@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} } @article{SagollaLoehmannsroebenHille2013, author = {Sagolla, Kristina and L{\"o}hmannsr{\"o}ben, Hans-Gerd and Hille, Carsten}, title = {Time-resolved fluorescence microscopy for quantitative Ca2+ imaging in living cells}, series = {Analytical \& bioanalytical chemistry}, volume = {405}, journal = {Analytical \& bioanalytical chemistry}, number = {26}, publisher = {Springer}, address = {Heidelberg}, issn = {1618-2642}, doi = {10.1007/s00216-013-7290-6}, pages = {8525 -- 8537}, year = {2013}, abstract = {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.}, 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} } @article{SchulzeWehrholdHille2018, author = {Schulze, Sven and Wehrhold, Michel and Hille, Carsten}, title = {Femtosecond-Pulsed laser written and etched fiber bragg gratings for fiber-optical biosensing}, series = {Sensors}, volume = {18}, journal = {Sensors}, number = {9}, publisher = {MDPI}, address = {Basel}, issn = {1424-8220}, doi = {10.3390/s18092844}, pages = {20}, year = {2018}, abstract = {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.}, language = {en} } @article{TechenHilleDoscheetal.2012, author = {Techen, Anne and Hille, Carsten and Dosche, Carsten and Kumke, Michael Uwe}, title = {Fluorescence study of drug-carrier interactions in CTAB/PBS buffer model systems}, series = {Journal of colloid and interface science}, volume = {377}, journal = {Journal of colloid and interface science}, publisher = {Elsevier}, address = {San Diego}, issn = {0021-9797}, doi = {10.1016/j.jcis.2012.03.063}, pages = {251 -- 261}, year = {2012}, abstract = {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.}, language = {en} } @article{WessigBehrendsKumkeetal.2016, author = {Wessig, Pablo and Behrends, Nicole and Kumke, Michael Uwe and Eisold, Ursula and Meiling, Til and Hille, Carsten}, title = {Two-photon FRET pairs based on coumarin and DBD dyes}, series = {RSC Advances}, volume = {6}, journal = {RSC Advances}, publisher = {Royal Society of Chemistry}, address = {Cambridge}, issn = {2046-2069}, doi = {10.1039/c6ra03983a}, pages = {33510 -- 33513}, year = {2016}, abstract = {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.}, language = {en} }