Fast fluorescence lifetime imaging of calcium in living cells.

A fast fluorescence lifetime imaging (FLIM) system is developed that can acquire images at a rate of hundreds of frames per second. The FLIM system is based on a wide-field microscope equipped with a time-gated intensified CCD detector and a pulsed laser. The time-gated detector acquires the signals from two time gates simultaneously and is therefore insensitive to movements of the specimen and photo-bleaching. The system is well suited for quantitative biological FLIM experiments and its performance is evaluated in calcium imaging experiments on beating neonatal rat myocytes. Several calcium sensitive dyes are characterized and tested for their suitability for fast FLIM experiments: Oregon Green Bapta-1 (OGB1), Oregon Green Bapta-2 (OGB2), and Oregon Green Bapta-5N (OGB5N). Overall the sensitivity range of these dyes is shifted to low calcium concentrations when used as lifetime dyes. OGB1 and OGB2 behave very similarly and can be used for FLIM-based calcium imaging in the range 1 to approximately 500 nM and OGB5N can be used up to 3 microM. The fast FLIM experiments on the myocytes could be carried out at a 100-Hz frame rate. During the beating of the myocytes a lifetime change of about 20% is observed. From the lifetime images a rest calcium level of about 65 nM is found.

[1]  J. Berlin,et al.  Ca2+ transients in cardiac myocytes measured with high and low affinity Ca2+ indicators. , 1993, Biophysical journal.

[2]  K. Svoboda,et al.  Estimating intracellular calcium concentrations and buffering without wavelength ratioing. , 2000, Biophysical journal.

[3]  Anthony W. Parker,et al.  Application of fluorescence lifetime imaging microscopy to the investigation of intracellular PDT mechanisms , 1997 .

[4]  T Takamatsu,et al.  High temporal resolution video imaging of intracellular calcium. , 1990, Cell calcium.

[5]  Marco Canepari,et al.  Imaging neuronal calcium fluorescence at high spatio-temporal resolution , 1999, Journal of Neuroscience Methods.

[6]  R. Tsien,et al.  Fluorescence ratio imaging: a new window into intracellular ionic signaling , 1986 .

[7]  R. Winslow,et al.  Cardiac Ca2+ dynamics: the roles of ryanodine receptor adaptation and sarcoplasmic reticulum load. , 1998, Biophysical journal.

[8]  D Thomas,et al.  A comparison of fluorescent Ca2+ indicator properties and their use in measuring elementary and global Ca2+ signals. , 2000, Cell calcium.

[9]  Hans C. Gerritsen,et al.  High frame rate fluorescence lifetime imaging , 2003 .

[10]  J. Lakowicz.  Principles of fluorescence spectroscopy , 1983 .

[11]  R. Tsien,et al.  A new generation of Ca2+ indicators with greatly improved fluorescence properties. , 1985, The Journal of biological chemistry.

[12]  Y. K. Levine,et al.  Quantitative pH imaging in cells using confocal fluorescence lifetime imaging microscopy. , 1995, Analytical biochemistry.

[13]  H Szmacinski,et al.  Fluorescence lifetime imaging of calcium using Quin-2. , 1992, Cell calcium.

[14]  R. Tsien,et al.  Cytoplasmic pH and free Mg2+ in lymphocytes , 1982, The Journal of cell biology.

[15]  Govindjee,et al.  Fluorescence Lifetime Imaging (FLI) in Real-Time - a New Technique in Photosynthesis Research , 2000, Photosynthetica.

[16]  W. Webb,et al.  Two-photon-excitation fluorescence imaging of three-dimensional calcium-ion activity. , 1994, Applied optics.

[17]  Y. K. Levine,et al.  Confocal fluorescence lifetime imaging of free calcium in single cells , 1994, Journal of Fluorescence.

[18]  H. Gerritsen,et al.  Multiple Time-Gate Module for Fluorescence Lifetime Imaging , 2001 .

[19]  Georges Wagnières,et al.  Instrumentation for real-time fluorescence lifetime imaging in endoscopy , 1999 .

[20]  Sytsma,et al.  Time‐gated fluorescence lifetime imaging and microvolume spectroscopy using two‐photon excitation , 1998 .

[21]  Hans C. Gerritsen,et al.  Fluorescence lifetime imaging of free calcium in single cells , 1994 .

[22]  W. Lederer,et al.  Spatial non-uniformities in [Ca2+]i during excitation-contraction coupling in cardiac myocytes. , 1994, Biophysical journal.

[23]  H T van der Voort,et al.  Imaging properties in two-photon excitation microscopy and effects of refractive-index mismatch in thick specimens. , 1999, Applied optics.

[24]  Hans C. Gerritsen,et al.  Fluorescence lifetime imaging using a confocal laser scanning microscope , 1992 .

[25]  Justin Teissié,et al.  Fluorescence imaging in the millisecond time range of membrane electropermeabilisation of single cells using a rapid ultra-low-light intensifying detection system , 1998, European Biophysics Journal.

[26]  D. Bers,et al.  Intrinsic cytosolic calcium buffering properties of single rat cardiac myocytes. , 1994, Biophysical journal.