TY  - JOUR
A1  - Lahn, Mattes
A1  - Dosche, Carsten
A1  - Hille, Carsten
T1  - Two-photon microscopy and fluorescence lifetime imaging reveal stimulus-induced intracellular Na+ and Cl- changes in cockroach salivary acinar cells
JF  - American journal of physiology : Cell physiology
N2  - 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.
KW  - epithelial ion transport
KW  - Na+-K+-2Cl(-) cotransporter
KW  - MQAE
KW  - SBFI
KW  - 2P cross section
Y1  - 2011
U6  - https://doi.org/10.1152/ajpcell.00320.2010
SN  - 0363-6143
VL  - 300
IS  - 6
SP  - C1323
EP  - C1336
PB  - American Chemical Society
CY  - Bethesda
ER  - 
TY  - JOUR
A1  - Sagolla, Kristina
A1  - Löhmannsröben, Hans-Gerd
A1  - Hille, Carsten
T1  - Time-resolved fluorescence microscopy for quantitative Ca2+ imaging in living cells
JF  - Analytical & bioanalytical chemistry
N2  - 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.
KW  - Fluorescence lifetime
KW  - TCSPC
KW  - Two-photon excitation
KW  - 2P cross section
KW  - Epithelial ion transport
KW  - OGB-1
Y1  - 2013
U6  - https://doi.org/10.1007/s00216-013-7290-6
SN  - 1618-2642
VL  - 405
IS  - 26
SP  - 8525
EP  - 8537
PB  - Springer
CY  - Heidelberg
ER  -