Metodi per l’analisi di immagini AFM

Prof. Claudio Rivetti Dipartimento di Biochimica e Biologia Molecolare Università degli Studi di Parma

Corso di perfezionamento in TECNICHE DI MICROSCOPIA A FORZA ATOMICA - Parma 29 febbraio 2012 Sommario

Che cosa è un’immagine e come viene rappresentata

Tipi di immagine: RGB, Indexed, Grayscale, Black and white

Formato dei file di immagini

Filtri e trasformazioni per elaborare immagini AFM

Riconoscimento e misura di oggetti

Software disponibili Che cosa è un’immagine R G B R 79 R 141 R 112 G 46 G 109 G 78 B 16 B 21 B 20

R 112 R 248 R 241 G 80 G 216 G 184 B 48 B 104 B 104

R 56 R 207 R 241 G 24 G 116 G 148 B 8 B 60 B 48 2563 = 16.777.216 colori 256 colori

Color Table Index R G B i 240 i 220 i 215 1 125 43 55 2 230 56 90 3 22 220 102 4 34 130 179 5 55 133 140 i 80 i 201 i 221 6 140 21 77 7 150 28 61 8 140 10 14 … … … … 254 8 89 98 i 4 i 208 i 180 255 220 100 199 256 11 202 251 I 54 I 114 I 85

I 87 I 219 I 195

I 33 I 141 I 172 0 1 0

0 11

0 1 0 Profondità del colore dell’immagine

La profondità di colore (color depth) è la quantità di bit necessari per rappresentare il colore di un singolo pixel in un'immagine

1 bit 4 bit

1 bit 21 = 2 colori

2 bit 22 = 4 colori

4 bit 24 = 16 colori

8 bit 28 = 256 colori

12 bit 212 = 4096 colori 8 bit 24 bit 16 bit 216 = 65536 colori

24 bit 224 = 16.777.216 colori B

G

R I Un’immagine grayscale può essere resa a colori utilizzando una scala di falsi colori Salvare un immagine su disco in un file

Contiene informazioni relative Header all’immagine

Contiene la matrice o le matrici (nel caso di RGB) di numeri che rappresentano l’immagine Data

La dimensione del file dipende, dalla dimensione dell’immagine, dal tipo di immagine (RGB, grayscale, indexed) dai byte utilizzati per rappresentare il valore dei pixel.

La matrice dell’immagine può essere compressa per ridurre la dimensione del file. In questo caso la dimensione del file dipende anche dal soggetto dell’immagine Immagine di 400x266 pixels => 400 x 266 x 3 = 319200 byte = 305 KB

120 KB 32 KB

Diverso grado di compressione

120 KB 20 KB

Stesso grado di compressione Compressione Lossless (non viene persa informazione) Tipi di compressione: Compressione Lossy (viene persa dell’informazione)

Lossless

Lossy Formati immagine

Format name: TIFF (Tagged Image ) Extension: tif Type: bitmapped Compression algorithm: CCITT-RLE, CCITT-FAX3, CCITT-FAX4, LZW, OJPEG, JPEG, CCITT-RLEW, Packbits, Deflate Color depth: up to 48 bits Platforms: all

Format name: JPEG (Joint Photographic Experts Group) Format name: PNG (Portable Network Graphic) Extension: jpg, , jfif,jfl Extension: png Type: bitmapped Type: bitmapped Compression algorithm: JPEG (lossy) Compression algorithm: Deflate (lossless) Color depth: 24 bits Color depth: up to 48 bits Platforms: all Platforms: all

Format name: GIF (Graphics Interchange Format) Format name: RAW (Image raw data) Extension: gif Extension: any (binary or ascii) Type: bitmapped Type: bitmapped Compression algorithm: LZW (lossless) Compression algorithm: without compression Color depth: up to 8 bits Color depth: any Platforms: all Platforms: all

Format name: Bitmap ( Bitmap) PROPRIETARY FORMAT Extension: bmp, dib, rle Type: bitmapped Compression algorithm: RLE (lossless), without compression Color depth: up to 32 bits Platforms: Windows, OS/2 Inoltre esiste la possibilità di catturare direttamente l’immagine sullo schermo in un buffer.

Per esempio in windows lo si ottiene premendo il tasto ‘Stamp’.

TIFF Export e JPEG Export salvano uno screen capture.

ASCII Export può salvare anche i valori in nm. ASCII export

\Exported image units: nm 3.281598 1.116525 1.606810 1.035802 4.647939 2.760723 3.861953 4.885859 4.732911 3.286696 5.914014 1.869372 2.209258 1.655244 4.350539 5.982841 4.503488 5.616614 4.591858 2.418288 2.900076 2.319721 1.451313 5.747470 4.732911 6.007482 1.113126 2.884781 1.241433 2.254293 1.142866 2.622219 4.800038 3.256106 1.713025 1.697730 2.787064 1.690082 5.922511 1.035802 3.726848 4.823830 2.735231 2.710590 2.447178 2.578884 1.155612 6.848700 2.315472 4.477146 4.515384 8.369689 3.704756 1.767406 3.414153 2.307825 1.410526 3.618935 6.316779 3.704756 4.996322 9.686747 9.533798 1.654394 1.073190 1.099531 5.396538 6.506265 3.900190 2.319721 5.829042 9.941661 1.350197 2.373253 8.505643 4.075231 5.706683 2.028269 1.650146 8.038300 1.319607 2.134483 4.928345 3.273101 5.965846 4.941091 1.262676 1.355295 6.058465 4.393025 1.167508 1.215092 2.323970 4.494141 1.886366 1.783551 2.268738 2.710590 7.842866 5.287774 5.326861 5.365948 4.873963 2.257692 1.419873 2.974001 4.435510 4.274065 5.493405 5.536741 4.517083 2.968053 7.061129 2.256842 1.684134 4.673430 7.987318 3.341078 4.985276 3.443893 2.294230 1.240583 6.593786 6.092454 9.559290 6.551300 6.338871 9.975650 5.055802 1.359543 1.010311 6.211414 1.283069 5.242739 9.346861 4.077781 2.444629 2.404692 1.302613 2.005327 1.753811 6.525808 1.263526 7.180089 2.669803 4.962333 9.780215 1.459810 1.410526 1.893164 2.374952 4.715916 9.873684 3.514420 2.405542 1.828586 7.214077 1.461509 6.313380 5.608117 1.516741 9.006975 3.986011 2.883931 1.781852 6.797717 6.389854 1.662042 1.622105 1.079138 6.228408 3.081915 1.983234 2.477768 1.911008 2.406392 2.370704 1.272873 2.243247 2.931515 4.700621 3.590894 4.075231 3.497425 1.858326 7.511477 3.568801 1.220190 1.461509 4.633494 2.994394 1.886366 1.309410 1.264375 6.891186 1.130120 9.610273 1.476804 2.542346 1.448763 1.948396 8.539632 2.914521 1.321306 2.285732 1.214242 6.984654 2.189715 1.096132 1.064692 5.030311 4.715916 9.720735 9.414838 1.082536 1.031554 3.380165 1.225289 1.724071 2.222853 2.192264 2.160824 1.598313 1.283069 6.440837 2.416588 2.389398 1.831985 1.804794 7.163094 3.721750 9.295878 8.497146 6.015980 3.754889 2.133633 2.105593 1.546481 2.049512 2.021471 1.462359 9.270387 3.832213 3.289245 2.193963 1.630602 1.597464 1.565174 1.532885 1.500596 9.091947 6.083957 3.169436 2.604375 2.039315 2.005327 1.440266 1.406278 8.420672 1.292416 2.337565 3.580697 3.008840 4.030197 3.458339 2.356259 7.239569 1.529486 9.125935 8.981484 3.444743 2.877134 1.780152 1.744464 1.708776 6.117945 4.588459 1.138618 1.125022 4.658985 3.554356 2.980799 1.875320 2.363906 1.790349 1.217641 2.379201 3.075967 2.922169 1.828586 2.327368 2.827001 1.733418 1.171756 1.671389 8.284718 1.345098 4.031896 2.928117 1.824337 1.253329 1.212543 1.096132 5.998985 1.737666 1.722372 1.456411 3.534813 8.437666 2.719087 7.621940 1.784401 1.744464 9.210907 9.074952 2.639214 2.069055 1.499746 1.461509 1.423272 1.385885 1.348497 2.693595 2.566138 2.575485 2.008725 1.973887 1.407977 1.903361 2.693595 2.948510 3.305390 8.480152 3.279898 2.187165 1.624654 2.125986 3.483830 6.542803 9.516804 7.689917 2.260241 2.701243 1.079138 2.112391 2.083500 2.054610 9.644261 4.061636 3.449841 7.018643 3.103158 1.464908 8.888015 1.906760 2.693595 2.260241 3.483830 4.673430 8.072289 3.504223 1.875320 2.369854 2.334166 1.236335 6.704248 1.165808 1.099531 2.147229 Formato dei file Nanoscope

\*File list \Version: 0x05300003 \Date: 01:05:57 PM Fri Mar 13 2009 00111001 => 57 => 9 \Start context: OL \Data length: 40960 \Navigator note: col(-5), row(4) \Engage X Pos: -19783.4 um \Engage Y Pos: -42151.3 um \*Equipment list \Description: Extended MultiMode \Controller: IIIA \Microscope: MultiMode \Extender: Basic \Tip Exchange: None \Vision: None \Zoom System: Motorized Header \Scanner file: 2211e.scn \Profile name: claudio \*Scanner list \Scanner type: AFM ù Ù \Serial number: 2211E \Piezo size: E 249 217 \File name: 2211e.scn \Motor direction: Reverse \Retracted offset der: 0.7 \Extended offset der: 6.15 \Allow rotation: Allow ùÙ = 1111100111011001 = 63961 \Piezo cal: 440 \X sensitivity: 22.6356 \X derate: 0.03101 \X mag: 1.3 ... CTRL-Z

Image

EOF \*File list \Xs-Yf coupling: 0.005002 \Profiler profiling: Disabled \Version: 0x05300003 \Xs-Yf coupling derating: -1.354e-005 \Profiler length goal: 500 \Date: 01:05:57 PM Fri Mar 13 2009 \Ys-Xf coupling: 0.01305 \Profiler length actual: 0.001 \Start context: OL \Ys-Xf coupling derating: 3.866e-006 \Profiler speed: 1 \Data length: 40960 \X offset sens: 35 \Profiler scan time: 0.001 \Navigator note: col(-5), row(4) \Y offset sens: 35 \Profiler resolution: 0.001 \Engage X Pos: -19783.4 um \Bias derate: 0 \Profiler x position: 0 \Engage Y Pos: -42151.3 um \Z min: -220 \Profiler y position: 0 \*Equipment list \@Sens. Zscan: V 8.792694 nm/V \Profiler z position: 0 \Description: Extended MultiMode \@Sens. Current: V 10.00000 nA/V \Profiler profiler position: 0 \Controller: IIIA \@Sens. Potential: V 1.000000 \Profiler tilt angle: 0 \Microscope: MultiMode \*Ciao scan list \Profiler direction: Forward \Extender: Basic \Parameter select: Main \Profiler axis: Aux \Tip Exchange: None \Operating mode: Image \Profiler ramp-up ideal-distance multiplier: 25 \Vision: None \Scan size: 2000 nm \Profiler ramp-up total-distance minimum: 2 \Zoom System: Motorized \X offset: 2500 nm \Profiler ramp-up total-distance goal: 10 \Scanner file: 2211e.scn \Y offset: 2500 nm \Profiler backlash-removal distance: 10 \Profile name: claudio \Rotate Ang.: 0 \Profiler backlash-removal speed: 33 \*Scanner list \Samps/line: 512 \Profiler num profile axis display points 3D: 512 \Scanner type: AFM \Lines: 512 \Gain start: 3.2 \Serial number: 2211E \Y disable: Enabled \Gain end: 32 \Piezo size: E \Aspect ratio: 1:1 \Gain incr: 3.2 \File name: 2211e.scn \Bidirectional scan: Disabled \Pro. Gain factor: 1.5 \Motor direction: Reverse \Scan line shift: 0 \Max shift: 0.02 \Retracted offset der: 0.7 \Scan rate: 1.96888 \Lines/gain: 8 \Extended offset der: 6.15 \Tip velocity: 7.8755 \Gain offset: 0.95 \Allow rotation: Allow \Minimum scan rate: 0.02 \Auto Gain: Disabled \Piezo cal: 440 \Lift rate: 4 \Start size: 3.82082e-005 nm \X sensitivity: 22.6356 \X drift: 0 \Pass 2 size: 3.82082e-005 nm \X derate: 0.03101 \Y drift: 0 \Pass 3 size: 3.82082e-005 nm \X mag: 1.3 \Step XY size: 300 \Pass 4 size: 3.82082e-005 nm \X mag1: 0.6 \Cycles: 10 \Feature scan angle: 0 \X arg: 2.8 \Step XY period: 0.005 \Feature Scan rate: 1 \X round: 0 \Step size: 3 \Feature step size: 0.138889 \Orthogonality: 0.00872665 \Units: Metric \Feature setpoint: 0.8 \Y sensitivity: 23.9532 \Color table: 12 \Feature threshold: 2000 \Y derate: 0.03922 \Scope dualtrace: Dual \Pass delay: 5 \Y mag: 1.3 \Auto X Sep: 171.107 \Z offset: 0 \Y mag1: 0.6 \Auto Y Sep: 1576.52 \Z tolerance: 29014.3 \Y arg: 2.5 \Auto pattern: Square \Engage X offset: 0 nm \X slow sensitivity: 24.828 \Auto number: 5 \Engage Y offset: 0 nm \X slow derate: 0.0388 \Capture direction: Down \Engage step size: 0.138889 \Y fast sensitivity: 21.906 \Capture prelines: 50 \Engage Z sens.: 12.5 \Y fast derate: 0.03035 \Engage Setpoint: 1 \Engage Z avg.: 10 \X slow-fast coupling: 0.07335 \Min int. gain: 3 \Engage Z delay: 0.5 \X slow-fast coupling derating: 7.846e-006 \Parm update retract: Disabled \Height engage: Disabled \Y slow-fast coupling: 0.07341 \Drive feedback: Disabled \Current sens.: High 1 pA/V \Y slow-fast coupling derating: 2.584e-006 \Drive time: 10 \Extended Current sens: High 1 pA/V \Fast cal freq: 2.44141 \Drive setpoint: 0.9 \Bias limit: 12 V \Slow cal freq: 4.76837 \Drive gain: 32 \AFM Current Sens.: High 1 nA/V \SECPM sens: 1000 mV/V \@TR mode: S [] "Disabled" \Nanopot gain: 1x \@TUNA calibration: S [] "Off" \Current range: 200 nA \@2:Zmod: S [] "Disabled" \HF current: 1 \@3:Zmod: S [] "Disabled" linked \HF freq.: 100 \@2:SPMFeedbackList: S [SPMFb] "Amplitude" \HF Start freq.: 100 \@3:SPMFeedbackList: S [SPMFb] "Amplitude" linked \HF End freq.: 100 \@2:InputFbList: S [] "Off" \HF Step freq.: 100 \@3:InputFbList: S [] "Off" linked \HF power: -30 \@2:Capacitance feedback: S [] "Off" \HF Start power: -30 \@3:Capacitance feedback: S [] "Off" \HF End power: -30 \@2:TR mode: S [] "Disabled" \HF Step power: 0 \@3:TR mode: S [] "Disabled" \HF RF: Off \@2:TRCouplingCheck: S [] "" \HF AM: Off \@3:TRCouplingCheck: S [] "" \HF lower limit: -20 \@2:DriveAttenState: S [DriveAttenOff] "Off" \HF upper limit: 0 \@3:DriveAttenState: S [DriveAttenOff] "Off" \HF int. gain: 5 \@Sens. Deflection: V 1.000000 \HF prop. gain: 5 \@Sens. Ebias: V 1000.000 mV/V \HF engage: Disabled \@Sens. TUNA DC test bias: V 1.000000 V/V \HF current capture: 0 \@Sens. Friction: V 1.000000 \HF display: Current \@Sens. Amplitude: V 1.000000 \MR preamp gain: 1000 \@Sens. Phase: V 1.000000 \MSM bias voltage: 0 \@Sens. Frequency: V 1.000000 \MSM resistance: 150 \@Sens. dC/dV: V 1.000000 \MSM bias delay: 1 \@Sens. Fdback bias: V 1.000000 \MSM test current: 3 \@Sens. In 0: V 1.000000 \MSM max sensor resist: 200 \@Sens. Thermal: V 1.000000 \MSM min sensor resist: 10 \@Sens. Resistance: V 1.000000 \MSM enable: Enabled \@Sens. High Frequency: V 1.000000 \MSM Probe Delay: 2 \@Sens. TR Amplitude: V 1.000000 \Engage MRR: -1 \@Sens. TR Phase: V 1.000000 \Scan MRR: -1 \@Sens. TR Vert Defl: V 1.000000 \XY Closed Loop: Off \@Sens. Aux B: V 1.000000 \FourPt Current: 1 mA cal \@Sens. Aux : V 1.000000 \FourPt IShunt: On (1 Mohm) \@Sample period: V (0.1000000 us/LSB) 16.00000 us \FourPt Gain: 1 mV/unit \@Z center: V [Sens. Zscan] (0.006713867 V/LSB) -0.006713867 V \FourPt Mplexer: (I, V, I, V) \@1:Z limit: V [Sens. Zscan] (0.006713867 V/LSB) 440.0000 V \FourPt MMode: Surface detect \@1:DeflectionLimit: V (20.00000 V/LSB) 20.00000 V \FourPt FdbkGain: 0.1x \@1:FM igain: V (1.000000 1/LSB) 0 \FourPt LaserGain: 30 micron \@1:FM pgain: V (1.000000 1/LSB) 0 \FourPt Lock-in Freq: 651 Hz \@1:AmplitudeLimit: V (20.00000 V/LSB) 2.500000 V \FourPt IntegrationTime: Short \@1:TRAmplitudeLimit: V (20.00000 V/LSB) 2.500000 V \FourPt SDThreshold: 10 \@Lift start height: V [Sens. Zscan] (0.006713765 V/LSB) 0 V \Analog2 High Voltage: 0 \@Lift scan height: V [Sens. Zscan] (0.006713765 V/LSB) 0 V \Analog22 High Voltage: 0 \@Drive height: V [Sens. Zscan] (0.006713765 V/LSB) 0.5592841 V \@InterleaveList: S [InterleaveOffMode] "Disabled" \@2:AFMSetDeflection: V (0.0003051758 V/LSB) 1.825000 V \@LinearizeList: S [LinearizeOffMode] "modeLinearizeOff" \@3:AFMSetDeflection: V (0.0003051758 V/LSB) 1.000000 V linked \@MicroscopeList: S [TMMode] "Tapping" \@2:TMSetAmplitude: V (0.0003051758 V/LSB) 1.581726 V \@Capacitance feedback: S [] "Off" \@3:TMSetAmplitude: V (0.0003051758 V/LSB) 2.000000 V linked \@MSMSource: S [MSMCurrentMode] "Current" \@2:TMSetDeflection: V (0.0003051758 V/LSB) 0 V \@3:TMSetDeflection: V (0.0003051758 V/LSB) 1.000000 V linked \@2:TMSetPhase: V (0.005493164 º/LSB) 0 º \@3:TMSetPhase: V (0.005493164 º/LSB) 0 º \@2:STMSetCurrent: V [Sens. Current] (0.0003051758 V/LSB) 10.00000 V \@3:STMSetCurrent: V [Sens. Current] (0.0003051758 V/LSB) 10.00000 V \@2:TRSetAmplitude: V (0.0003051758 V/LSB) 0 V \@3:TRSetAmplitude: V (0.0003051758 V/LSB) 0 V \@2:TRSetPhase: V (0.005493164 º/LSB) 0 º \@3:TRSetPhase: V (0.005493164 º/LSB) 0 º \@2:SetTRVertDefl: V (0.0003051758 V/LSB) 0 V \@3:SetTRVertDefl: V (0.0003051758 V/LSB) 0 V \@2:SetTRVertDeflMM: V (0.001220703 V/LSB) 0 V \@3:SetTRVertDeflMM: V (0.001220703 V/LSB) 0 V \@2:SECPMSetPotential: V [Sens. Potential] (0.0003051758 V/LSB) 2.000000 V \@3:SECPMSetPotential: V [Sens. Potential] (0.0003051758 V/LSB) 2.000000 V \@2:TROnAnalog3: V (0.0003051758 V/LSB) 6.000000 V \@3:TROnAnalog3: V (0.0003051758 V/LSB) 6.000000 V \@2:TROffAnalog3: V (0.0003051758 V/LSB) 1.000000 V \@3:TROffAnalog3: V (0.0003051758 V/LSB) 1.000000 V \@2:Drive frequency: V (0.0000058208 kHz/LSB) 348.6384 kHz \@3:Drive frequency: V (0.0000058208 kHz/LSB) 73.21000 kHz linked \@2:Drive phase: V (0.005493164 º/LSB) 38.16101 º \@3:Drive phase: V (0.005493164 º/LSB) 129.1992 º linked \@2:Drive amplitude: V (0.6103516 mV/LSB) 30.00000 mV \@3:Drive amplitude: V (0.6103516 mV/LSB) 200.0000 mV linked \@2:Cap. feedback setpt: V (0.0003051758 V/LSB) 0 V \@3:Cap. feedback setpt: V (0.0003051758 V/LSB) 0 V \@2:Bias Frequency: V (0.0000058208 kHz/LSB) 0 kHz \@3:Bias Frequency: V (0.0000058208 kHz/LSB) 0 kHz \@2:AC bias ampl: V (0.0003051758 V/LSB) 0 V \@3:AC bias ampl: V (0.0003051758 V/LSB) 0 V \@2:DC bias: V (0.0003662109 V/LSB) 0 V \@3:DC bias: V (0.0003662109 V/LSB) 0 V \@2:Lock-in phase: V (0.005493164 º/LSB) 90.00000 º \@3:Lock-in phase: V (0.005493164 º/LSB) 90.00000 º \@2:Stray cap. adj.: V (0.0003051758 V/LSB) 0 V \@3:Stray cap. adj.: V (0.0003051758 V/LSB) 0 V \@2:Bias: V (0.3051758 mV/LSB) 0 mV \@3:Bias: V (0.3051758 mV/LSB) 0 mV \@2:Analog 1: V (0.0003051758 V/LSB) 0 V \@3:Analog 1: V (0.0003051758 V/LSB) 0 V \@2:Analog 2: V (0.0003662109 V/LSB) 0 V \@3:Analog 2: V (0.0003662109 V/LSB) 0 V \@2:TR Drive frequency: V (0.0000058208 kHz/LSB) 0 kHz \@3:TR Drive frequency: V (0.0000058208 kHz/LSB) 0 kHz \@2:TR Drive phase: V (0.005493164 º/LSB) 0 º \@3:TR Drive phase: V (0.005493164 º/LSB) 0 º \@2:TR Drive amplitude: V (0.6103516 mV/LSB) 0 mV \@3:TR Drive amplitude: V (0.6103516 mV/LSB) 0 mV \@2:TUNA DC test bias: V [Sens. TUNA DC test bias] (0.0003051758 V/LSB) 0 V \@3:TUNA DC test bias: V [Sens. TUNA DC test bias] (0.0003051758 V/LSB) 0 V \Check for active tip: Enabled \@2:SPMFbIgain: V (0.03125000 1/LSB) 0.2000000 \XY Threshold: 50 \@3:SPMFbIgain: V (0.03125000 1/LSB) 2.000000 linked \X IGain: 6 \@2:SPMFbPgain: V (0.03125000 1/LSB) 1.000000 \X PGain: 6 \@3:SPMFbPgain: V (0.03125000 1/LSB) 4.000000 linked \Y IGain: 6 \@2:SPMFbSgain: V (0.0000305176 1/LSB) 0 \Y PGain: 6 \@3:SPMFbSgain: V (0.0000305176 1/LSB) 0 linked \*Ciao image list \@2:Tip Drive phase: V (0.005493164 º/LSB) 0 º \Data offset: 40960 \@3:Tip Drive phase: V (0.005493164 º/LSB) 0 º \Data length: 524288 \@2:MSM lock-in phase: V (0.005493164 º/LSB) 90.00000 º \Bytes/pixel: 2 \@3:MSM lock-in phase: V (0.005493164 º/LSB) 90.00000 º \Start context: OL \@2:STMFbIgain: V (0.03125000 1/LSB) 1.000000 \Data type: AFM \@3:STMFbIgain: V (0.03125000 1/LSB) 1.000000 \Plane fit: -443.246 0.261719 1380.22 1 \@2:STMFbPgain: V (0.03125000 1/LSB) 0 \Frame direction: Down \@3:STMFbPgain: V (0.03125000 1/LSB) 0 \Stage X: 0 \@2:STMFbSgain: V (0.0000305176 1/LSB) 0 \Stage Y: 0 \@3:STMFbSgain: V (0.0000305176 1/LSB) 0 \Stage type: N/A \*Fast Scan list \Profile length: 0 \Phase Shift: 0 \Profile speed [um/s]: 0 \Fast Integral Gain: 0 \Samps/line: 512 \Fast Proportional Gain: 0 \Number of lines: 512 \Fast Interleave Igain: 0 \Aspect ratio: 1:1 \Fast Interleave Pgain: 0 \Scan size: 2 2 ~m \Contact Gain: 123 \Scan line: Main \Q-Control Gain: 0 \Line direction: Trace \QC Interleave Gain: 0 \Highpass: 0 \QC Lift Gain: 0 \Lowpass: 0 \Spare IGain: 6 \Realtime planefit: Line \Spare PGain: 6 \Offline planefit: Offset \Preamp Gain: 0 \Valid data start X: 0 \Preamp Offset: 0 \Valid data start Y: 0 \High Pass Filter: 1 \Valid data len X: 512 \Low Pass Filter: 1 \Valid data len Y: 512 \All Pass Filter: 1 \Tip x width correction factor: 1 \Interleave Mode: Disabled \Tip y width correction factor: 1 \Lift Mode: Disabled \Tip x width correction factor sigma: 1 \QC Interleave Mode: Disabled \Tip y width correction factor sigma: 1 \QC Lift Mode: Disabled \@2:Image Data: S [Height] "Height" \Closed Loop: Disabled \@Z magnify: C [2:Z scale] 0.06967584 \Tapping Engage Fast I Gain: 10 \@2:Z scale: V [Sens. Zscan] (0.006713765 V/LSB) 5.223309 V \Tapping Engage Fast P Gain: 10 \@2:Z offset: V [Sens. Zscan] (0.006713765 V/LSB) 0 V \Contact Engage Fast I Gain: 5 \*File list end \Contact Engage Fast P Gain: 5 \Tapping Minimum Scanning I Gain: 0.1 \Tapping Minimum Scanning P Gain: 0.1 \Contact Minimum Scanning I Gain: 0.1 \Contact Minimum Scanning P Gain: 0.1 \Tapping Maximum Scan Rate: 3 \Contact Maximum Scan Rate: 4 \Determine Contact Gain: Enabled Conversione dei numeri a 16 bit dell’immagine in nanometri

Z scale (V) h (nm) = LSB ¯ ¯ Sens. Zscan (nm/V) 65536

\*File list \Version: 0x05300003 \Date: 01:05:57 PM Fri Mar 13 2009 \Start context: OL \Data length: 40960 5.223309 (V) \Navigator note: col(-5), row(4) h = 5000 ¯ ¯ 8.792694 (nm/V) \Engage X Pos: -19783.4 um 65536 \Engage Y Pos: -42151.3 um \*Equipment list \Description: Extended MultiMode \Controller: IIIA \Microscope: MultiMode h = 3.5039 nm \Extender: Basic ... \Bias derate: 0 \Z min: -220 \@Sens. Zscan: V 8.792694 nm/V \@Sens. Current: V 10.00000 nA/V \@Sens. Potential: V 1.000000 \Samps/line: 512 \Number of lines: 512 \Aspect ratio: 1:1 \Scan size: 2 2 ~m

... \@2:Image Data: S [Height] "Height" \@Z magnify: C [2:Z scale] 0.06967584 \@2:Z scale: V [Sens. Zscan] (0.006713765 V/LSB) 5.223309 V \@2:Z offset: V [Sens. Zscan] (0.006713765 V/LSB) 0 V ... Matlab script per leggere e visualizzare un’immagine AFM

>> HEADER_SIZE = 40960; 3.0 nm >> IMAGE_SIZE = 512*512;

>> fp = fopen('sample.000', 'r'); >> H = fread(fp, HEADER_SIZE, 'char'); >> H=setstr(H)'; >> I = fread(fp, IMAGE_SIZE, 'short', 'vaxd'); >> I = rot90(I); >> fclose(fp);

>> I = I * (5.223309/65536) * 8.792694;

>> imshow(I); >> 0.0 nm Filtri e trasformazioni delle immagini AFM

Highpass filter Ogni pixel dell’immagine viene sostituito con la media delle differenze tra in valore di quel pixel e gli otto pixel confinanti.

Lowpass filter Ogni pixel dell’immagine viene sostituito con il valore medio dei 3x3 pixel che lo circondano incluso il pixel stesso.

Mediana Ogni pixel dell’immagine viene sostituito con la mediana dei 3x3 pixel che lo circondano incluso il pixel stesso.

Flattening Ogni scan line viene “raddrizzata” mediante fitting con un polinomio di ordine 0, 1, 2 o 3.

Tip deconvolution L’effetto di allargamento dell’oggetto causato dalla dimensione della punta viene sottratto utilizzando l’immagine della punta.

Averaging Oggetti simili e orientati nella stesso modo possono essere sovrapposti per per aumentare il rapporto segnale rumore. Lowpass filter

8 7 2

9 6 1

534

5

>> B = blkproc(I,[M N],FUN) Filtri per la riduzione del noise: Lowpass, mediana, etc.

Prima Dopo

Scelta del grado del polinomio per il flattening y = axn + bx(n-1) + cx(n-2) …

2 2

1 3 0

Sottrazione dell’effetto della punta dall’immagine

Tip

Tip convolution Sottrazione dell’effetto della punta dall’immagine

Tip

Tip deconvolution Averaging Riconoscimento e misura di oggetti

Conteggio di particelle

Analisi dei profili

Misure di volume

Misure di distanza

Misure di lunghezza del DNA Image segmentation

>> [H,I] = loadAFMimage('sample.000'); >> BW = im2bw(I,1); >> C=imcontour(BW,1); >>

Misura del volume a nm b 8.0 nm 12.0

8.0

4.0 4.0

0.0 20 120 100 40 0.0 60 80 80 60 nm 40 nm 100 20 c 120 nm d 8.0

4.0

0.0 e f

g Stechiometria di proteine multimeriche Digitization of the DNA backbone and chaincode determination

Eight-connected chaincode

1 2 2 3 1

4 0

5 7 6

3 4

Chaincode string: 100111210077756

ne = 6 no = 9 nc = 6 AFM image 3x3 median filter Binary image

Automated DNA fragments recognition and sizing through AFM image processing Ficarra, E.; Benini, L.; Macii, E.; Zuccheri, G.; Information Technology in Biomedicine, IEEE Transactions on (2005) 9:508 - 517 Algorithms for length determination of digitized contours

I

The Freeman estimator LF = ne + 2no =1.000ne +1.414no

II 2 2 The MPO estimator LMPO = ()ne + no + ne

III

The Kulpa estimator LK = 0.948ne +1.343no

IV

The corner count estimator LC = 0.980ne +1.406no − 0.091nc

V n−1 2 2 The n-point moving polynomial fitting LPF = ∑ (xi+1 − xi ) + (yi+1 − yi ) i=1

VI P + P2 −16A The edge chord algorithm L = ECA 4 Simulation of DNA molecules in two dimensions

Chain of rigid rods θ σ = l P

l = 3.4Å P = 53nm

l= 3.4 Å θ Skeletonized chain -20 -10 0 10 20

AFM-like image

Tip convolution FreemanError 4.1% MPO Error -2.6% Kulpa Error -1.2% -omDNA B-form -omDNA B-form DNA B-form Simulated molecules measured with an automatic procedure

Simulated molecules measured with a manual procedure

Real DNA molecules measured with a manual procedure Corner countError -0.4% Polynomial fit Error 0.0% ECA Error -1.2% -omDNA B-form DNA B-form -omDNA B-form

300 320 340 360 380 400 300 320 340 360 380 400 300 320 340 360 380 400

DNA contour length DNA contour length DNA contour length DNA 1054 bp long (B-form contour length: 358.4 nm) Contour Length Error CV Method (nm) (%) (%) A. Simulated polymers: “automatic” measurements. N=192

LF 373.2 4.1 1.1

LMPO 349.1 -2.6 0.9

LK 354.1 -1.2 1.1 LC 356.8 -0.4 1.0 Best performing algorithms... LPF 358.4 0.0 0.8 ECA 354.0 -1.2 0.8 The n-point moving polynomial fitting B. Simulated polymers: “manual” measurements. N=198 n−1 LF 377.8 5.4 1.1 2 2 LPF = ∑ (xi+1 − xi ) + (yi+1 − yi ) LMPO 353.3 -1.4 0.9 i=1 LK 358.4 0.0 1.1

LC 361.2 0.8 1.0

LPF 363.7 1.5 0.9 The corner count estimator ECA 358.7 0.1 0.9 L = 0.980n +1.406n − 0.091n C. DNA molecules: “manual” measurements. N=305 C e o c

LF 351.8 -1.8 3.2

LMPO 329.1 -8.2 3.1

LK 333.5 -6.9 3.1

LC 336.0 -6.2 3.2

LPF 338.1 -5.6 3.1 ECA 334.5 -6.7 3.1 LF = ne + 2no =1.000ne +1.414no

The DNA estimator ab L(ne ,no )= ane + bno

LF = ne + 2no =1.000ne +1.414no

LK = 0.948LF = 0.948ne +1.343no

L C f = LF

LDNA = C f (1.000ne +1.414no ) cd

2 3 1

4 0

5 7 6 160 200 efAverage = 0.964 ±0.010

120 150

80 100 Frequency Frequency 40 50

0 0 640 660 680 700 720 0.92 0.94 0.96 0.98 1.00 1.02 1.04 Contour length (nm) Correction factor

160 g

120 L C f = LF

80

LDNA = C f (1.000ne +1.414no ) Frequency 40

LDNA = 0.964ne +1.363no 0 640 660 680 700 720 Contour length (nm) The correction factor represents the average of the distribution of Cf ± SD obtained by applying Eq. (4) to each of the one thousand DNA simulated with persistence length and image resolution indicated in the table. The computer simulated DNA was 2000 bp long (680 nm) except (*) where the length of the DNA was set to 200 bp (68 nm).

LDNA = C f (1.000ne +1.414no )

Software utilizzati per analisi di immagini AFM

Gwyddion Free, Unix, MacOS, Windows

WsxM Nanotech Electronica

ImageSXM Versione di NIHimage adattata per AFM

NIHimage Public domain image processing for MacOS

ImageJ Versione Java di NIHimage

SPIP Image Metrology

Matlab Mathworks Riferimenti Bibliografici

1. Russ JC. The Image Processing Handbook; 2007.

2. Freeman H. Boundary encoding and processing. In: Lipkin BS, Rosenfeld A, editors. Picture Processing and Psychopictorics. New York: Academic Press; 1970. p 241-266.

3. Dorst L, Smeulders AWM. Length estimators for digitized contours. Comput. Graphics Image Process. 1987;40:311-333.

4. Vossepoel AM, Smeulders AWM. Vector code probability and metrication error in the representation of straight lines of finite length. Comput. Graphics Image Process. 1982;20:347-364.

5. Rivetti C, Codeluppi S. Accurate length determination of DNA molecules visualized by : evidence for a partial B- to A-form transition on mica. Ultramicroscopy 2001;87:55-66.

6. Rivetti C. A simple and optimized length estimator for digitized DNA contours. Cytometry A. 2009;75:854-861.

7. Marek J, Demjenova E, Tomori Z, Janacek J, Zolotova I, Valle F, Favre M, Dietler G. Interactive measurement and characterization of DNA molecules by analysis of AFM images. Cytometry A 2005;63:87-93.

8. Sanchez-Sevilla A, Thimonier J, Marilley M, Rocca-Serra J, Barbet J. Accuracy of AFM measurements of the contour length of DNA fragments adsorbed on mica in air and in aqueous buffer. Ultramicroscopy 2002;92:151-158.

9. Spisz TS, Fang Y, Reeves RH, Seymour CK, Bankman IN, Hoh JH. Automated sizing of DNA fragments in atomic force microscope images. Med. Biol. Eng. Comput. 1998;36:667-672.

10. Kulpa Z. Area and perimeter measurement of blobs in discrete binary pictures. Comput. Graphics Image Process. 1977;6:434-454.

11. Ficarra E, Benini L, Macii E, Zuccheri G. Automated DNA fragments recognition and sizing through AFM image processing. IEEE Trans Inf Technol Biomed 2005;9:508-517.

12. Wyman C, Grotkopp E, Bustamante C, Nelson HCM. Determination of heat-shock transcription factor 2 stoichiometry at looped DNA complexes using scanning force microscopy. EMBO J. 1995;14:117-23.

13. Wyman C, Rombel I, North AK, Bustmante C, Kustu S. Unusual oligomerization required for activation of NtrC, a bacterial enhancer- binding protein. Science 1997;275:1658-1661.