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- Two-Photon Microscopy of Cells and Tissue


Figure 1. Localization of TPE. Distribution of fluorescein fluorescence in the x-z plane during single-photon (1P) excitation by focused 488-nm laser light (a) and during two-photon (2P) excitation using femtosecond pulses of 850-nm light (b). The sample was illuminated through the same objective lens during one- and two-photon excitation. White lines indicate plane of focus. Reprinted from Soeller C, Cannell MB. Two-photon microscopy: imaging in scattering samples and three-dimensionally resolved flash photolysis. Microsc Res Tech. 1999;47:182–195, with permission of Wiley-Liss, Inc, a subsidiary of the John Wiley & Sons, Inc ©1999.

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Figure 2. Simulated effects of excitation wavelength and numerical aperture on the dimensions of the TPE volume. a, Normalized distributions of laser intensity-squared in the x-y and x-z plane for three different numerical apertures of water-immersion objective lenses at an excitation wavelength of 850 nm. Intensities-squared at lateral (x,y,0) and axial (x,0,z) positions were calculated using an ellipsoidal Gaussian approximation to the diffraction limited focus7,11 and expressed as the fraction of the intensity-squared at the focal point [I(0,0,0)2=1]. Color-coded contour plots depict isointensity lines in the x-z and x-y plane at the levels of 0.1, 0.3, 0.5, 0.7, and 0.9 of I(0,0,0)2. Note different scales for each panel. b, Dependence of TPE volume on numerical aperture of the objective lens and illumination wavelength. Values were obtained by approximating the intensity-squared distribution as a three-dimensional Gaussian volume.12 For all calculations, it is assumed that the objective lens is uniformly illuminated (overfilled) and that no saturation of the fluorescence excitation process occurs. NA indicates numerical aperture.

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Figure 3. Simultaneous TPE of fluorophores with disparate one-photon-absorption spectra. A Langendorff-perfused transgenic mouse heart expressing EGFP (peak one-photon excitation wavelength, {approx}490 nm) in a mosaic pattern was loaded with the calcium-sensitive dye rhod-2 (peak one-photon excitation wavelength of the Ca2+ bound form, {approx}550 nm). White line in full-frame image (a) denotes position of line-scan. Periodic increases in rhod-2 fluorescence in (b) correspond to action potential-evoked [Ca2+]i transients. Plots of the spatially averaged fluorescence intensities as function of time for an EGFP-expressing and nonexpressing cardiomyocyte (c) show cyclic changes in fluorescence only in the red channel, indicative of effective color separation in the detection path of the microscope.

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Figure 4. Applications of TPE-induced photolysis. a through d, Reprinted from Jacobs et al,48 with permission of the Association for Research in Vision and Ophthalmology ©2004. Functional consequences of gap junction redistribution in the lens. a and b, Connexin46 gap junctions (red) in the lens periphery are primarily located at the broad site of the hexagonally shaped lens epithelial cells (a), whereas gap junction plaques in cells at the center (b) appear to be randomly dispersed around the entire cell periphery. Green indicates wheat germ agglutinin staining of cell membrane. c and d, In a lens loaded with nonfluorescent CMNB-caged fluorescein, TPE-induced photolysis at a stationary spot in the periphery (c) and center (d) of the lens causes release of green fluorescent fluorescein within minuscule volumes around the focal point. Simultaneous monitoring of the time course of diffusion of uncaged fluorescein reveals anisotropic dye diffusion in the periphery but an approximately isotropic diffusion pattern in the center of the lens. Cell borders (red) are visualized with wheat germ agglutinin. e through g, Reprinted from DelPrincipe et al,8 with permission of the Nature Publishing Group (http://www.nature.com/) ©1999. Demonstration of refractoriness of Ca2+-induced Ca2+ release in a ventricular cardiomyocyte. e, Two highly localized (full width at half maximum=2 µm) Ca2+ transients are elicited by TPE of the photolyzable Ca2+ chelator DM-nitrophen. Changes in intracellular calcium levels associated with each pulse were recorded with the fluorescent calcium indicator fluo-3 and were largely attributable to local Ca2+ release. Note the similarity in the spatial and temporal characteristics of the two consecutive transients despite the short TPE pulse interval (300 ms). f, Surface plot showing two localized Ca2+ transients of equal amplitude elicited by two two-photon photolysis events at an interval of 300 ms, followed by a global Ca2+ signal that was triggered by UV-flash photolysis of caged Ca2+. The first two-photon photolysis signal after the global signal (arrow) was markedly suppressed. g, Spatially averaged temporal Ca2+ profiles before and during a global Ca2+ transient demonstrating slow recovery of TPE-induced Ca2+ signals.

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Figure 5. Applications of TPE fluorescence microscopy. a and b, Reprinted from Kleinfeld et al,26 with permission of the National Academy of Sciences, USA ©1998. a, Measurement of blood flow in an individual capillary in the cortex of an anesthetized rat. Red blood cells appear as dark spots in the fluorescently labeled serum. The change in position of a particular red blood cell is indicated by the series of arrows. b, Line-scan mode image obtained by repeatedly scanning along the central longitudinal axis of the capillary in (b). {Delta}x is the spatial dimension of the line scan, whereas {Delta}t is the time dimension, and {Delta}x/{Delta}t is the instantaneous blood flow velocity. c, Image collected from the kidney of a living rat. Rats were infused with a 3000 MW Cascade Blue dextran 24 hours before imaging. At the time of imaging, the rats were given Hoechst 33342, a 3000 MW Texas Red dextran, and a 500 000 MW Fluorescein dextran. The latter labels the plasma volume. Capillary blood flow is apparent from the shadows of circulating cells moving in the green-fluorescing volume of the blood. The 3000 MW dextran filter into Bowman space and travel through different tubule segments where they are secreted. The Cascade Blue dextran is localized toward the basal side of proximal tubule cells (PT) within mature lysosomes (thin arrow). The Texas Red dextran is localized to the early endosomes below the apical region of the cells (thick arrow). Distal tubules (DT) do not accumulate these dextrans within the cells. Their intratubular concentrations and, thus, fluorescence intensities, increase as water is reabsorbed. Nuclei of all cells are blue. Asterisk indicates white blood cell. The 100-second time series, from which this image was selected, is shown in the movie in the online data supplement. Image c and movie from the online data supplement were provided by K.W. Dunn, R. Sandoval, and B. Molitoris (Indiana University Center for Biological Microscopy, Indianapolis). d, Reprinted from Helmchen et al,4 with permissionof the Nature Publishing Group (http://www.nature.com/) ©1999. Spatial profile of dendritic [Ca2+]i transients ({Delta}F/F) associated with action potential bursts in a layer 5 pyramidal neuron filled with the fluorescent Ca2+ indicator Calcium Green-1. Left panel shows coronal side-projection of the indicator-filled neuron. Arrows indicate the depth for the Calcium Green-1 fluorescence recordings shown in the right panel.

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Figure 6. TPE fluorescence imaging of [Ca2+]i transients in individual cardiomyocytes within a Langendorff-perfused mouse heart. a and b, Stimulation-evoked single myocyte [Ca2+]i transients in the intact left ventricle of a mouse heart loaded with the fluorescent Ca2+ indicator rhod-2. a, Full-frame mode image during electrical stimulation at 1 Hz. Increases in rhod-2 fluorescence along the entire length of the horizontal scan line are apparent in the middle of the image, corresponding to a depolarization-induced increase in [Ca2+]i that occurred when the scan was approximately halfway across the field of vision. The stimulated [Ca2+]i transients appeared to rise quite uniformly as a result of intra- and intercellularly synchronized activation of SR calcium release. Red arrowheads, endothelial cells. b, Transverse line-scan mode images of action potential-evoked [Ca2+]i transients in three cardiomyocytes located at 10, 150, and 200 µm from the epicardial surface during stimulation at 1 Hz. Images were obtained by using a Zeiss LSM-510 Meta laser scanning microscope modified for two-photon illumination. The excitation beam (810 nm, {approx}100 fs, 82 MHz) was scanned through a x40, 1.2 numerical aperture water-immersion objective. Note the rapid and spatially uniform rise in [Ca2+]i in all three cells. c, Time courses of spatially averaged rhod-2 fluorescence from the cells in (b). d, Plots of normalized rhod-2 fluorescence as function of time derived from traces shown in (c).

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Figure 7. TPE Ca2+ imaging in hearts carrying cellular grafts. a through e, Simultaneous imaging of rhod-2 and EGFP fluorescence in a nontransgenic mouse following transplantation of EGFP-expressing fetal cardiomyocytes. Red (rhod-2) and green (EGFP) fluorescent signals were superimposed. a, Full-frame mode image obtained during continuous stimulation at 2 Hz. Ripple-like wavefronts correspond to action potential-evoked [Ca2+]i transients. b, Line-scan mode image of the region in (a) demarcated by the white line. The line scan encompasses 7 cardiomyocytes. Cells 1, 4, and 7 are host (EGFP-negative) cardiomyocytes and cells 2, 3, 5, and 6 are EGFP-expressing donor cardiomyocytes. The preparation was paced at the rate indicated. Spont. indicates spontaneous [Ca2+]i transient. c, Spatially integrated traces of the changes in rhod-2 (red) and EGFP (green) fluorescence for cardiomyocytes No. 1 (host) and No. 2 (donor). Tracings were recorded during pacing at 2 and 4 Hz as indicated. d, Normalized and superimposed tracings of electrically evoked changes in rhod-2 fluorescence as a function of time from host (squares and triangles) and donor (circles) cardiomyocytes. e, Full-frame images taken from the same heart as in (a) at the depths indicated. Preparation was paced continuously at 2 Hz. f, Full-frame mode images of a rhod-2 loaded following transplantation of EGFP-expressing skeletal myoblasts. The heart was stimulated at a rate of 4 Hz. Asterisk denotes noncoupled donor cell. g, Line-scan images depicting [Ca2+]i transients at 2 and 4 Hz along the white line in (f). h, Superimposed tracings of normalized changes in rhod-2 fluorescence as a function of time from the EGFP-positive myotube (green traces) and neighboring host EGFP-negative cardiomyocytes, respectively.

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