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

"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


•A. Control. Sequential vs selective scanning

Sequential

SelectiveMother Image


•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.


•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)


B. Image Processing. - Background suppression

Mother Image

Without background

Top-Hat


B. Image Processing. - Binarization (Adaptive thresholding)

Without background

Triangle’s method

Thresholded image

Thresholding


B. Image Processing. - Binarization (Centroid calculation)

Thresholding image (X-Y positions)

Centroid Calculation


B. Image Processing. - Scanning strategies method

Particledensity>τ

Particledensity>τ

Mother image

Sequential scanning

NoYes

Selective scanning

System setupSystem setup


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.


• 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”


• ADIAC extensions:

- Multiple slide scanning

- Automatic objective’s change

- Problem: option not available in some microscopes

- Pollen counting and identification

Documents

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