D. Neff, expts done This report shows 2rh window origami (two rhodamine labeled staples/origami). Massud annealed and stored this origami at 4 deg. C for ~ 1 week before dialysis and deposition shown here.

this annealing run was dialyzed before imaging through a 25nm pore membrane against 1x TAE buffer (30 mins) I am unsure of the staple:plasmid:rhodamine ratios used in origami synthesis all images taken with the ixon3 (photon count capable) CCD and TM AFM AFM and fluor. images are of the very same coverslip, AFM was NOT on fluor microscope stage for spot analysis 9 pixels per spot (a 3x3 pixel area around the spot center) were averaged each frame background plots look smoother because many more pixels were averaged than for spot (point source) analysis

Preparing 2-rhodamine labeled DNA origami sample for fluorescence microscope (Neff proceedure ) Clean glass coverslip Make light scratch on one side of glass coverslip with diamond scribe Sonicate in ethanol for 15 minutes Rinse with UV-treated ddH 2 O and dry with N 2 Irradiate with UV light for 10 minutes Place 1 µL of 2-rhodamine labeled origami and 9µL UV-treated ddH 2 O on coverslip (may have to adjust amount of origami depending on concentration but make sure you have a total of 10µL of liquid) Let stand at room temperature for 10 minutes Rinse coverslip one time with UV-treated ddH 2 O and dry with N 2 Image using fluorescence microscope. Filter set 2 ex max 560, em max Hg lamp 100x oil objective pixel = 129nm imaging params with ixon3 are indicated beside images Preparing 2-rhodamine labeled DNA origami sample for fluorescence microscope (Dawn's original) Clean glass coverslip 1.Make light scratch on one side of glass coverslip with diamond scribe 2.Sonicate in ethanol for 15 minutes 3.Rinse with UV-treated ddH 2 O and dry with N 2 4.Irradiate with UV light for 10 minutes Place 1 µL of 2-rhodamine labeled origami and 9µL UV-treated ddH 2 O on coverslip (may have to adjust amount of origami depending on concentration but make sure you have a total of 10µL of liquid) Let stand at room temperature for 10 minutes Rinse coverslip with UV-treated ddH 2 O and dry with N 2 Image using fluorescence microscope. 1.Filter set x oil objective 3.100msec exposure time and EM gain of 2500 for the Roleramgi camera

33 um total 1090 um 2 48 point sources so here there are ~1 origami / 23um 2 display counts of um time.150 seconds time 180 seconds Filename: 150ms-50em-256x256center.sif Date and Time: Wed Jul 06 16:48: Software Version: Temperature (C): -75 Model: DU897_BV Data Type: Counts Acquisition Mode: Frame Transfer Trigger Mode: Internal Exposure Time (secs): 0.15 Cycle Time (secs): Frequency (Hz): Frame Transfer Mode: Kinetics Number in Series: 1200 Readout Mode: Image {left,right,bottom,top}: 128,383,128,383 Horizontal Binning: 1 Vertical Binning: 1 Horizontally flipped: false Vertically flipped: false Clockwise rotation: false Anti-Clockwise rotation: false Vertical Shift Speed (usecs): 0.5 Pixel Readout Rate (MHz): 10 Baseline offset: 0 Number of prescans: 0 Baseline Clamp: ON Clock Amplitude: Normal Output Amplifier: Electron Multiplying EM Gain level: 50 Serial Number: 5762 Pre-Amplifier Gain: 5 SR163: Wavelength (nm): 500 Grating Groove Density (l/mm):200 Count Convert Mode: Counts Spurious Noise Filter Mode: No Filter Photon counted: false Data Averaging Filter Mode: No Filter

plots for spots indicated in last slide (150ms-50em-256x256center.tif)

first 75 seconds of same plot as last slide (150ms-50em-256x256center.tif)

total 384 um 2 13 point sources so here there are ~1 origami / 30um 2 24 um 16 um display photons of time.120 seconds time 40 seconds 16 um Filename: 120ms-100em.tif Date and Time: Wed Jul 06 16:42: Software Version: Temperature (C): -75 Model: DU897_BV Data Type: Not available Acquisition Mode: Frame Transfer Trigger Mode: Internal Exposure Time (secs): 0.12 Cycle Time (secs): Frequency (Hz): Frame Transfer Mode: Kinetics Number in Series: 330 Readout Mode: Full Resolution Image Horizontal Binning: 1 Vertical Binning: 1 Horizontally flipped: false Vertically flipped: false Clockwise rotation: false Anti-Clockwise rotation: false Vertical Shift Speed (usecs): 0.5 Pixel Readout Rate (MHz): 10 Baseline offset: 0 Number of prescans: 0 Baseline Clamp: ON Clock Amplitude: Normal Output Amplifier: Electron Multiplying EM Gain level: 100 Serial Number: 5762 Pre-Amplifier Gain: 5 SR163: Wavelength (nm): 500 Grating Groove Density (l/mm):200 Count Convert Mode: Photons Sensitivity: Detection Wavelength (nm): 630 Spurious Noise Filter Mode: No Filter Photon counted: false Data Averaging Filter Mode: No Filter background

plots for spots indicated in last slide (120ms-100em.tif)

total area in these 3 scans; 49 um um um 2 =149um 2 total # origami in this area = 11 so we see about one origami / 15um 2 AFM of same coverslip as preceeding slides