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1.
Figure 6

Figure 6. From: Oxygen Microscopy by Two-Photon-Excited Phosphorescence.

Diagram of the imaging system. APD1 and APD2: avalanche photodiodes; F1–F4: short-pass (SP), long-pass (LP) and notch filters.

Olga S. Finikova, et al. Chemphyschem. ;9(12):1673-1679.
2.
Figure 1

Figure 1. From: Oxygen Microscopy by Two-Photon-Excited Phosphorescence.

Two-photon-enhanced oxygen probe PtP-C343 consisting of phosphorescent Pt meso-tetraarylporphyrin (PtP, red), several coumarin-343 units (C343, blue), polyarylglycine dendrimer (black) and peripheral oligoethylene-glycol residues (green). Arrows in the cartoon depict excitation of the C343 antenna via 2PA (brown), FRET (yellow) and phosphorescence of PtP-core (red).

Olga S. Finikova, et al. Chemphyschem. ;9(12):1673-1679.
3.
Figure 3

Figure 3. From: Oxygen Microscopy by Two-Photon-Excited Phosphorescence.

Top: photophysical processes occurring in PtP-C343. a) calculated dependence of the fraction of excited triplet state molecules (PT1) in the laser focal volume on the number of high repetition rate pulses (840 nm, 110 fs, 76 MHz) at different average laser powers; b) calculated dependence of PT1 in the focal volume on the average laser power under continuous train of high repetition rate pulses. The inset shows the region where the phosphorescence is proportional to the square of the excitation flux.

Olga S. Finikova, et al. Chemphyschem. ;9(12):1673-1679.
4.
Figure 4

Figure 4. From: Oxygen Microscopy by Two-Photon-Excited Phosphorescence.

Imaging of heterogeneously oxygenated objects. a) A section of the glass capillary, about 200 μm from the tip, as seen through the eyepiece of the microscope. b) Integrated phosphorescence intensity image (50 × 50 pixel2) acquired under 2P excitation. c) Emission spectra from the areas 1 and 2 marked in image (b). d) Phosphorescence decays (CPC—counts-per-channel) in individual pixels in the regions 1 and 2 (1 μs gate, 100 gates per pixel, 50 ms pixel dwell time) and their fits to single-exponentials. Phosphorescence lifetime images of e) the capillary and f) the capillary tip. g) Stack of pO2 images obtained by raster-scanning in z-axial planes (10 gates per pixel).

Olga S. Finikova, et al. Chemphyschem. ;9(12):1673-1679.
5.
Figure 2

Figure 2. From: Oxygen Microscopy by Two-Photon-Excited Phosphorescence.

Properties of nanoprobe PtP-C343. a) Absorption and emission (λex = 460 nm, arbitrary intensity units) spectra of the probe and reference chromophores: PtP and C343. b) 2PA-induced emission spectra (excitation: λex = 840 nm, 110 fs, 76 MHz rep. rate) at different excitation powers and power dependence (inset) for phosphorescence of a reference Pt porphyrin (PtP) without the antenna (black squares), PtP-C343 phosphorescence (white circles) and PtP-C343 fluorescence (white triangles). The data points [phosphorescence intensity (I), normalized by concentration, vs excitation power (P)], were fit to quadratic functions: I(P)=aP2 (solid line). The phosphorescence deviates from the quadratic law at higher incident powers (see text and ). The fluorescence rises quadratically throughout the entire power range examined. c) Phosphorescence lifetime (τ) vs pO2 calibration plots in pure buffer (black) and in cell growth medium containing 3 % of serum albumin (25 °C, pH 7.2) (white). Inset: 2PA-induced emission spectra (λex = 840 nm) of the probe at air saturation (black) and in deoxygenated solution (gray), showing that only the phosphorescence signal (λmax = 680 nm) responds to the change in oxygen pressure.

Olga S. Finikova, et al. Chemphyschem. ;9(12):1673-1679.
6.
Figure 5

Figure 5. From: Oxygen Microscopy by Two-Photon-Excited Phosphorescence.

Images of fixed (a–e) and live (f–i) human umbilical vein endothelial cells (EC) after induced co-internalization of 2 μm latex microspheres and PtP-C343. 87 ± 3 % of the total number of microspheres were internalized, of which 86 ±7 % contained PtP-C343. 2P excitation was the same as in the experiments (e, f) in . a) Overlaid phase-contrasted and conventional fluorescence images show several cells with nuclei stained by a blue fluorescent dye (DAPI). The cell marked by the square contains several vesicles, where microspheres (white) are surrounded by the probe solution (red). b) Magnification (3.75-fold) of the perinuclear region of a selected cell. Small vesicles containing the probe, but no microspheres, appear as reddish dots around the nucleus. c) The same cell viewed through the eye-piece of the two-photon imaging microscope. The scanned area (square) and the nucleus (dashed line) are marked. d) Integrated intensity image of phosphorescence (50 × 50 pixels2). e) Phosphorescence lifetime image. f) Nucleus of a cell surrounded by internalized micro-spheres. g) Integrated intensity image of phosphorescence and the emission spectra (to the left) collected from the areas 1–3 (marked by the squares). h) Phosphorescence lifetime image. i) pO2 image calculated from the lifetime image (h).

Olga S. Finikova, et al. Chemphyschem. ;9(12):1673-1679.

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