Gabriel Cristóbal, José Luis Pech-Pacheco & José I. Uzquiano Instituto de Óptica, CSIC,...

"Development of automatic slide scanning "Development of automatic slide scanning

methods for diatom detection"methods for diatom detection"

Gabriel Cristóbal, José Luis Pech-Pacheco & José I. Uzquiano

Instituto de Óptica, CSIC, Madrid, Spain

http://www.iv.optica.csic.es

OverviewOverview

• I. Motivation / Objectives• II. Equipment• III. Control and Image Processing• IV. Discussion / Problems

I. Motivation / ObjectivesI. Motivation / Objectives

• Automatic slide scanning in lighting microscopy “Sequential scanning vs selective scanning”• Background suppression• Diatom detection and localization•Remote slide scanning (telemicroscopy)•Autofocusing

II. Computer EquipmentII. Computer Equipment

• 2 Pentium PC (II & III) with Windows / Linux - 128 M RAM, 14+9 Gbytes disk, networked•1 Frame Grabber Scion (lg-3) + ImagePC Soft (Formerly NIH)•1 Hitachi CCD camera + Pulnix CCD camera (8 bpp, B/W)•1 Videoconference Logitech camera for inspection•1 SUN Enterprise 450 with Solaris 2.6 - 2 Processors (400 Mhz) + 512 M + 9 Gbytes - SUN AEG Research Grant (x2)

•1 Zeiss Axiophot microscope + 2 objective sets•1 Prior X-Y-Z positioning stage (Resol. = 0.1 µm)•1 Stage Controller•1 X-Y Joystick

II. Microscopy EquipmentII. Microscopy Equipment

II. Microscopy EquipmentII. Microscopy Equipment

System configuration for automatic slide scanning

•A. Control

macro ’Contro Prior';var i:integer; ch:string; {actually one char}begin OpenSerial('com1:,9600 baud,no parity,eight data,one stop');

for i:=1 to 10 do begin PutSerial(’r',chr(13)); wait(1); capture; ch:=GetSerial; ShowMessage('char="',ch,'" ); end;end;

III. Control / Image ProcessingIII. Control / Image Processing

•A. Control. -Example of a control algorithm

III. Control / Image ProcessingIII. Control / Image Processing

•A. Control. Sequential vs selective scanning

Sequential

SelectiveMother Image

III. Control / Image ProcessingIII. Control / Image Processing

•A. Control. -Calibration & Computational costs

Objectives Resolution Area in microns # images Time

5x 0.2972 2584.11 x 1938.08 100 1 min. 40 sec. 10x 0.6023 1275.02 x 956.27 400 6 min. 39 sec. 20x 1.1874 676.75 x 485.06 1600 36 min. 0 sec. 50x 3.0610 250.75 x 188.06 10000 2 hrs. 46 min.

III. Control / Image ProcessingIII. Control / Image Processing

•Background suppression - Top-hat algorithms•Binarization - Adaptive thresholding. Triangle’s method - Morphological operators - Centroid calculation (X-Y positions)

B. Image Processing (low resolution mother image)

III. Control / Image ProcessingIII. Control / Image Processing

B. Image Processing. - Background suppression

Mother Image

Without background

Top-Hat

III. Control / Image ProcessingIII. Control / Image Processing

B. Image Processing. - Binarization (Adaptive thresholding)

Without background

Triangle’s method

Thresholded image

Thresholding

III. Control / Image ProcessingIII. Control / Image Processing

B. Image Processing. - Binarization (Centroid calculation)

Thresholding image (X-Y positions)

Centroid Calculation

III. Control / Image ProcessingIII. Control / Image Processing

B. Image Processing. - Scanning strategies method

Particledensity>τ

Particledensity>τ

Mother image

Sequential scanning

NoYes

Selective scanning

System setupSystem setup

III. Control / Image ProcessingIII. Control / Image Processing

System setup and example of automatic slide scanning

Automatic slide scanning

IV. Discussion / ProblemsIV. Discussion / Problems

• Scanning area from slides. Retangular vs circular

preparations. Some standards?

• Broken / agglomerates of particles.

• Geometric invariances. Scale and Mellin transforms.

Correlation measures

• Symmetry.

IV. Discussion / ProblemsIV. Discussion / Problems

• BibTex database of diatom references@article{casasent76a,

author = “Casasent, D. and Psaltis, D.”,

title = “Position, rotation and scale invariant optical correlation”,

journal = “Appl. Opt.”

year = “1976”,

volume = “15”,

number = “7”,

pages = “1795-1799”

IV. Discussion / ProblemsIV. Discussion / Problems

• ADIAC extensions:

- Multiple slide scanning

- Automatic objective’s change

- Problem: option not available in some microscopes

- Pollen counting and identification