Towards the Prediction of Antimicrobial Efficacy for Hydrogen Bonded, Self‐Associating Amphiphiles

Abstract Herein we report 50 structurally related supramolecular self‐associating amphiphilic (SSA) salts and related compounds. These SSAs are shown to act as antimicrobial agents, active against model Gram‐positive (methicillin‐resistant Staphylococcus aureus) and/or Gram‐negative (Escherichia coli) bacteria of clinical interest. Through a combination of solution‐state, gas‐phase, solid‐state and in silico measurements, we determine 14 different physicochemical parameters for each of these 50 structurally related compounds. These parameter sets are then used to identify molecular structure‐physicochemical property‐antimicrobial activity relationships for our model Gram‐negative and Gram‐positive bacteria, while simultaneously providing insight towards the elucidation of SSA mode of antimicrobial action.

a -Original SSA underwent reaction during crystallisation process.   a -could not be calculated due to compound solubility.  3.18 6.36 a Ampicillin a -End point of experiment predicted due to compound solubility. b -MIC50 determination not possible due to compound solubility. c -SSA unstable in solution. d -Clouding of media observed upon addition of SSA solution to bacterial culture, MIC50 data should be treated with caution. F -Compound failed initial screening.

Experimental
General remarks: A positive pressure of nitrogen and oven dried glassware were used for all reactions. All solvents and starting materials were purchased from known chemical suppliers or available stores and used without any further purification unless specifically stipulated. The NMR spectra were obtained using a Burker AV2 400 MHz or AVNEO 400 MHz spectrometer. The data was processed using ACD Labs, MestReNova or Topspin software. NMR Chemical shift values are reported in parts per million (ppm) and calibrated to the centre of the residual solvent peak set (s = singlet, br = broad, d = doublet, t = triplet, q = quartet, m = multiplet). Tensiometry measurements were undertaken using the Biolin Scientific Theta Attension optical tensiometer. The data was processed using Biolin OneAttension software. A Hamilton (309) syringe was used for the measurements. The melting point for each compound was measured using Stuart SMP10 melting point apparatus. High resolution mass spectrometry was performed using a Bruker microTOF-Q mass spectrometer and spectra recorded and processed using Bruker's Compass Data Analysis software. Infrared spectra were obtained using a Shimadzu IR-Affinity-1 model Infrared spectrometer. The data are analysed in wavenumbers (cm -1 ) using IRsolution software. DLS and Zeta Potential studies were carried out using Anton Paar Litesizer TM 500 and processed using KalliopeTM Professional or using a Malvern Zetasizer Nano ZS. Cellular growth curve measurements obtained using Thermo Scientific Multiscan Go 1510-0318C plate reader and recorded using the SkanIt Software 4.0 and a Clariostar plater reader using MARS data analysis software.
Mass Spectrometry: Approximately 1 mg of each compound was dissolved in 1 mL of methanol. This solution was further diluted 100-fold before undergoing analysis where 10 μL of each sample was then injected directly into a flow of 10 mM ammonium acetate in 95 % water (flow rate = 0.02 mL/min).
Tensiometry Studies: All the samples were prepared in an EtOH: H2O (1:19) solution. All samples underwent an annealing process in which the various solutions were heated to approximately 40 °C before being allowed to cool to room temperature, allowing each sample to reach a thermodynamic minimum. All samples were prepared through serial dilution of the most concentrated sample. Three surface tension measurements were obtained for each sample at a given concentration, using the pendant drop method.
The average values were then used to calculate the critical micelle concentration (CMC).

DLS Studies:
All vials used for preparing the samples were clean dry. All solvents used were filtered to remove any particulates that may interfere with the results obtained. Samples of differing concentrations were obtained through serial dilution of a concentrated solution. All samples underwent an annealing process, in which they were heated to 40 °C before being allowed to cool to 25 °C. A series of 9 or 10 runs were recorded at 25 °C.
Zeta Potential Studies: All vials used for preparing the samples were clean dry. All solvents used were filtered to remove any particulates that may interfere with the results obtained. All samples underwent an annealing process in which the various solutions were heated to approximately 40 °C before cooling to room temperature, allowing each sample to reach a thermodynamic minimum. The final zeta potential value given is an average of the number of experiments conducted at 25 °C.
Single Crystal X-ray Studies: A suitable crystal of each amphiphile was selected and mounted on a Rigaku Oxford Diffraction Supernova diffractometer. Data were collected using Cu Kα radiation at 100 K or 293 K as necessary due to crystal instability at lower temperatures. Structures were solved with the ShelXT or ShelXS structure solution programs via Direct Methods and refined with ShelXL by Least Squares minimisation. Olex2 was used as an interface to all ShelX programs (CCDC 1866274-1866275).

Biological experiments 1 Bacteria experimental
Preparation of antimicrobial compounds for screening: Compounds were dissolved in 5 % ethanol to make up 20 mM solutions on the day of experiment.
Preparation of antimicrobial compounds for MIC50 calculations: Compounds were dissolved into 5 % ethanol to make up solutions on the day of experiment. Eight concentrations ranging were made up using 5 % ethanol.
Preparation of bacterial plates: Sterile LB agar plates were streaked using the desired bacteria (either Escherichia coli or USA300 Methicillin-Resistant Staphylococcus aureus) then incubated at 25°C overnight.
Preparation of Inoculum: An initial culture was made up by inoculating LB media (5 mL) with at least 4 single colonies of the desired bacteria under sterile conditions and incubating at 37 °C overnight. The following day, a subculture was made up using LB (5 mL) and the initial culture (50 µL), then incubated at 37°C until the culture had reached an optical density (OD) of 0.4 at 600 nm. Optical Density was adjusted using sterile dH2O to equal 0.5 McFarland standard (10 7 -10 8 cfu/mL), then a 1:10 dilution was carried out using sterile dH2O (900 µL) and the McFarland adjusted suspension (100 µL). A final dilution (1:100) was carried using the 1:10 suspension (150 µL) and LB (14.85 mL) before use to achieve a final cell concentration of 10 5 cfu/mL. Preparation of 96 well microplate for screening: 20 mM solutions of each compound to be tested were made up using 5 % ethanol. The 1:100 cell suspension (150 µL) was pipetted into the wells. Compound solutions (30 µL) were added into 6 wells on the plate so that 14 compounds could be screened on each plate. The final screening concentration for each compound was 3.3 mM in the well. These were incubated for 20 hours in a plate reader, with optical density readings being taken at 600 nm every 15 minutes. Optical density readings were plotted against time to produce growth curves. Compounds that inhibited growth by 10 % or more were taken forward for MIC50 calculations.
Preparation of 96 well microplate for MIC50: The 1:100 cell suspension (150 µL) was dispensed into individual wells under sterile conditions. Compounds (30 µL) were added to the wells to equal a total volume of 180 µL in the wells. Six repeats of each concentration for each compound were created each plate. The plates were sealed using parafilm, then incubated at 37°C in a microplate reader for 18-25 hours. Optical density readings were taken at 600 nm every 15 min. Data was used to generate growth curves.
Calculation of MIC50: Growth curves were plotted using OD600 optical density readings in Microsoft ® Excel ® 2013. OD600 optical density readings 900 minutes from the start of each of these growth cultures for each concentration of drug were plotted in Origin ® 2015. The resultant curve was normalized and fitted using the Boltzmann fit to define the MIC50 values for each drug.

Competitive binding assay (E. coli and MRSA)
Cultures: An initial culture was created by inoculating LB media (5 mL) with at least 4 single colonies of desired bacteria under sterile conditions and then incubating at 37 °C overnight. The following day, the initial culture (100 µL) was used to inoculate fresh LB media (10 mL) and incubated at 37 °C to form a secondary culture. This was grown to mid log phase (~ OD 0.4), then each culture was separated into four 1.5 mL Eppendorfs under sterile conditions (500 µL in each). In the Eppendorf with no compound or FM 4-64 present, only the cell culture, Fresh LB (100 µL) and 5% ethanol (100 µL, v/v) was added (total volume 700 µL). In the Eppendorf with only FM 4-64 present, the cell culture (500 µL), fresh LB (100 µL) 5% ethanol (100 µL) and FM 4-64 (0.7 µL from a stock solution of 5 ugmL -1 ) were added. For the Eppendorf with only compound 39 present, the cell culture (500 µL), fresh LB (100 µL) and compound 39 (100 µl, 5 mM) dissolved in 5% ethanol. For the Eppendorf with both compound 39 and FM 4-64 present, the culture (500 µL), fresh LB (100 µL) were added, then FM 4-64 was added before adding compound 39 (100 µL) roughly a minute after. These Eppendorfs were incubated for 30 minutes at 25 °C, then microscopy samples (6-8 µL) were taken.
Microscopy: Cells were mounted onto the agarose LB pads under coverslips as described previously. 8 Samples were visualised using an Olympus IX71 microscope with UAPON 150x 1.45 NA TIRF lens mounted on a PIFOC z-axis focus drive (Physik Instrumente, Karlsruhe, Germany), and illuminated using LED light sources (Cairn Research Ltd, Faversham, UK) with appropriate filters (Chroma, Bellows Falls, VT). Samples were visualised using a Zyla 4.2 CMOS camera (Andor), and the system was controlled with Metamorph software (Molecular Devices). Images were analysed using Metamorph and Fiji software. 9

Chemical Synthesis
Compound 1: This compound was synthesised in line with our previously published methods. Proton NMR were found to match our previously published values. 2  Compound 16: Aminomethane sulfonic acid (0.44 g, 4.00 mM) was added to a stirring solution of 4-(Trifluoromethyl) phenyl isothiocyanate (0.82 g, 4.00 mM) in anhydrous pyridine (20 mL) under an inert atmosphere. The mixture was heated to 60 °C overnight. The pyridinium salt was then removed by filtration as a white solid with a yield of 78 % (1.23 g, 3.14 mM). The pyridinium salt (0.78 g, 2.00 mM) was dissolved in a solution of tetramethylammonium (TMA) hydroxide pentahydrate (0.19 g) and H2O (0.90 g) to give the pure product as a white solid with a yield of 98 % (0. Compound 40: Aminomethane sulfonic acid (0.16 g, 1.50 mM) was dissolved in tetrabutylammonium in methanol (1.50 mL, 1.50 mM) with excess methanol to aid dissolving before being taken to complete dryness. Tetrabutylammonium aminomethane sulfonate (1.50 mM) was dissolved in chloroform (12.5 mL) and left overnight in a sealed vial with 1-isocyanatobutane (0.15 g, 1.50 mM). The resulting solution was taken to dryness, re-dissolved in ethyl acetate (15 mL) and extracted with water (15 mL). The pure product was obtained by flash chromatography 100 % ethyl acetate followed by 100 % methanol. The methanol fraction was taken to dryness to give the pure product as an opaque viscous oil with a yield of 63 % (0.28 g, 0.62 mM); Melting Point: 131 °C; 1  Compound 41: Tetrabutylammonium in methanol (1.50 mL, 1.50 mM) was added to 2-aminoethane-1sulfonic acid (0.19 g, 1.50 mM), excess methanol was added to aid solubility before being taken to complete dryness. Tetrabutylammonium 2-aminoethane-1-sulfonate (1.50 mM) was dissolved in pyridine (2.0 mL) and left overnight in a sealed vial with 1-isocyanatobutane (0.15 g, 1.50 mM). The resulting solution was taken to dryness, re-dissolved in ethyl acetate (15 mL) and extracted with water (15 mL). The pure product was obtained by flash chromatography 100 % ethyl acetate followed by 100 % methanol. The methanol fraction was taken to dryness to give the pure product as an opaque viscous oil with a yield of 73 % (0.31 g, 0.67 mM); Melting Point: 135 °C; 1  Compound 42: Tetrabutylammonium in methanol (1.50 mL, 1.50 mM) was added to 3-aminopropane-1sulfonic acid (0.21 g, 1.50 mM), excess methanol was added to aid solubility before being taken to complete dryness. Tetrabutylammonium 3-aminopropane-1-sulfonate (1.50 mM) was dissolved in pyridine (2.0 mL) and left overnight in a sealed vial with 1-isocyanatobutane (0.15 g, 1.50 mM). The resulting solution was taken to dryness, re-dissolved in ethyl acetate (15 mL) and extracted with water (15 mL). The pure product was obtained by flash chromatography 100 % ethyl acetate followed by 100 % methanol. The methanol fraction was taken to dryness to give the pure product as a cream oil with a yield of 56 % (0.27 g, 0.56 mM); Melting Point: 145 °C; 1  Compound 43: Aminomethane sulfonic acid (0.16 g, 1.50 mM) was dissolved in tetrabutylammonium in methanol (1.50 mL) with excess methanol to aid dissolving before being taken to complete dryness. Tetrabutylammonium aminomethane sulfonate (1.50 mM) was dissolved in chloroform (12.5 mL) and left overnight in a sealed vial with 1-isothiocyanatobutane (0.18 mL, 1.50 mM). The resulting solution was taken to dryness, re-dissolved in ethyl acetate (15 mL) and extracted with water (15 mL). The pure product was obtained by flash chromatography 100 % ethyl acetate followed by 100 % methanol. The methanol fraction was taken to dryness to give the pure product as an opaque viscous oil with a yield of 32 % (0. Compound 44: Tetrabutylammonium in methanol (1.50 mL, 1.50 mM) was added to 2-aminoethane-1sulfonic acid (0.19 g, 1.50 mM), excess methanol was added to aid solubility before being taken to complete dryness. Tetrabutylammonium 2-aminoethane-1-sulfonate (1.50 mM) was dissolved in pyridine (2.0 mL) and left overnight in a sealed vial with 1-isothiocyanatobutane (0.18 mL, 1.50 mM). The resulting solution was taken to dryness, re-dissolved in ethyl acetate (15 mL) and extracted with water (15 mL). The pure product was obtained by flash chromatography 100 % ethyl acetate followed by 100 % methanol. The methanol fraction was taken to dryness to give the pure product as an opaque viscous oil with a yield of 64 % (0. Compound 45: Tetrabutylammonium in methanol (1.50 mL, 1.50 mM) was added to 3-Aminopropane-1sulfonic acid (0.21 g, 1.50 mM), excess methanol was added to aid solubility before being taken to complete dryness. Tetrabutylammonium 3-aminopropane-1-sulfonate (1.50 mM) was dissolved in pyridine (2.0 mL) and left overnight in a sealed vial with 1-isothiocyanatobutane (0.18 mL, 1.50 mM). The resulting solution was taken to dryness, re-dissolved in ethyl acetate (15 mL) and extracted with water (15 mL). The pure product was obtained by flash chromatography 100 % ethyl acetate followed by 100 % methanol. The methanol fraction was taken to dryness to give the pure product as a cream solid with a yield of 58 % (0.29 g, 0.59 mM); Melting Point: 137 °C; 1

Self-association constant calculation
Compound 40 -Dilution study in DMSO-d 6                                 Overview Table S6 -Summary of average intensity particle size distribution data. Error = standard error of the mean.

Surface Tension and Stability Data
Zeta Potential Figure                  Overview Single crystal X-ray structures                                                           Figure S166 -E. coli DH10B growth curves created from an average of 6 optical density readings in the presence of compounds 9* and 10.                                                                                   Additional physicochemical -antimicrobial activity relationships identified by an exhaustive search of models    Figure S289 -Intensity profile of line scan generated for cell 2, Figure S287. Figure S290 -Intensity profile of line scan generated for cell 3, Figure S287. Figure S291 -Intensity profile of line scan generated for cell 4, Figure S287. Figure S292 -Intensity profile of line scan generated for cell 5, Figure S287. Figure S293 -Intensity profile of line scan generated for cell 6, Figure S287. Figure S294 -Intensity profile of line scan generated for cell 7, Figure S287. Figure S295 -Intensity profile of line scan generated for cell 8, Figure S287. Figure S296 -Intensity profile of line scan generated for cell 9, Figure S287. Figure S297 -Intensity profile of line scan generated for cell 10, Figure S287. Figure S298 -Intensity profile of line scan generated for cell 11, Figure S287. Figure S299 -Intensity profile of line scan generated for cell 12, Figure S287.                Figure S287. Figure S315 -Intensity profile of line scan generated for cell 28, Figure S287. Figure S316 -Intensity profile of line scan generated for cell 29, Figure S287. Figure S317 -Intensity profile of line scan generated for cell 30, Figure S287. Figure S318 -Intensity profile of line scan generated for cell 31, Figure S287. Figure S319 -Intensity profile of line scan generated for cell 32, Figure S287. Figure S320 -Intensity profile of line scan generated for cell 33, Figure S287. Figure S321 -Intensity profile of line scan generated for cell 34, Figure S287. Figure S322 -Intensity profile of line scan generated for cell 35, Figure S287. Figure S323 -Intensity profile of line scan generated for cell 36, Figure S287. Figure S324 -Intensity profile of line scan generated for cell 37, Figure S287. Figure S325 -Intensity profile of line scan generated for cell 38, Figure S287. Figure S326 -Intensity profile of line scan generated for cell 39, Figure S287. Figure S327 -Intensity profile of line scan generated for cell 40, Figure S287. Figure S328 -Intensity profile of line scan generated for cell 41, Figure S287. Figure S329 -Intensity profile of line scan generated for cell 42, Figure S287.    8 Figure S333 -Intensity profile of line scan generated for cell 2, Figure S331. Figure S334 -Intensity profile of line scan generated for cell 3, Figure S331. Figure S335 -Intensity profile of line scan generated for cell 4, Figure S331. Figure S336 -Intensity profile of line scan generated for cell 5, Figure S331. Figure S337 -Intensity profile of line scan generated for cell 6, Figure S331. Figure S338 -Intensity profile of line scan generated for cell 7, Figure S331. Figure S339 -Intensity profile of line scan generated for cell 8, Figure S331. Figure S340 -Intensity profile of line scan generated for cell 9, Figure S331.                              10 Figure S372 -Intensity profile of line scan generated for cell 2, Figure S370. Figure S373 -Intensity profile of line scan generated for cell 3, Figure S370. Figure S376 -Intensity profile of line scan generated for cell 6, Figure S370. Figure S377 -Intensity profile of line scan generated for cell 7, Figure S370. Figure S378 -Intensity profile of line scan generated for cell 8, Figure S370. Figure S379 -Intensity profile of line scan generated for cell 9, Figure S370. Figure S380 -Intensity profile of line scan generated for cell 10, Figure S370.                      Figure S401 -Intensity profile of line scan generated for cell 1, Figure S400. Figure S402 -Intensity profile of line scan generated for cell 2, Figure S400. Figure S403 -Intensity profile of line scan generated for cell 3, Figure S400. Figure S404 -Intensity profile of line scan generated for cell 4, Figure S400. Figure S405 -Intensity profile of line scan generated for cell 5 , Figure S400. Figure S406 -Intensity profile of line scan generated for cell 6, Figure S400. Figure S407 -Intensity profile of line scan generated for cell 7, Figure S400. Figure S408 -Intensity profile of line scan generated for cell 8, Figure S400. Figure S409 -Intensity profile of line scan generated for cell 9, Figure S400. Figure S410 -Intensity profile of line scan generated for cell 10, Figure S400. Figure S411 -Intensity profile of line scan generated for cell 11, Figure S400. Figure S412 -Intensity profile of line scan generated for cell 12, Figure S400. Figure S413 -Intensity profile of line scan generated for cell 13, Figure S400. Figure S414 -Intensity profile of line scan generated for cell 14, Figure S400. Figure S415 -Intensity profile of line scan generated for cell 15, Figure S400. Figure S416 -Intensity profile of line scan generated for cell 16, Figure S400. Figure S417 -Intensity profile of line scan generated for cell 17, Figure S400. Figure S418 -Intensity profile of line scan generated for cell 18, Figure S400. Figure S419 -Intensity profile of line scan generated for cell 19, Figure S400. Figure S420 -Intensity profile of line scan generated for cell 20, Figure S400. Figure S421 -Intensity profile of line scan generated for cell 21, Figure S400. Figure S422 -Intensity profile of line scan generated for cell 22, Figure S400. Figure S423 -Intensity profile of line scan generated for cell 23, Figure S400. Figure S424 -Intensity profile of line scan generated for cell 24, Figure S400. Figure S425 -Intensity profile of line scan generated for cell 25, Figure S400. Figure S426 -Intensity profile of line scan generated for cell 26, Figure S400. Figure S427 -Intensity profile of line scan generated for cell 27, Figure S400. Figure S428 -Intensity profile of line scan generated for cell 28, Figure S400. Figure S429 -Intensity profile of line scan generated for cell 29, Figure S400. Figure S430 -Intensity profile of line scan generated for cell 30, Figure S400. Figure S431 -Intensity profile of line scan generated for cell 31, Figure S400. Figure S432 -Intensity profile of line scan generated for cell 32, Figure S400. Figure S433 -Intensity profile of line scan generated for cell 33, Figure S400. Figure S434 -Intensity profile of line scan generated for cell 34, Figure S400. Figure S435 -Intensity profile of line scan generated for cell 35, Figure S400. Figure S436 -Intensity profile of line scan generated for cell 36, Figure S400. Figure S437 -Intensity profile of line scan generated for cell 37, Figure S400.   Figure S439 -Intensity profile of line scan generated for cell 1, Figure S438. Figure S440 -Intensity profile of line scan generated for cell 2, Figure S438. Figure S441 -Intensity profile of line scan generated for cell 3, Figure S438. Figure S442 -Intensity profile of line scan generated for cell 4, Figure S438. Figure S443 -Intensity profile of line scan generated for cell 5, Figure S438. Figure S444 -Intensity profile of line scan generated for cell 6, Figure S438. Figure S445 -Intensity profile of line scan generated for cell 7, Figure S438. Figure S446 -Intensity profile of line scan generated for cell 8, Figure S438. Figure S447 -Intensity profile of line scan generated for cell 9, Figure S438. Figure S448 -Intensity profile of line scan generated for cell 10, Figure S438. Figure S449 -Intensity profile of line scan generated for cell 11, Figure S438.   Figure S451 -Intensity profile of line scan generated for cell 1, Figure S450. Figure S452 -Intensity profile of line scan generated for cell 2, Figure S450. Figure S453 -Intensity profile of line scan generated for cell 3, Figure S450. Figure S454 -Intensity profile of line scan generated for cell 4, Figure S450. Figure S455 -Intensity profile of line scan generated for cell 5 , Figure S450. Figure S456 -Intensity profile of line scan generated for cell 6, Figure S450. Figure S457 -Intensity profile of line scan generated for cell 7, Figure S450. Figure S458 -Intensity profile of line scan generated for cell 8, Figure S450. Figure S459 -Intensity profile of line scan generated for cell 9, Figure S450. Figure S460 -Intensity profile of line scan generated for cell 10, Figure S450. Figure S461 -Intensity profile of line scan generated for cell 11, Figure S450.   Figure S463 -Intensity profile of line scan generated for cell 1, Figure S462. Figure S464 -Intensity profile of line scan generated for cell 2, Figure S462. Figure S465 -Intensity profile of line scan generated for cell 3, Figure S462. Figure S466 -Intensity profile of line scan generated for cell 4, Figure S462. Figure S467 -Intensity profile of line scan generated for cell 5, Figure S462. Figure S468 -Intensity profile of line scan generated for cell 6, Figure S462. Figure S469 -Intensity profile of line scan generated for cell 7, Figure S462. Figure S470 -Intensity profile of line scan generated for cell 8, Figure S462. Figure S471 -Intensity profile of line scan generated for cell 9, Figure S462. Figure S472 -Intensity profile of line scan generated for cell 10, Figure S462.  13 Figure S475 -Intensity profile of line scan generated for cell 2, Figure S473. Figure S476 -Intensity profile of line scan generated for cell 3, Figure S473. Figure S477 -Intensity profile of line scan generated for cell 4, Figure S473. Figure S478 -Intensity profile of line scan generated for cell 5, Figure S473. Figure S479 -Intensity profile of line scan generated for cell 6, Figure S473. Figure S480 -Intensity profile of line scan generated for cell 7, Figure S473. Figure S481 -Intensity profile of line scan generated for cell 8, Figure S473. Figure S482 -Intensity profile of line scan generated for cell 9, Figure S473. Figure S483 -Intensity profile of line scan generated for cell 1, Figure S473.0

Overview
Intensity profile of line scan generated for cell 11, Figure S473. Figure S484 -Intensity profile of line scan generated for cell 12, Figure S473. Figure S485 -Intensity profile of line scan generated for cell 13, Figure S473.    Figure S488 -Intensity profile of line scan generated for cell 2, Figure S486. Figure S489 -Intensity profile of line scan generated for cell 3, Figure S486. Figure S490 -Intensity profile of line scan generated for cell 4, Figure S486. Figure S491 -Intensity profile of line scan generated for cell 5, Figure S486. Figure S492 -Intensity profile of line scan generated for cell 6, Figure S486. Figure S493 -Intensity profile of line scan generated for cell 7, Figure S486. Figure S494 -Intensity profile of line scan generated for cell 8, Figure S486. Figure S495 -Intensity profile of line scan generated for cell 9, Figure S486. Figure S496 -Intensity profile of line scan generated for cell 10, Figure S486. Figure S497 -Intensity profile of line scan generated for cell 11, Figure S486. Figure S498 -Intensity profile of line scan generated for cell 12, Figure S486. Figure S499 -Intensity profile of line scan generated for cell 13, Figure S486.   8 Figure S502 -Intensity profile of line scan generated for cell 2, Figure S500. Figure S503 -Intensity profile of line scan generated for cell 3, Figure S500. Figure S504 -Intensity profile of line scan generated for cell 4, Figure S500. Figure S505 -Intensity profile of line scan generated for cell 5, Figure S500. Figure S506 -Intensity profile of line scan generated for cell 6, Figure S500. Figure S507 -Intensity profile of line scan generated for cell 7, Figure S500. Figure S508 -Intensity profile of line scan generated for cell 8, Figure S500. Figure S509 -Intensity profile of line scan generated for cell 9, Figure S500.  8 Figure S512 -Intensity profile of line scan generated for cell 2, Figure S510. Figure S513 -Intensity profile of line scan generated for cell 3, Figure S510. Figure S514 -Intensity profile of line scan generated for cell 4, Figure S510. Figure S515 -Intensity profile of line scan generated for cell 5, Figure S510. Figure S516 -Intensity profile of line scan generated for cell 6, Figure S510. Figure S517 -Intensity profile of line scan generated for cell 7, Figure S510.   290 Figure S522 -Intensity profile of line scan generated for cell 2, Figure S520. Figure S523 -Intensity profile of line scan generated for cell 3, Figure S520. Figure S524 -Intensity profile of line scan generated for cell 4, Figure S520. Figure S525 -Intensity profile of line scan generated for cell 5, Figure S520. Figure S526 -Intensity profile of line scan generated for cell 6, Figure S520. Figure S527 -Intensity profile of line scan generated for cell 7, Figure S520. Figure S528 -Intensity profile of line scan generated for cell 8, Figure S520. Figure S529 -Intensity profile of line scan generated for cell 9, Figure S520. Figure S530 -Intensity profile of line scan generated for cell 10, Figure S520.   Figure S531. Figure S533 -Intensity profile of line scan generated for cell 2, Figure S531. Figure S534 -Intensity profile of line scan generated for cell 3, Figure S531. Figure S535 -Intensity profile of line scan generated for cell 4, Figure S531. Figure S536 -Intensity profile of line scan generated for cell 5, Figure S531. Figure S537 -Intensity profile of line scan generated for cell 6, Figure S531. Figure S538 -Intensity profile of line scan generated for cell 7, Figure S531. Figure S539 -Intensity profile of line scan generated for cell 8, Figure S531. Figure S540 -Intensity profile of line scan generated for cell 9, Figure S531. Figure S541 -Intensity profile of line scan generated for cell 10, Figure S531.                                                                 Figure S608 -Intensity profile of line scan generated for cell 2, Figure S606. Figure S609 -Intensity profile of line scan generated for cell 3, Figure S606. Figure S610 -Intensity profile of line scan generated for cell 4, Figure S606. Figure S611 -Intensity profile of line scan generated for cell 5, Figure S606. Figure S612 -Intensity profile of line scan generated for cell 6, Figure S606. Figure S613 -Intensity profile of line scan generated for cell 7, Figure S606. Figure S614 -Intensity profile of line scan generated for cell 8, Figure S606. Figure S615 -Intensity profile of line scan generated for cell 9, Figure S606.    Figure S618 -Intensity profile of line scan generated for cell 2, Figure S616. Figure S619 -Intensity profile of line scan generated for cell 3, Figure S616. Figure S620 -Intensity profile of line scan generated for cell 4, Figure S616. Figure S621 -Intensity profile of line scan generated for cell 5, Figure S616. Figure S622 -Intensity profile of line scan generated for cell 6, Figure S616. Figure S623 -Intensity profile of line scan generated for cell 7, Figure S616. Figure S624 -Intensity profile of line scan generated for cell 8, Figure S616. Figure S625 -Intensity profile of line scan generated for cell 9, Figure S616.    Figure S628 -Intensity profile of line scan generated for cell 2, Figure S626. Figure S629 -Intensity profile of line scan generated for cell 3, Figure S626. Figure S630 -Intensity profile of line scan generated for cell 4, Figure S626. Figure S631 -Intensity profile of line scan generated for cell 5, Figure S626. Figure S632 -Intensity profile of line scan generated for cell 6, Figure S626.     Figure S634. Figure S637 -Intensity profile of line scan generated for cell 3, Figure S634. Figure S638 -Intensity profile of line scan generated for cell 4, Figure S634. Figure S639 -Intensity profile of line scan generated for cell 5, Figure S634. Figure S640 -Intensity profile of line scan generated for cell 6, Figure S634. Figure S641 -Intensity profile of line scan generated for cell 7, Figure S634. Figure S642 -Intensity profile of line scan generated for cell 8, Figure S634. Figure S643 -Intensity profile of line scan generated for cell 9, Figure S634.    Figure S646 -Intensity profile of line scan generated for cell 2, Figure S644. Figure S647 -Intensity profile of line scan generated for cell 3, Figure S644. Figure S648 -Intensity profile of line scan generated for cell 4, Figure S644. Figure S649 -Intensity profile of line scan generated for cell 5, Figure S644. Figure S650 -Intensity profile of line scan generated for cell 6, Figure S644. Figure S651 -Intensity profile of line scan generated for cell 7, Figure S644. Figure S652 -Intensity profile of line scan generated for cell 8, Figure S644. Figure S653 -Intensity profile of line scan generated for cell 9, Figure S644. Figure S654 -Intensity profile of line scan generated for cell 10, Figure S644. Figure S655 -Intensity profile of line scan generated for cell 11, Figure S644. Figure S656 -Intensity profile of line scan generated for cell 12, Figure S644.     Figure S658. Figure S661 -Intensity profile of line scan generated for cell 3, Figure S658. Figure S662 -Intensity profile of line scan generated for cell 4, Figure S658. Figure S663 -Intensity profile of line scan generated for cell 5, Figure S658. Figure S664 -Intensity profile of line scan generated for cell 6, Figure S658. Figure S665 -Intensity profile of line scan generated for cell 7, Figure S658. Figure S666 -Intensity profile of line scan generated for cell 8, Figure S658. Figure S667 -Intensity profile of line scan generated for cell 9, Figure S658.    Figure S671 -Intensity profile of line scan generated for cell 2, Figure S669. Figure S672 -Intensity profile of line scan generated for cell 3, Figure S669. Figure S673 -Intensity profile of line scan generated for cell 4, Figure S669. Figure S674 -Intensity profile of line scan generated for cell 5, Figure S669. Figure S675 -Intensity profile of line scan generated for cell 6, Figure S669. Figure S676 -Intensity profile of line scan generated for cell 7, Figure S669. Figure S677 -Intensity profile of line scan generated for cell 8, Figure S669. Figure S678 -Intensity profile of line scan generated for cell 9, Figure S669. Figure S679 -Intensity profile of line scan generated for cell 10, Figure S669. Figure S680 -Intensity profile of line scan generated for cell 11, Figure S669. Figure S681 -Intensity profile of line scan generated for cell 12, Figure S669.             Figure S693 -Intensity profile of line scan generated for cell 2, Figure S691. Figure S694 -Intensity profile of line scan generated for cell 3, Figure S691. Figure S695 -Intensity profile of line scan generated for cell 4, Figure S691. Figure S696 -Intensity profile of line scan generated for cell 5, Figure S691. Figure S697 -Intensity profile of line scan generated for cell 6, Figure S691. Figure S698 -Intensity profile of line scan generated for cell 7, Figure S691. Figure S699 -Intensity profile of line scan generated for cell 8, Figure S691. Figure S700 -Intensity profile of line scan generated for cell 9, Figure S691.    Figure S701. Figure S704 -Intensity profile of line scan generated for cell 3, Figure S701. Figure S705 -Intensity profile of line scan generated for cell 4, Figure S701. Figure S706 -Intensity profile of line scan generated for cell 5, Figure S701. Figure S707 -Intensity profile of line scan generated for cell 6, Figure S701. Figure S708 -Intensity profile of line scan generated for cell 7, Figure S701. Figure S709 -Intensity profile of line scan generated for cell 8, Figure S701. Figure S710 -Intensity profile of line scan generated for cell 9, Figure S701.    Figure S712. Figure S715 -Intensity profile of line scan generated for cell 3, Figure S712. Figure S716 -Intensity profile of line scan generated for cell 4, Figure S712. Figure S717 -Intensity profile of line scan generated for cell 5, Figure S712. Figure S718 -Intensity profile of line scan generated for cell 6, Figure S712. Figure S719 -Intensity profile of line scan generated for cell 7, Figure S712. Figure S720 -Intensity profile of line scan generated for cell 8, Figure S712. Figure S721 -Intensity profile of line scan generated for cell 9, Figure S712. Figure S722 -Intensity profile of line scan generated for cell 10, Figure S712.    12 13 Figure S727 -Intensity profile of line scan generated for cell 2, Figure S725. Figure S728 -Intensity profile of line scan generated for cell 3, Figure S725. Figure S729 -Intensity profile of line scan generated for cell 4, Figure S725. Figure S730 -Intensity profile of line scan generated for cell 5, Figure S725. Figure S731 -Intensity profile of line scan generated for cell 6, Figure S725. Figure S732 -Intensity profile of line scan generated for cell 7, Figure S725. Figure S733 -Intensity profile of line scan generated for cell 8, Figure S725. Figure S734 -Intensity profile of line scan generated for cell 9, Figure S725. Figure S735 -Intensity profile of line scan generated for cell 10, Figure S725. Figure S736 -Intensity profile of line scan generated for cell 11, Figure S725. Figure S737 -Intensity profile of line scan generated for cell 12, Figure S725.    Figure S739. Figure S741 -Intensity profile of line scan generated for cell 2, Figure S739. Figure S742 -Intensity profile of line scan generated for cell 3, Figure S739. Figure S743 -Intensity profile of line scan generated for cell 4, Figure S739. Figure S744 -Intensity profile of line scan generated for cell 5, Figure S739. Figure S745 -Intensity profile of line scan generated for cell 6, Figure S739. Figure S746 -Intensity profile of line scan generated for cell 7, Figure S739. Figure S747 -Intensity profile of line scan generated for cell 8, Figure S739. Figure S748 -Intensity profile of line scan generated for cell 9, Figure S739. Figure S749 -Intensity profile of line scan generated for cell 10, Figure S739. Figure S750 -Intensity profile of line scan generated for cell 11, Figure S739. Figure S751 -Intensity profile of line scan generated for cell 12, Figure S739. Figure S752 -Intensity profile of line scan generated for cell 13, Figure S739.           Figure S762. Figure S764 -Intensity profile of line scan generated for cell 2, Figure S762. Figure S765 -Intensity profile of line scan generated for cell 3, Figure S762. Figure S766 -Intensity profile of line scan generated for cell 4, Figure S762. Figure S767 -Intensity profile of line scan generated for cell 5, Figure S762. Figure S768 -Intensity profile of line scan generated for cell 6, Figure S762. Figure S769 -Intensity profile of line scan generated for cell 7, Figure S762. Figure S770 -Intensity profile of line scan generated for cell 8, Figure S762. Figure S771 -Intensity profile of line scan generated for cell 9, Figure S762. Figure S772 -Intensity profile of line scan generated for cell 10, Figure S762. Figure S773 -Intensity profile of line scan generated for cell 11, Figure S762. Figure S774 -Intensity profile of line scan generated for cell 12, Figure S762. Figure S775 -Intensity profile of line scan generated for cell 13, Figure S762.                       Figure S797 -Intensity profile of line scan generated for cell 2, Figure S795. Figure S798 -Intensity profile of line scan generated for cell 3, Figure S795. Figure S799 -Intensity profile of line scan generated for cell 4, Figure S795. Figure S800 -Intensity profile of line scan generated for cell 5, Figure S795. Figure S801 -Intensity profile of line scan generated for cell 6, Figure S795. Figure S802 -Intensity profile of line scan generated for cell 7, Figure S795. Figure S803 -Intensity profile of line scan generated for cell 8, Figure S795. Figure S804 -Intensity profile of line scan generated for cell 9, Figure S795.                Figure S820 -Intensity profile of line scan generated for cell 2, Figure S818. Figure S821 -Intensity profile of line scan generated for cell 3, Figure S818. Figure S822 -Intensity profile of line scan generated for cell 4, Figure S818. Figure S823 -Intensity profile of line scan generated for cell 5, Figure S818. Figure S824 -Intensity profile of line scan generated for cell 6, Figure S818. Figure S825 -Intensity profile of line scan generated for cell 7, Figure S818. Figure S826 -Intensity profile of line scan generated for cell 8, Figure S818. Figure S827 -Intensity profile of line scan generated for cell 9, Figure S818. Figure S828 -Intensity profile of line scan generated for cell 10, Figure S818. Figure S829 -Intensity profile of line scan generated for cell 11, Figure S818. Figure S830 -Intensity profile of line scan generated for cell 12, Figure S818.