Dietzel S , Pircher J , Nekolla AK , Gull M , Brändli AW , Pohl U , Rehberg M .

	Figure 2. THG line scanning in the mouse cremaster muscle.(a) Scheme of the process. Erythrocytes passing by cause a signal in each of the four sequentially scanned lines, recorded at time points T1 to T4. Lines are put together in an x-t-representation (bottom). Here, each erythrocyte moving along the scanned line shows a continuously advancing position, resulting in a streak of signal with a measurable angle. The orientation of the individual lines is the same in all following x-t-representations. (b) x-t-representation from a capillary, (c–f): Comparison of various evaluations on the same 30 µm cremaster venule. (c) x-t-representation. (d) Fourier-transformation of the x-t-representation which is partly shown in c. The calculated average velocity in this example was 1.5 mm/s. (e) Comparison of velocities measured with THG line scan visualized in C (1.56 mm/s±0.22 s.d.) and with fluorescent beads and a camera (1.65 mm/s±0.50 s.d., Figure S1) in the same venule. Horizontal lines indicate mean values. Double line scans (ds) scanned two parallel lines alternately, with ds-cen more centrally (1.56 mm/s±0.17) and ds-per more peripherally (1.41 mm/s±0.12). Note higher velocity in ds-cen (p = 0.037, t-test). (f) LS-PIV measurements with the line scanning data set visualized in C. Two of seven seconds continuous run are shown with a data point every 16 ms. Average speed was 1.3 mm/s±0.07 s.d. The inset shows the position of the scan line (red) in this venule, visualized by THG. (g) LS-PIV measurements at the center of a 50 µm arteriole from the same cremaster muscle with a data point every 3 ms. Mean velocity over 11.9 seconds (19895 data points) was 2.35 mm/s, heart rate was ∼250 beats/min.
	Figure 3. THG microscopy in the mouse ear.(a,b) Intravital imaging in different animals with arteriole (A), venule (V) and nerve fiber (N) running in parallel. In addition to the THG signal (cyan) SHG (red) is displayed. The arrow points to a typical linear vessel wall signal found only in arterioles. Scale bars 20 µm. The declining THG signal strength in more axial parts of a vessel are easiest to recognize in the arteriole in (a) and the venule in (b). (c–e) Flow velocity profile measurements by THG shifted line scan in the venule shown in b. (c) Clippings from x-t-representations of 12 parallel scan lines spaced 4.4 µm from each other. Lines 1, 11 and 12 were located in the vessel wall and thus show no movement. Note the shallower angles (faster velocity) and decreased intensity in the central scans. (d) Measurements derived from c by manual evaluation of individual streaks (circles) with mean values (horizontal lines) and mean values from LS-PIV calculations (diamonds). Scan lines 2 (0 µm) to 10 (35.2 µm) from (c) are included. Manually measured velocities revealed significant differences between the scan lines (p<0.0001, ANOVA) with significant differences between all direct neighbors (p<0.05 or smaller, post hoc Newman-Keuls test). For scan line 2 (0 µm), only a part of the x-t-representation showed blood flow. Apparently this line was close to the vessel wall so that slight movement could shift it outside. No reasonable LS-PIV average could thus be obtained. LS-PIV evaluations of the other scan lines confirmed significantly different blood flow velocities in general (p<0.0001; ANOVA, n = 88 for each line) and for all direct neighbors (****), except for the two central lines. (e) Measurements by manual evaluation for the arteriole shown in (b) Velocity measurements were significantly different between 0 and 4.4 µm lines (p = 0.0075) and between 8.8 and 13.2 µm lines (p = 0.0129). (f) LS-PIV results for the same arteriole. Only systolic maxima and diastolic minima (circles) and the mean values over all 3.2-millisecond-spaced 179 measurements (line) are shown. In the first scan line systole and diastole could not be identified reliably, therefore only the mean is given. Velocity differences between neighboring scan lines were all significant (p<0.0001) except between 13.2 and 17.6 µm (p = 0.87).
	Figure 4. THG line scan measurement of the blood flow velocity in the tail artery of a stage 45 Xenopus laevis larvae (4 days post fertilization).(a) xt-representation showing 680 lines of the recording, representing the first 0.46 s of the graph in (b). (b) LS-PIV results from the two seconds depicted show values oscillating between 4 mm/s systolic and 0.9 mm/s diastolic. The higher peaks are determined by ventricular heart contractions. Arrows mark the first three secondary peaks (see main text). Calculation of the heart rate yielded a value of about 200 beats per minute, the vessel diameter was 45 µm.
	Figure 5. Visualization of the cell free layer in the cremaster muscle by THG with mirror-enhanced signals[29]. (a) RBCs are excluded from a gap near the vessel wall. Boxed area magnified on the right. 1, vessel wall. 2, THG free gap. 3, RBC tracks. (b–d) Image of the same area after injection of fluorescently labeled 40 kDa dextran. THG in gray (b,c), dextran fluorescence in green (c,d). The THG free gap is unchanged in width. Contrary to the erythrocytes, this small dextran infiltrates the cell free layer. Scale bar 20 µm. A movie of this vessel is shown in Movie S1.
	Figure 1. Intravital THG imaging of blood flow in the mouse cremaster muscle.Images were scanned with lines from top to bottom and line addition from right to left. Arrows indicate direction of blood flow. All scale bars 20 µm. (a) Combined SHG (red) and THG (cyan) image with mirror-enhanced signals [29]. In the left vessel, RBCs flow with the direction of added scan lines, therefore some are elongated to intense streaks. Image brightness was adjusted to allow simultaneous visualization of low and high intensities with a gamma value of 2. (b) Flow in this capillary was with the scan direction from right to left, therefore RBCs appear elongated. (c) Flow in this capillary was against scan direction, from left to right, RBCs therefore appear much shorter (compare scale bars). (d) RBCs flowing in a ∼25 µm vessel. RBC shapes in the image are optically deformed by the relation of blood flow velocity and the scanning process (see main text). (e) THG recording of RBCs suitable for blood flow velocity measurement. Scheme on top illustrates the principle with scanlines 0–3, see Methods for details. In the example shown scan speed was 800 lines per second, the temporal distance between two lines was thus 1.25 ms. Pixel size was 0.31 µm. Blood flow velocity was determined to be 0.75 mm/s.

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