D. Neff, expts done 7-6-2011This 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 (Dawn's original)•Clean glass coverslip

1.Make light scratch on one side of glass coverslip with diamond scribe2.Sonicate in ethanol for 15 minutes3.Rinse with UV-treated ddH2O and dry with N2

4.Irradiate with UV light for 10 minutes1.Place 1 µL of 2-rhodamine labeled origami and 9µL UV-treated ddH2O on coverslip (may have to adjust amount

of origami depending on concentration but make sure you have a total of 10µL of liquid)2.Let stand at room temperature for 10 minutes3.Rinse coverslip with UV-treated ddH2O and dry with N2

4.Image using fluorescence microscope. 1.Filter set 22.100x oil objective3.100msec exposure time and EM gain of 2500 for the Roleramgi camera

33 um

33 um

total 1090 um2

48 point sources so here there are ~1 origami / 23um2

display 1500-4500 counts of 65536

33 um

time .150 seconds

time 180 seconds

Filename: 150ms-50em-256x256center.sifDate and Time: Wed Jul 06 16:48:46 2011Software Version: 4.18.30004.0Temperature (C): -75Model: DU897_BVData Type: CountsAcquisition Mode: Frame TransferTrigger Mode: InternalExposure Time (secs): 0.15Cycle Time (secs): 0.15027Frequency (Hz): 6.6547Frame Transfer Mode: KineticsNumber in Series: 1200Readout Mode: Image{left,right,bottom,top}: 128,383,128,383Horizontal Binning: 1Vertical Binning: 1Horizontally flipped: falseVertically flipped: falseClockwise rotation: falseAnti-Clockwise rotation: falseVertical Shift Speed (usecs): 0.5Pixel Readout Rate (MHz): 10Baseline offset: 0Number of prescans: 0Baseline Clamp: ONClock Amplitude: NormalOutput Amplifier: Electron MultiplyingEM Gain level: 50Serial Number: 5762Pre-Amplifier Gain: 5SR163:Wavelength (nm): 500Grating Groove Density (l/mm):200Count Convert Mode: CountsSpurious Noise Filter Mode: No FilterPhoton counted: falseData 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 um2

13 point sources so here there are ~1 origami / 30um2

24 um

16 um

display 0-1500 photons of 65536

time .120 seconds

time 40 seconds

16 um

2

13

Filename: 120ms-100em.tifDate and Time: Wed Jul 06 16:42:25 2011Software Version: 4.18.30004.0Temperature (C): -75Model: DU897_BVData Type: Not availableAcquisition Mode: Frame TransferTrigger Mode: InternalExposure Time (secs): 0.12Cycle Time (secs): 0.12027Frequency (Hz): 8.3146Frame Transfer Mode: KineticsNumber in Series: 330Readout Mode: Full Resolution ImageHorizontal Binning: 1Vertical Binning: 1Horizontally flipped: falseVertically flipped: falseClockwise rotation: falseAnti-Clockwise rotation: falseVertical Shift Speed (usecs): 0.5Pixel Readout Rate (MHz): 10Baseline offset: 0Number of prescans: 0Baseline Clamp: ONClock Amplitude: NormalOutput Amplifier: Electron MultiplyingEM Gain level: 100Serial Number: 5762Pre-Amplifier Gain: 5SR163:Wavelength (nm): 500Grating Groove Density (l/mm):200Count Convert Mode: PhotonsSensitivity: 12.08Detection Wavelength (nm): 630Spurious Noise Filter Mode: No FilterPhoton counted: falseData Averaging Filter Mode: No Filter

background

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

total area in these 3 scans; 49 um2 + 36um2 + 64 um2=149um2

total # origami in this area = 11 so we see about one origami / 15um2

AFM of same coverslip as preceeding slides