@article{RoderHille2018, author = {Roder, Phillip and Hille, Carsten}, title = {Local tissue manipulation via a force- and pressure-controlled AFM micropipette for analysis of cellular processes}, series = {Scientific reports}, volume = {8}, journal = {Scientific reports}, publisher = {Nature Publ. Group}, address = {London}, issn = {2045-2322}, doi = {10.1038/s41598-018-24255-9}, pages = {9}, year = {2018}, abstract = {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.}, 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} } @misc{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 = {Postprints der Universit{\"a}t Potsdam : Mathematisch-Naturwissenschaftliche Reihe}, journal = {Postprints der Universit{\"a}t Potsdam : Mathematisch-Naturwissenschaftliche Reihe}, number = {1073}, issn = {1866-8372}, doi = {10.25932/publishup-47269}, url = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus4-472692}, pages = {22}, 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 µg/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{BruunHille2019, author = {Bruun, Kristina and Hille, Carsten}, title = {Study on intracellular delivery of liposome encapsulated quantum dots using advanced fluorescence microscopy}, series = {Scientific reports}, volume = {9}, journal = {Scientific reports}, publisher = {Nature Publ. Group}, address = {London}, issn = {2045-2322}, doi = {10.1038/s41598-019-46732-5}, pages = {15}, year = {2019}, abstract = {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.}, 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{GuentherKlaussToroNahuelpanetal.2019, author = {G{\"u}nther, Erika and Klauß, Andr{\´e} and Toro-Nahuelpan, Mauricio and Sch{\"u}ler, Dirk and Hille, Carsten and Faivre, Damien}, title = {The in vivo mechanics of the magnetotactic backbone as revealed by correlative FLIM-FRET and STED microscopy}, series = {Scientific reports}, volume = {9}, journal = {Scientific reports}, publisher = {Nature Publ. Group}, address = {London}, issn = {2045-2322}, doi = {10.1038/s41598-019-55804-5}, pages = {9}, year = {2019}, abstract = {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.}, language = {en} } @article{KhademHilleLoehmannsroebenetal.2017, author = {Khadem, S. M. J. and Hille, Carsten and L{\"o}hmannsr{\"o}ben, Hans-Gerd and Sokolov, Igor M.}, title = {Spot variation fluorescence correlation spectroscopy by data post-processing}, series = {Scientific reports}, volume = {7}, journal = {Scientific reports}, publisher = {Nature Publ. Group}, address = {London}, issn = {2045-2322}, doi = {10.1038/s41598-017-05672-8}, pages = {1 -- 9}, year = {2017}, abstract = {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.}, 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{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} } @article{KhademHilleLoehmannsroebenetal.2016, author = {Khadem, S. M. J. and Hille, Carsten and L{\"o}hmannsr{\"o}ben, Hans-Gerd and Sokolov, Igor M.}, title = {What information is contained in the fluorescence correlation spectroscopy curves, and where}, series = {Physical review : E, Statistical, nonlinear and soft matter physics}, volume = {94}, journal = {Physical review : E, Statistical, nonlinear and soft matter physics}, publisher = {American Physical Society}, address = {College Park}, issn = {2470-0045}, doi = {10.1103/PhysRevE.94.022407}, pages = {8}, year = {2016}, language = {en} } @misc{WessigHilleKumkeetal.2016, author = {Wessig, Pablo and Hille, Carsten and Kumke, Michael Uwe and Meiling, Till Thomas and Behrends, Nicole and Eisold, Ursula}, title = {Two-photon FRET pairs based on coumarin and DBD dyes}, url = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus4-394445}, 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} } @article{KlaussKoenigHille2015, author = {Klauß, Andr{\´e} and Koenig, Marcelle and Hille, Carsten}, title = {Upgrade of a Scanning Confocal Microscope to a Single-Beam Path STED Microscope}, series = {PLoS one}, volume = {10}, journal = {PLoS one}, number = {6}, publisher = {PLoS}, address = {San Fransisco}, issn = {1932-6203}, doi = {10.1371/journal.pone.0130717}, pages = {27}, year = {2015}, abstract = {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