Supplementary data set
A non-invasive method for the in vivo tracking of high-speed vesicle transport in mouse neutrophils
Kenji Kikushima, Sayaka Kita and Hideo Higuchi*
*Department of Physics, Graduate School of Science, The University of Tokyo,
7-3-1 Bunkyo, Tokyo 113-0033, Japan
Correspondence and requests for materials should be addressed to H.H. (email: ).
Supplementary Movie 1
A flowing neutrophil in a blood vessel in the absence of inflammation.
The images were taken at 30 msec/frame and are shown at 90 msec/frame without averaging (one-third of the original speed). The width of the image is 69 m.
Supplementary Movie 2
A reconstructed 3D image of neutrophils bound to a blood vessel in response to inflammation.
A scanning session of 40 confocal images taken with 1.0-μm movements of the objective in 4.5 swas repeated 5 times. The images are shown at 0.2 sec/frame (22.5 times the original speed). The width of the image is 114 m.
Supplementary Movie 3
A three-dimensional rotation showing the distribution of neutrophils around blood vessels.
One set of confocal images shown in Video 2 was rotated 120° around the x-axis.
Supplementary Movie 4
Neutrophil migration in the interstitium following the induction of inflammation.
Fluorescent images of a neutrophil with bright vesicles moving 36 μm below the skin surface were captured at 100 msec/frame and averaged over successive ten images. The images are shown at 100 msec/frame (ten times the original speed). The width of the image is 22.3 m.
Supplementary Movie 5
Vesicle movement within a neutrophil in the interstitium.
Fluorescent images of vesicles within a neutrophil 50 m below the skin surface were obtained at 13.3 msec/frame. The images were not averaged and shown at the original speed. The width of the image is 28.5 m.
Supplementary Movie 6
A reconstructed 3D image of vesicles in a neutrophil.
A scanning session of nine confocal images taken with 1.0 m movements of the objective in 1.1 swas repeated60 times. The images are shown at 100 msec/frame (11 times the original speed). The depth of the image is 28.5 m, and the height of the image is 9 m.
Figures
Supplementary Figure S1: An additional example of the high-speed tracking of vesicles within an interstitial neutrophil.
(a) A fluorescent image of vesicles in a neutrophil. The images were captured at 14.4 msec/frame. Each image shown here was averaged over 50successive images.The vesicles were designated Vesicles 1–3. The bar indicates 10 m. (b) A 2D trace of each vesicle over 5.4 seconds for Vesicle 1 and 14.3 seconds for Vesicle 2 and 3. The fluorescent images were averaged five times to get clear images of the vesicles brighter than 40 photons/vesicle, and the two-dimensional trace of each vesicle was tracked using the FIONA method.Parameters for the fitting are available on Supplementary Table S1. (c) Speed of each vesicle. The positions of 5 successive points were averaged, and the velocity was calculated from the distance between these two positions.
SupplementaryFigure S2: Histogram ofmaximum velocitiesofindividual vesicles in the interstitial neutrophils.
21 vesicles in 6 interstitial neutrophils were analyzed. The velocity of each vesicle was calculated by the same method as Fig. 7 and Supplementary Fig. S1.
SupplementaryFigure S3: Continuous confocal images of the interstitium neutrophil (the same in Fig. 8) from shallow to deep in the skin with 1 um intervals in the focal position.
Each image was averaged by 15 successive images of same z-position to estimate the shape of the cell (white line). The focalposition of the optical slice started and ended in the middle of the cell because we needed to get both high spatial (1 um in z-axis) and temporal resolution (1.1 s intervals) to track the position of the vesicles by reducing the number of the optical slices. Width of each image: 28.5 um.
Method to determine the cell shape:Outlines of cell shape (solid white lines) were determined by the positions where intensity increased clearly brighter than that of background. The intensity inside the cell even at distance of a few micrometers away from bright vesicles was higher than that of background. The intensity of these bright vesicles should negligibilitycontribute to the distant intensity because standarddeviations (~0.3 m) of Gaussian were much shorter than a few micrometers. Therefore, we speculate that, besides a few bright vesicles, there were many small vesicles containing small number of QDs in the interstitial neutrophils, and thatQDs in the small vesicles of which intensity are too low to analyze the center of vesicles by FIONA contribute to the distant intensity. By this method, we could confirm that the bright vesicles we analyzed were located within the cell.
SupplementaryFigure S4: Neutrophils forming a cluster around a blood vessel after the application of a depilatory cream.
A transillumination image (a) and a fluorescent image (b) of blood vessels 40 m below the skin surface 5 min after the application of a depilatory cream. The images were captured at 30 msec/frame, and the fluorescent image (b) was averaged over successive 41 images. The transillumination image (a) was not averaged. Bar: 10 m.
Supplementary Figure S5: Effect of the averaging process on the displacement of vesicle.
Vesicle 1 of Fig. 6 were tracked on the original non-averaged images and on the five times averaged images.The displacement of the vesicle along the axis rotated to the vesicle movement was shown. Orange lines: displacement on the original non-averaged images (same as Fig. 6f). Black lines: displacement on thefive times averaged images.
Supplementary Table S1
Data for 2D-Gaussian fitting
SDs: (x, y) (pixel) / Regression coefficient: (r2) / Height of the peak:A (photons per pixel (223 nm)2) / Base line:B (photons per pixel (223 nm)2) / Number of photon: V
Fig. 4h (averaged by 5 times)
(1.52, 1.59) / 0.973 / 4.06 / 1.78 / 61.7
Fig. 6 (without averaging)
Vesicle 1 / (1.20, 1.09) / 0.981 / 197.10 / 21.91 / 1619.8
Vesicle 2 / (1.19, 1.21) / 0.939 / 120.60 / 14.79 / 1091.1
Vesicle 3 / (1.39, 1.61) / 0.962 / 102.49 / 24.16 / 1441.1
Fig. 7(averaged by 4 times)
Vesicle 1 / (1.81, 1.65) / 0.930 / 5.85 / 1.27 / 109.8
Vesicle 2 / (1.49, 1.30) / 0.923 / 4.96 / 1.15 / 60.4
Vesicle 3 / (1.43, 1.21) / 0.916 / 6.65 / 1.37 / 72.3
Vesicle 4 / (1.78, 1.29) / 0.895 / 3.95 / 1.17 / 57.0
Vesicle 5 / (2.52, 1.64) / 0.910 / 4.41 / 1.27 / 114.5
Fig. S1 (averaged by 5 times)
Vesicle 1 / (1.28, 1.29) / 0.926 / 4.11 / 1.24 / 42.7
Vesicle 2 / (1.98, 1.98) / 0.920 / 5.24 / 0.96 / 129.1
Vesicle 3 / (1.30, 1.68) / 0.899 / 4.23 / 1.20 / 57.9
Data were fitted with 2D Gaussian function:
where (x0, y0) denotes the center position of the Gaussian, (xy) is SDs in x- and y- axis, A is height of the peak, and B is base line. Photon number of Gaussian (V) is calculated by
V = 2Axy.
Width of one pixel corresponds to 223 nm.
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