Nature Methods Quantitative dynamic footprinting microscopy reveals mechanisms of neutrophil rolling Prithu Sundd, Edgar Gutierrez, Maria K Pospieszalska, Hong Zhang, Alexander Groisman & Klaus Ley Supplementary igure 1 Dependence of the evanescent wave intensity on the penetration depth. Supplementary igure 2 Supplementary igure 3 Estimation of o. Supplementary igure 4 Supplementary igure 5 Supplementary igure 6 Supplementary igure 7 Supplementary igure 8 Supplementary igure 9 Supplementary igure 10 Supplementary igure 11 Supplementary igure 12 Supplementary igure 13 Supplementary igure 14 Supplementary igure 15 Supplementary Table 1 Supplementary Note 1 ootprints of live and fixed neutrophils. Auto-perfused microfluidic device for studying neutrophil footprints with qd microscopy. Assembly of the magnetic clamp. P-selectin site densities. Adhesion specificity. mage thresholding of Di-labeled neutrophils. ootprints of rolling neutrophils identify locations of stressed and compressed bonds. Microvilli are preserved in the footprints of rolling neutrophils. Coordinates of microvilli tips in the footprints of rolling neutrophils. Comparison of the z positions of microvilli tips in the footprints of GP-expressing and Di-stained neutrophils. Location of the microvilli tips in the footprint of a rolling neutrophil. ootprints reveal anchorage points of long tethers at the rear of rolling neutrophils. Anchorage points of long tethers at the rear of rolling neutrophils. Calibration of the microfluidic device. Theory of qd microscopy. Note: Supplementary Videos 1 3 are available on the Nature Methods website.
SUPPLEMENTARY NORMATON SUPPLEMENTARY GURES AND LEGENDS Supplementary igure 1 Dependence of the evanescent wave intensity on the penetration depth. The intensity of the evanescent wave, E, decays exponentially with distance z above the cover slip and is defined by the penetration depth, d(θ) (Eq. 1-2 in Supplementary Note). E0 is the intensity of the evanescent wave at the interface (z = 0). θ 1 and θ 2 are representative of incidence angles greater than the critical angle, θ c = 64.33⁰ for the glass (n 1 =1.52)-cell (n 2 = 1.37) TR interface. n 1 and n 2 refractive index of glass and cell, respectively. 1
Supplementary igure 2 ootprints of live and fixed neutrophils. qd images of a live rolling neutrophil (wall shear stress 6 dyn/cm 2 ) and a fixed neutrophil (in static assay) stained with membrane dye Di were thresholded (similar to igs. 2d-i) to reveal footprints at different z distances from the cover slip as shown on the right. At 35 nm from the cover slip, the contact area for both live as well as fixed neutrophil is very small. Bar 10 µm. 2
Supplementary igure 3 Estimation of Δ o. Mouse bone marrow neutrophils were fixed and stained with Di. TR images were captured at incidence angles of θ = 68, 69, 70, and 71⁰. (θ), the intensity of the brightest pixel in the TR image, which is closest to the cover slip, was measured for all the four angles. Plotting ln[ (θ)/t(θ)] as a function of 1/d(θ) results in a straight line with slope equal to the z position (Δ o ) of the point of closest approach between the neutrophil and the cover slip (for details refer to Online Methods). A value of Δ o = 25 nm was used in all calculations. T(θ) is the transmission factor for linearly polarized incident laser and d(θ) is the penetration depth of the evanescent wave. 3
Supplementary igure 4 Auto-perfused microfluidic device for studying neutrophil footprints with qd microscopy. The region of interest (shown in black) on the cover slip (a) was coated with a solution containing 2 µg/ml of recombinant murine P-selectin-c and blocked with 1% casein. A custom designed PDMS chip with 9 identical flow channels (b) was placed on the cover slip (c) and assembled using a magnetic clamp (shown in Supplementary ig. 5; for details refer to Online Methods). The left carotid artery of an anesthetized mouse was cannulated using PE10 catheter (shown in red). The other end of the catheter was connected to the device inlet (c). The outlet of the device was connected by tubing to a reservoir filled with PBS which was raised or lowered to adjust the wall shear stress in the flow channels. Scale bar 10 mm. Mouse and outlet reservoir not drawn to scale. 4
Supplementary igure 5 Assembly of the magnetic clamp. The microfluidic chip and the cover slip were sealed together using the magnetic clamp 1. Scale bar 10 mm. 1. Tkachenko, E., Gutierrez, E., Ginsberg, M.H. & Groisman, A. Lab Chip 9, 1085-1095 (2009). 5
Supplementary igure 6 P-selectin site densities. P-selectin site densities on the cover slips used in microfluidic devices were measured using radio-immuno assay (for details refer to Online Methods). P-selectin site density plotted as a function of the coating concentration. Black diamonds are the individual data points and the blue line is the Langmuir-reundlich isotherm 2 ; inset- plot is magnified to show the site density at smaller concentrations. Each data point represents at least three separate experiments with each experiment consisting of triplicates. The error bars are s.e.m. 2. errante, E.A., Pickard, J.E., Rychak, J., Klibanov, A. & Ley, K. J. Control Release 140, 100-107 (2009). 6
Supplementary igure 7 Adhesion specificity. The number of rolling or arrested GPexpressing neutrophils on P-selectin (20 sites/µm 2 ) were counted for 10s and averaged for two fields of view (OV) to calculate the fraction of rolling (grey) or arrested (black) cells. The number on top of each bar is the total number (n) of neutrophils in that group. * indicates significant difference from P-selectin without mab treatment (α = 0.01). Wall shear stress 6 dyn/cm 2. 7
Supplementary igure 8 mage thresholding of Di-labeled neutrophils. The intensity histogram of the qd image of a Di labeled neutrophil rolling on P-selectin thresholded to reveal footprints at different z distances (35, 50, 75, and 100 nm) from the cover slip. or details refer to Online Methods. A fluorescence threshold value is estimated for any pixel in the qd image at a selected distance z above the cover slip (Eq. 8b in Supplementary Note). The position of the Min slider (yellow line) in the intensity histogram is fixed at this value to create a thresholded qd image showing membrane features that are within distance z above the cover slip. 8
Supplementary igure 9 ootprints of rolling neutrophils identify locations of stressed and compressed bonds. ootprints of two rolling GP-expressing neutrophils (flowing in x-direction) encoded as 2D color maps where regions in the footprint within 175 nm (a, b) or 100 nm (c, d) of the cover slip are encoded using the indicated color scheme. 3D surface maps (e, f) show the x and z co-ordinates of individual microvilli tips in the footprint. a, c, e and b, d, f represent cell 1 and 2, respectively. P-selectin 20 molecules/µm 2. Wall shear stress 6 dyn/cm 2. 9
Supplementary igure 10 Microvilli are preserved in the footprints of rolling neutrophils. A time series of 3D surface maps of the footprint of a GP expressing neutrophil rolling on P- selectin. Only three microvilli are labeled. Microvillus #2 is on the back side of the cell at t = 1.1 s and is therefore not visible. A microvillus was defined as a conical protrusion longer than 25 nm in z-direction. P-selectin 20 molecules/µm 2. Wall shear stress 6 dyn/cm 2. Panels a, c, and f also shown in igure 3. 10
Supplementary igure 11 Coordinates of microvilli tips in the footprints of rolling neutrophils. Raw data showing z positions of microvilli tips in the footprints of GP-expressing (open circles; n = 5 cells) and Di-stained (closed diamonds; n = 5 cells) neutrophils rolling on P- selectin plotted as a function of x position form the center of the cell. Sampled within 2 µm on each side of the center of the footprint in y direction. (a) Wall shear stress 6 dyn/cm 2. (b) Wall shear stress 8 dyn/cm 2. P-selectin 20 molecules/µm 2. The dashed line represents the unstressed length (~70 nm) of the P-selectin-PSGL-1 bond. 11
Supplementary igure 12 Comparison of the z positions of microvilli tips in the footprints of GP-expressing and Di-stained neutrophils. The z positions of microvilli tips reported in igure 3e were binned into the leading area (front, 2 µm < x < 4 µm; n GP = 9; n Di = 8) and rear (- 4 µm < x < - 2 µm; n GP = 18; n Di = 13). The average was computed for both GP and Di in each interval and compared using two-tailed students t-test with unequal variances and α = 0.05. Dashed line denotes the P-selectin-PSGL-1 equilibrium bond length ~ 70 nm. Wall shear stress 6 dyn/cm 2. P-selectin 20 molecules/µm 2. The GP and Di method report the same z distances in the front and rear of the footprint. The distance at the rear is significantly larger (P < 0.05) than in the front. Error bars are s.e.m. 12
Supplementary igure 13 Location of the microvilli tips in the footprint of a rolling neutrophil. (a-b) Positions of the microvilli tips in the footprint of two rolling neutrophils calculated by ETMA 3 ; wall shear stress 0.5 dyn/cm 2 and P-selectin site density 150 molecules/μm 2. Since the shear stress is low, the cell does not deform significantly resulting in a small footprint (~3 µm 2 ). (c-d) Positions of the microvilli tips measured from qd images of two GP-expressing neutrophils rolling in whole blood; wall shear stress 6 dyn/cm 2 and P-selectin 20 molecules/μm 2. The color bars encode the z position (nm) of each microvillus tip above the substrate. The microvilli at the rear (z distance > 70 nm) are held in place by stretched bonds. 13
The cell deforms significantly resulting in a larger footprint (12 ± 1 µm 2 (mean ± s.e.m; n = 8)). Black arrows show the direction of rolling. 3. Pospieszalska, M.K. & Ley, K. Cellular and Molecular Bioengineering 2, 207-217 (2009). 14
Supplementary igure 14 ootprints reveal anchorage points of long tethers at the rear of rolling neutrophils. (a, b) ootprints of two Di-stained neutrophils rolling (along x-direction) on P-selectin encoded as color maps showing anchorage points of long tethers at the rear of the footprint. The color bar encodes the z distance above the cover slip. (c, d) ootprints of the two Di-stained neutrophils denoted as 3D surface maps. Tether anchorage points are denoted with black arrows. a, c and b, d represent cell 1 and 2, respectively. Wall shear stress 8 dyn/cm 2. P- selectin 20 molecules/µm 2. 15
Supplementary igure 15 Anchorage points of long tethers at the rear of rolling neutrophils. (a) Processed qd images of a Di-stained neutrophil rolling on P-selectin from left to right at 8 dyn/cm 2 reveal anchorage points (red dots marked with white arrows) of long tethers at the rear. These results are also shown in Supplementary Video 2. (b) Processed qd images of a GPexpressing neutrophil rolling on P-selectin from left to right at 6 dyn/cm 2 reveal the anchorage points (green dots marked with white arrows) of long tethers at the rear of the neutrophil (for details on image processing refer to Online Methods). P-selectin 20 molecules/µm 2. Scale bar 5 µm. 16
ΔP (cm water) Τ w (dyn/cm 2 ) Q device (µl/min) 6.8 2 1.2 13.7 4 2.4 20.6 6 3.6 27.4 8 4.8 Supplementary Table 1 Calibration of the microfluidic device. ΔP is the pressure difference across the inlet and outlet, Τ w is the wall shear stress, and Q is the volumetric flow rate through the device. 17
SUPPLEMENTARY NOTE Theory of qd microscopy. The theory of TR microscopy and the estimation of cell-substrate separation distances from 2D TR images have been described previously 1,2. When a light beam propagating through a medium of refractive index n 1 (e.g. glass cover slip) meets an interface with an another medium of refractive index n 2 < n 1 (e.g. cell), it undergoes total internal reflection into the high refractive index medium for incidence angles (θ 1 or θ 2 in ig. 1a) greater 1 than the critical angle, sin n n c 2 1. Through total internal reflection, the incident light establishes an evanescent wave in the low refractive index (n 2 ) medium which decays exponentially with distance z from the interface (Supplementary ig. 1) as shown in Eq. 1. E z z d e (1) EO E z is the intensity of the evanescent wave at a distance z above the interface, EO is the intensity at the interface (z = 0), and d is the penetration depth of the evanescent wave, which depends on the wavelength of the incident light, λ, as well as the angle of incidence, θ, as shown in Eq. 2 and Supplementary igure 1. d 2 2 2 n n 4 sin 1 2 1 2 (2) The penetration depth is the characteristic of the evanescent wave and is the z distance above the glass cover slip at which the intensity of the evanescent wave drops to 1/e times of that at the interface. t varies from 50 to 200nm for a glass (n 1 =1.52) to cell (n 2 = 1.37) interface but never exceeds the wavelength, λ, of the incident light. As a result, fluorochromes in or close to the plasma membrane are excited to emit fluorescence while the rest of the cell remains dark. Thus, the TR image of a neutrophil with either GP in the cytoplasm or Di in the plasma membrane will show fluorescent regions of neutrophil membrane within ~200 nm above the cover slip. 18
(a) LysM-GP neutrophil with GP in the cytoplasm. The fluorescence intensity,, at any point on the membrane of the neutrophil at a distance z = Δ from the cover slip (refer ig. 1b) can be expressed as Eq. 3a. z d AT cze dz (3a) A is an experimental constant, T is the transmission factor for linearly or s-polarized incident light, and c z is the concentration of GP in the cytoplasm 1. A neutrophil in close proximity to a cover slip (as shown in ig. 1b) is a TR system with four different layers which are the glass cover slip, aqueous buffer layer between the neutrophil and the cover slip, the plasma membrane, and the cytoplasm, each with a different refractive index. t has been shown previously that a simple two layer model using the refractive index of the glass cover slip, n 1 = 1.52 and the cell cytoplasm, n 2 =1.37 (neglecting the plasma membrane and the aqueous buffer layer) results in negligible errors 3. Thus, a simple two layer model consisting of the neutrophil in contact with the cover slip was used to estimate Δ. f the GP concentration in the cytoplasm is uniform i.e. z c c, then at any angle of incidence, c where 2 4cos 1 n n 2 1 2 T and Eq. 3a simplifies to Eq.4a. o 64., c 33 which can be linearized to ln T d AT cd e (4a), d d Thus, a plot ofln T d against d ln Ac (5a) 1 for different values of c will be a straight line with the slope equal to -Δ. To measure intensity (θ) at different incidence angles θ, the 19
neutrophil must be stationary so that TR images of the neutrophil can be recorded at the same position at different values of θ. Since rolling neutrophils are not stationary, an alternative approach was used (see below) 2. The qd images of rolling neutrophils were recorded at an incidence angle of θ = 70⁰. n a qd image, the pixel with the maximum intensity was considered to be the closest to the cover slip and this distance was denoted by Δ o (as shown in ig. 1b). Substituting Δ = Δ o + δ in Eq.4a yields Eq.6a. f δ = 0, then Δ = Δ o and o d AT cd e max Dividing Eq. 6a with Eq. 7a results in Eq. 8a. (6a), substituting this in Eq. 6a yields Eq. 7a. o d AT cd e max (7a) d e max (8a) Writing Eq. 8a in linear form and substituting δ = Δ - Δ o yields Eq. 9a. max O d ln (9a) (b) Neutrophils with Di in the plasma membrane. The fluorescence intensity at any point on the membrane of the neutrophil at a distance z = Δ from the cover slip (refer ig. 1b) can be expressed as Eq. 3b. t 2 z d AT cze dz t 2 (3b) 20
A is an experimental constant, T is the transmission factor for linearly or s-polarized incident light, t is the thickness of the plasma membrane and c z is the concentration of Di in the plasma membrane 1. f the Di concentration is uniform i.e. cz c and 2 4cos 1 n n 2 1 2 T, then Eq. 3b simplifies to Eq. 4b. d AT cte (4b) Treatment of Eq. 4b similar to Eq. 4a yields a similar set of equations as shown below. ln T f Δ = Δ o + δ such that max d ln when δ = 0, then Act d e (5b) max (8b) d max O ln (9b) 21
References 1. Stock, K. et al. J. Microsc. 211, 19-29 (2003). 2. Truskey, G., Burmeister, J., Grapa, E. & Reichert, W. J. Cell Sci. 103, 491-499 (1992). 3. Olveczky, B.P., Periasamy, N. & Verkman, A.S. Biophys. J. 73, 2836-2847 (1997). 22