Refine
Year of publication
Language
- English (25) (remove)
Is part of the Bibliography
- yes (25)
Keywords
- Confocal microscopy (2)
- Fluorescence imaging (2)
- Fluorescence spectroscopy (2)
- Fluorescent probes (2)
- acinar-cells (2)
- aptamers (2)
- cockroach salivary-glands (2)
- fiber Bragg gratings (2)
- fiber etching (2)
- fiber-optical sensors (2)
- hela-cells (2)
- intracellular na+ (2)
- lifetime microscopy (2)
- nanostructure fabrication (2)
- periplaneta-americana (2)
- rat (2)
- sodium green (2)
- ultra-fast laser inscription (2)
- ventricular myocytes (2)
- 2,10-Bis-(3-aminopropyloxy)dibenzo[aj]perylene-8,16-dione (1)
- 2P cross section (1)
- 5-Hexadecanoylaminofluorescein (1)
- C-reactive (1)
- C-reactive protein (1)
- Fluorescence anisotropy (1)
- Fluorescence correlation spectroscopy (1)
- Hexadecyltrimethylammonium bromide (1)
- MQAE (1)
- Na+-K+-2Cl(-) cotransporter (1)
- SBFI (1)
- Single-molecule FRET (1)
- acid (1)
- biomarker (1)
- conformational-changes (1)
- cytosolic sodium (1)
- epithelial ion transport (1)
- microscopy (1)
- proteins (1)
- resonance energy-tansfer (1)
Institute
- Institut für Chemie (25) (remove)
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.
Two-photon fluorescence lifetime imaging of intracellular chloride in cockroach salivary glands
(2009)
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.
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.
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.
ANG-2 for quantitative Na+ determination in living cells by time-resolved fluorescence microscopy
(2014)
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.
ANG-2 for quantitative Na+ determination in living cells by time-resolved fluorescence microscopy
(2014)
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.
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.
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.
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.