evaluated with the well-known organic fluorescent dye rhodamine 6G and the AlexaFluor647-labeled antibody goat anti-rat IgG as an example of a larger biological compound. After micropipette usage, specific cleaning procedures were tested. Furthermore, a storage method is described, at which the AFM micropipettes could be stored for a few hours up to several days without drying out or clogging of the microchannel. In summary, the rapid, versatile and cost-efficient frontloading and cleaning procedure for the repeated usage of a single AFM micropipette is beneficial for various application situations from specific surface modifications through to local manipulation of living cells, and provides a simplified and faster handling for already known experiments with fluid force microscopy.}, language = {en} } @article{MutigKahlSaritasetal.2011, author = {Mutig, Kerim and Kahl, Thomas and Saritas, Turgay and Godes, Michael and Persson, Pontus and Bates, James and Raffi, Hajamohideen and Rampoldi, Luca and Uchida, Shinichi and Hille, Carsten and Dosche, Carsten and Kumar, Satish and Castaneda-Bueno, Maria and Gamba, Gerardo and Bachmann, Sebastian}, title = {Activation of the Bumetanide-sensitive Na+, K+,2Cl(-) Cotransporter (NKCC2) Is Facilitated by Tamm-Horsfall Protein in a Chloride-sensitive Manner}, series = {The journal of biological chemistry}, volume = {286}, journal = {The journal of biological chemistry}, number = {34}, publisher = {American Society for Biochemistry and Molecular Biology}, address = {Bethesda}, issn = {0021-9258}, doi = {10.1074/jbc.M111.222968}, pages = {30200 -- 30210}, year = {2011}, abstract = {Active transport of NaCl across thick ascending limb (TAL) epithelium is accomplished by Na+, K+,2Cl(-) cotransporter (NKCC2). The activity of NKCC2 is determined by vasopressin (AVP) or intracellular chloride concentration and includes its amino-terminal phosphorylation. Co-expressed Tamm-Horsfall protein (THP) has been proposed to interact with NKCC2. We hypothesized that THP modulates NKCC2 activity in TAL. THP-deficient mice (THP-/-) showed an increased abundance of intracellular NKCC2 located in subapical vesicles (+47\% compared with wild type (WT) mice), whereas base-line phosphorylation of NKCC2 was significantly decreased (-49\% compared with WT mice), suggesting reduced activity of the transporter in the absence of THP. Cultured TAL cells with low endogenous THP levels and low base-line phosphorylation of NKCC2 displayed sharp increases in NKCC2 phosphorylation (+38\%) along with a significant change of intracellular chloride concentration upon transfection with THP. In NKCC2-expressing frog oocytes, co-injection with THP cRNA significantly enhanced the activation of NKCC2 under low chloride hypotonic stress (+112\% versus +235\%). Short term (30 min) stimulation of the vasopressin V2 receptor pathway by V2 receptor agonist (deamino-cis-D-Arg vasopressin) resulted in enhanced NKCC2 phosphorylation in WT mice and cultured TAL cells transfected with THP, whereas in the absence of THP, NKCC2 phosphorylation upon deamino-cis-D-Arg vasopressin was blunted in both systems. Attenuated effects of furosemide along with functional and structural adaptation of the distal convoluted tubule in THP-/- mice supported the notion that NaCl reabsorption was impaired in TAL lacking THP. In summary, these results are compatible with a permissive role for THP in the modulation of NKCC2-dependent TAL salt reabsorptive function.}, language = {en} } @article{RoderHille2014, author = {Roder, Phillip and Hille, Carsten}, title = {ANG-2 for quantitative Na+ determination in living cells by time-resolved fluorescence microscopy}, series = {Photochemical \& photobiological sciences}, volume = {13}, journal = {Photochemical \& photobiological sciences}, number = {12}, publisher = {Royal Society of Chemistry}, address = {Cambridge}, issn = {1474-905X}, doi = {10.1039/c4pp00061g}, pages = {1699 -- 1710}, year = {2014}, abstract = {Sodium ions (Na+) play an important role in a plethora of cellular processes, which are complex and partly still unexplored. For the investigation of these processes and quantification of intracellular Na+ concentrations ([Na+](i)), two-photon coupled fluorescence lifetime imaging microscopy (2P-FLIM) was performed in the salivary glands of the cockroach Periplaneta americana. For this, the novel Na+-sensitive fluorescent dye Asante NaTRIUM Green-2 (ANG-2) was evaluated, both in vitro and in situ. In this context, absorption coefficients, fluorescence quantum yields and 2P action cross-sections were determined for the first time. ANG-2 was 2P-excitable over a broad spectral range and displayed fluorescence in the visible spectral range. Although the fluorescence decay behaviour of ANG-2 was triexponential in vitro, its analysis indicates a Na+-sensitivity appropriate for recordings in living cells. The Na+-sensitivity was reduced in situ, but the biexponential fluorescence decay behaviour could be successfully analysed in terms of quantitative [Na+](i) recordings. Thus, physiological 2P-FLIM measurements revealed a dopamine-induced [Na+](i) rise in cockroach salivary gland cells, which was dependent on a Na+-K+-2Cl-cotransporter (NKCC) activity. It was concluded that ANG-2 is a promising new sodium indicator applicable for diverse biological systems.}, language = {en} } @article{RoderHille2014, author = {Roder, Phillip and Hille, Carsten}, title = {ANG-2 for quantitative Na+ determination in living cells by time-resolved fluorescence microscopy}, series = {Photochemical \& Photobiological Sciences}, volume = {12}, journal = {Photochemical \& Photobiological Sciences}, number = {13}, editor = {Hille, Carsten}, publisher = {The Royal Society of Chemistry}, address = {Cambridge}, issn = {1474-905X}, pages = {1699 -- 1710}, year = {2014}, abstract = {Sodium ions (Na+) play an important role in a plethora of cellular processes, which are complex and partly still unexplored. For the investigation of these processes and quantification of intracellular Na+ concentrations ([Na+]i), two-photon coupled fluorescence lifetime imaging microscopy (2P-FLIM) was performed in the salivary glands of the cockroach Periplaneta americana. For this, the novel Na+-sensitive fluorescent dye Asante NaTRIUM Green-2 (ANG-2) was evaluated, both in vitro and in situ. In this context, absorption coefficients, fluorescence quantum yields and 2P action cross-sections were determined for the first time. ANG-2 was 2P-excitable over a broad spectral range and displayed fluorescence in the visible spectral range. Although the fluorescence decay behaviour of ANG-2 was triexponential in vitro, its analysis indicates a Na+-sensitivity appropriate for recordings in living cells. The Na+-sensitivity was reduced in situ, but the biexponential fluorescence decay behaviour could be successfully analysed in terms of quantitative [Na+]i recordings. Thus, physiological 2P-FLIM measurements revealed a dopamine-induced [Na+]i rise in cockroach salivary gland cells, which was dependent on a Na+-K+-2Cl- cotransporter (NKCC) activity. It was concluded that ANG-2 is a promising new sodium indicator applicable for diverse biological systems.}, language = {en} } @misc{RoderHille2014, author = {Roder, Phillip and Hille, Carsten}, title = {ANG-2 for quantitative Na+ determination in living cells by time-resolved fluorescence microscopy}, publisher = {The Royal Society of Chemistry}, address = {Cambridge}, url = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus4-76851}, pages = {1699 -- 1710}, year = {2014}, abstract = {Sodium ions (Na+) play an important role in a plethora of cellular processes, which are complex and partly still unexplored. For the investigation of these processes and quantification of intracellular Na+ concentrations ([Na+]i), two-photon coupled fluorescence lifetime imaging microscopy (2P-FLIM) was performed in the salivary glands of the cockroach Periplaneta americana. For this, the novel Na+-sensitive fluorescent dye Asante NaTRIUM Green-2 (ANG-2) was evaluated, both in vitro and in situ. In this context, absorption coefficients, fluorescence quantum yields and 2P action cross-sections were determined for the first time. ANG-2 was 2P-excitable over a broad spectral range and displayed fluorescence in the visible spectral range. Although the fluorescence decay behaviour of ANG-2 was triexponential in vitro, its analysis indicates a Na+-sensitivity appropriate for recordings in living cells. The Na+-sensitivity was reduced in situ, but the biexponential fluorescence decay behaviour could be successfully analysed in terms of quantitative [Na+]i recordings. Thus, physiological 2P-FLIM measurements revealed a dopamine-induced [Na+]i rise in cockroach salivary gland cells, which was dependent on a Na+-K+-2Cl- cotransporter (NKCC) activity. It was concluded that ANG-2 is a promising new sodium indicator applicable for diverse biological systems.}, language = {en} } @article{JahnHille2014, author = {Jahn, Karolina and Hille, Carsten}, title = {Asante calcium green and asante calcium red-novel calcium indicators for two-photon fluorescence lifetime imaging}, series = {PLoS one}, volume = {9}, journal = {PLoS one}, number = {8}, publisher = {PLoS}, address = {San Fransisco}, issn = {1932-6203}, doi = {10.1371/journal.pone.0105334}, pages = {13}, year = {2014}, abstract = {For a comprehensive understanding of cellular processes and potential dysfunctions therein, an analysis of the ubiquitous intracellular second messenger calcium is of particular interest. This study examined the suitability of the novel Ca2+-sensitive fluorescent dyes Asante Calcium Red (ACR) and Asante Calcium Green (ACG) for two-photon (2P)-excited time-resolved fluorescence measurements. Both dyes displayed sufficient 2P fluorescence excitation in a range of 720-900 nm. In vitro, ACR and ACG exhibited a biexponential fluorescence decay behavior and the two decay time components in the ns-range could be attributed to the Ca2+-free and Ca2+-bound dye species. The amplitude-weighted average fluorescence decay time changed in a Ca2+-dependent way, unraveling in vitro dissociation constants K-D of 114 nM and 15 nM for ACR and ACG, respectively. In the presence of bovine serum albumin, the absorption and steady-state fluorescence behavior of ACR was altered and its biexponential fluorescence decay showed about 5-times longer decay time components indicating dye-protein interactions. Since no ester derivative of ACG was commercially available, only ACR was evaluated for 2P-excited fluorescence lifetime imaging microscopy (2P-FLIM) in living cells of American cockroach salivary glands. In living cells, ACR also exhibited a biexponential fluorescence decay with clearly resolvable short (0.56 ns) and long (2.44 ns) decay time components attributable to the Ca2+-free and Ca2+-bound ACR species. From the amplitude-weighted average fluorescence decay times, an in situ K-D of 180 nM was determined. Thus, quantitative [Ca2+](i) recordings were realized, unraveling a reversible dopamine-induced [Ca2+](i) elevation from 21 nM to 590 nM in salivary duct cells. It was concluded that ACR is a promising new Ca2+ indicator dye for 2P-FLIM recordings applicable in diverse biological systems.}, language = {en} } @article{KlaussConradHille2017, author = {Klauß, Andr{\´e} and Conrad, Florian and Hille, Carsten}, title = {Binary phase masks for easy system alignment and basic aberration sensing with spatial light modulators in STED microscopy}, series = {Scientific reports}, volume = {7}, journal = {Scientific reports}, publisher = {Nature Publ. Group}, address = {London}, issn = {2045-2322}, doi = {10.1038/s41598-017-15967-5}, pages = {11}, year = {2017}, language = {en} } @article{SassStoeckleinKlevesathetal.2019, author = {Sass, Stephan and St{\"o}cklein, Walter F. M. and Klevesath, Anja and Hurpin, Jeanne and Menger, Marcus and Hille, Carsten}, title = {Binding affinity data of DNA aptamers for therapeutic anthracyclines from microscale thermophoresis and surface plasmon resonance spectroscopy}, series = {The analyst : the analytical journal of the Royal Society of Chemistry}, volume = {144}, journal = {The analyst : the analytical journal of the Royal Society of Chemistry}, number = {20}, publisher = {Royal Society of Chemistry}, address = {Cambridge}, issn = {0003-2654}, doi = {10.1039/c9an01247h}, pages = {6064 -- 6073}, year = {2019}, abstract = {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.}, language = {en} }