@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}
}
@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 <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.},
  language  = {en}
}
@article{LahnDoscheHille2011,
  author    = {Lahn, Mattes and Dosche, Carsten and Hille, Carsten},
  title     = {Two-photon microscopy and fluorescence lifetime imaging reveal stimulus-induced intracellular Na+ and Cl- changes in cockroach salivary acinar cells},
  series = {American journal of physiology : Cell physiology},
  volume    = {300},
  journal   = {American journal of physiology : Cell physiology},
  number    = {6},
  publisher = {American Chemical Society},
  address   = {Bethesda},
  issn      = {0363-6143},
  doi       = {10.1152/ajpcell.00320.2010},
  pages     = {C1323 -- C1336},
  year      = {2011},
  abstract  = {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.},
  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{HilleLahnLoehmannsroebenetal.2009,
  author    = {Hille, Carsten and Lahn, Mattes and L{\"o}hmannsr{\"o}ben, Hans-Gerd and Dosche, Carsten},
  title     = {Two-photon fluorescence lifetime imaging of intracellular chloride in cockroach salivary glands},
  issn      = {1474-905X},
  doi       = {10.1039/B813797H},
  year      = {2009},
  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{HilleBergBresseletal.2008,
  author    = {Hille, Carsten and Berg, Maik and Bressel, Lena and Munzke, Dorit and Primus, Philipp and L{\"o}hmannsr{\"o}ben, Hans-Gerd and Dosche, Carsten},
  title     = {Time-domain fluorescence lifetime imaging for intracellular pH sensing in living tissues},
  doi       = {10.1007/s00216-008-2147-0},
  year      = {2008},
  abstract  = {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.},
  language  = {en}
}
@article{MareljaChowdhuryDoscheetal.2013,
  author    = {Marelja, Zvonimir and Chowdhury, Mita Mullick and Dosche, Carsten and Hille, Carsten and Baumann, Otto and L{\"o}hmannsr{\"o}ben, Hans-Gerd and Leimk{\"u}hler, Silke},
  title     = {The L-cysteine desulfurase NFS1 is localized in the cytosol where it provides the sulfur for molybdenum cofactor biosynthesis in humans},
  series = {PLoS one},
  volume    = {8},
  journal   = {PLoS one},
  number    = {4},
  publisher = {PLoS},
  address   = {San Fransisco},
  issn      = {1932-6203},
  doi       = {10.1371/journal.pone.0060869},
  pages     = {13},
  year      = {2013},
  abstract  = {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.},
  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{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}
}