Two-photon molecular excitation imaging of Ca2+transients in Langendorff-perfused mouse hearts

2003 ◽  
Vol 284 (6) ◽  
pp. C1654-C1668 ◽  
Author(s):  
Michael Rubart ◽  
Exing Wang ◽  
Kenneth W. Dunn ◽  
Loren J. Field
Keyword(s):  
Laser Scanning ◽  
Single Photon ◽  
Laser Scanning Microscopy ◽  
Fluorescent Indicators ◽  
Fura 2 ◽  
Two Photon ◽  
General Utility ◽  
Photon Excitation ◽  
Molecular Excitation ◽  
The Times

The ability to image calcium signals at subcellular levels within the intact depolarizing heart could provide valuable information toward a more integrated understanding of cardiac function. Accordingly, a system combining two-photon excitation with laser-scanning microscopy was developed to monitor electrically evoked [Ca2+]itransients in individual cardiomyocytes within noncontracting Langendorff-perfused mouse hearts. [Ca2+]itransients were recorded at depths ≤100 μm from the epicardial surface with the fluorescent indicators rhod-2 or fura-2 in the presence of the excitation-contraction uncoupler cytochalasin D. Evoked [Ca2+]i transients were highly synchronized among neighboring cardiomyocytes. At 1 Hz, the times from 90 to 50% ( t 90–50%) and from 50 to 10% ( t 50–10%) of the peak [Ca2+]i were (means ± SE) 73 ± 4 and 126 ± 10 ms, respectively, and at 2 Hz, 62 ± 3 and 94 ± 6 ms ( n = 19, P < 0.05 vs. 1 Hz) in rhod-2-loaded cardiomyocytes. [Ca2+]i decay was markedly slower in fura-2-loaded hearts ( t 90–50% at 1 Hz, 128 ± 9 ms and at 2 Hz, 88 ± 5 ms; t 50–10% at 1 Hz, 214 ± 18 ms and at 2 Hz, 163 ± 7 ms; n = 19, P < 0.05 vs. rhod-2). Fura-2-induced deceleration of [Ca2+]i decline resulted from increased cytosolic Ca2+ buffering, because the kinetics of rhod-2 decay resembled those obtained with fura-2 after incorporation of the Ca2+ chelator BAPTA. Propagating calcium waves and [Ca2+]i amplitude alternans were readily detected in paced hearts. This approach should be of general utility to monitor the consequences of genetic and/or functional heterogeneity in cellular calcium signaling within whole mouse hearts at tissue depths that have been inaccessible to single-photon imaging.

scholarly journals Assessment of Fluorochromes for Two-Photon Laser Scanning Microscopy of Biofilms

2002 ◽  
Vol 68 (2) ◽  
pp. 901-909 ◽  
Author(s):  
Thomas R. Neu ◽  
Ute Kuhlicke ◽  
John R. Lawrence
Keyword(s):  
Nucleic Acid ◽  
Laser Scanning ◽  
Single Photon ◽  
Selective Excitation ◽  
Laser Scanning Microscopy ◽  
Scanning Microscopy ◽  
Excitation Wavelength ◽  
Laser Microscopy ◽  
Two Photon ◽  
Photon Excitation

ABSTRACT A major limitation for the use of two-proton laser scanning microscopy (2P-LSM) in biofilm and other studies is the lack of a thorough understanding of the excitation-emission responses of potential fluorochromes. In order to use 2P-LSM, the utility of various fluorochromes and probes specific for a range of biofilm constituents must be evaluated. The fluorochromes tested in this study included classical nucleic acid-specific stains, such as acridine orange (AO) and 4",6"-diamidino-2-phenylindole (DAPI), as well as recently developed stains. In addition, stains specific for biofilm extracellular polymeric substances (EPS matrix components) were tested. Two-photon excitation with a Ti/Sapphire laser was carried out at wavelengths from 760 to 900 nm in 10-nm steps. It was found that autofluorescence of phototrophic organisms (cyanobacteria and green algae) resulted in strong signals for the entire excitation range. In addition, the coenzyme F420-related autofluorescence of methanogenic bacteria could be used to obtain images of dense aggregates (excitation wavelength, 780 nm). The intensities of the emission signals for the nucleic acid-specific fluorochromes varied. For example, the intensities were similar for excitation wavelengths ranging from 780 to 900 nm for AO but were higher for a narrower range, 780 to 810 nm, for DAPI. In selective excitation, fading, multiple staining, and combined single-photon-two-photon studies, the recently developed nucleic acid-specific fluorochromes proved to be more suitable regardless of whether they are intended for living or fixed samples. Probes specific for proteins and glycoconjugates allowed two-photon imaging of polymeric biofilm constituents. Selective excitation-emission was observed for Calcofluor White M2R (780 to 800 nm) and SyproOrange (880 to 900 nm). In addition, fluor-conjugated concanavalin A lectins were examined and provided acceptable two-photon emission signals at wavelengths ranging from 780 to 800 nm. Finally, CellTracker, a fluorochrome suitable for long-term labeling of microbial eucaryote cells, was found to give strong emission at wavelengths ranging from 770 to 810 nm. If fluorochromes have the same two-photon excitation cross section, they are suitable for multiple staining and multichannel recording. Generally, if an appropriate excitation wavelength and fluorochrome were used, it was possible to obtain more highly resolved images for thick biofilm samples with two-photon laser microscopy than with conventional single-photon laser microscopy. Due to its potential for higher resolution in light-scattering tissue-like material, such as biofilms, and extremely localized excitation, 2P-LSM is a valuable addition to conventional confocal laser scanning microscopy with single-photon excitation. However, further development of the method and basic research are necessary to take full advantage of nonlinear excitation in studies of interfacial microbial ecology.

scholarly journals Cardiovascular Imaging Using Two-Photon Microscopy

2008 ◽  
Vol 14 (6) ◽  
pp. 492-506 ◽  
Author(s):  
John A. Scherschel ◽  
Michael Rubart
Keyword(s):  
Laser Scanning ◽  
Single Photon ◽  
Second Harmonic ◽  
Multiphoton Microscopy ◽  
Cardiovascular Imaging ◽  
Laser Scanning Microscopy ◽  
Dye Transfer ◽  
Two Photon ◽  
Photon Excitation ◽  
Two Photon Excitation

AbstractTwo-photon excitation microscopy has become the standard technique for high resolution deep tissue and intravital imaging. It provides intrinsic three-dimensional resolution in combination with increased penetration depth compared to single-photon confocal microscopy. This article will describe the basic physical principles of two-photon excitation and will review its multiple applications to cardiovascular imaging, including second harmonic generation and fluorescence laser scanning microscopy. In particular, the capability and limitations of multiphoton microscopy to assess functional heterogeneity on a cellular scale deep within intact, Langendorff-perfused hearts are demonstrated. It will also discuss the use of two-photon excitation-induced release of caged compounds for the study of intracellular calcium signaling and intercellular dye transfer.

scholarly journals Preface: Multiphoton Microscopy

2008 ◽  
Vol 14 (6) ◽  
pp. 481-481
Author(s):  
Heide Schatten
Keyword(s):  
Penetration Depth ◽  
Laser Scanning ◽  
Single Photon ◽  
Multiphoton Microscopy ◽  
Laser Scanning Microscopy ◽  
Live Cells ◽  
Great Success ◽  
Two Photon ◽  
The Past ◽  
Photon Excitation

Two-photon excitation microscopy (also referred to as multiphoton laser scanning microscopy) has gained increasing popularity during the past few years because of the distinct advantages over single-photon microscopy, which includes increased penetration depth and low out-of-focus photobleaching and photodamage. It allows superior imaging of thick specimen compared to single-photon microscopy, and it excels at imaging live cells either single or within intact tissue. This highly valuable tool has been used with great success to gain important new insights into brain tissue, embryos, whole organs, entire animals, and it has been most useful in numerous other applications.

scholarly journals Single-photon-counting detector for increased sensitivity in two-photon laser scanning microscopy

2008 ◽  
Vol 33 (24) ◽  
pp. 2895 ◽  
Author(s):  
Richard K. P. Benninger ◽  
William J. Ashby ◽  
Elisabeth A. Ring ◽  
David W. Piston
Keyword(s):  
Laser Scanning ◽  
Single Photon ◽  
Photon Counting ◽  
Laser Scanning Microscopy ◽  
Scanning Microscopy ◽  
Single Photon Counting ◽  
Photon Counting Detector ◽  
Two Photon ◽  
Increased Sensitivity

Imaging Subcellular Changes in Living Mammalian Embryos Using 1047 Nm Two Photon Excitation Fluorescence Microscopy

1999 ◽  
Vol 5 (S2) ◽  
pp. 1060-1061
Author(s):  
J. M. Squirrell ◽  
D. L. Wokosin ◽  
B. D. Bavister ◽  
J. G. White
Keyword(s):  
Fluorescence Imaging ◽  
Laser Scanning ◽  
Prolonged Exposure ◽  
Laser Scanning Confocal Microscopy ◽  
Living Cells ◽  
Laser Scanning Microscopy ◽  
Scanning Confocal Microscopy ◽  
Two Photon ◽  
Photon Excitation ◽  
Mammalian Embryos

A major challenge for fluorescence imaging of living cells is maintaining viability during and following prolonged exposure to excitation illumination, especially when imaging over hours or even days, as when studying mammalian embryonic development. The use of specific fluorescently labeled components in living embryos promises to reveal the roles of organelles and molecules in a native and reproducible context. However, to gain a thorough understanding of dynamic biological systems, events of interest must be recorded as they occur, while limiting perturbations caused by the observation technique. Therefore, establishing long-term fluorescence imaging methods that maintain viability is critical for advancing our understanding of cell and developmental biology.One promising technique for imaging living cells is two photon laser scanning microscopy (TPLSM). The lower energy per photon and the restriction of fluorophore excitation to the imaged focal plane should reduce the total photodamage to thick specimens when compared to conventional laser scanning confocal microscopy (LSCM).

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