Calibration of Leica Scanscope AT2
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1 Calibration of Leica Scanscope AT2 Allen H. Olson, PhD Aperio epathology, Leica Biosystems ICC Medical Imaging Working Group 18 Nov 2013
2 Calibration of Leica Scanscope AT2 Overview Spectral Models for Scanner and Microscope Histological Stain Spectra (examples from literature) Construction of Color Transform Viewing of Digital Slides (ICC Profile using 3D LUT) Validation of Spectral Models (IT8.7 Film Target) Measuring Scanner Spectral Response Slide-Specific Color Profiling
3 Spectral Model for Scanner Dalsa Piranha PC-30 Camera < r ( f ), g ( f ), b ( f ) > LED Light Source L S ( f ) < R, G, B > Scanner Output < W > White Balance [T = 1.0] T( f ) Stain Transmission Spectra
4 Spectral Model for Microscope Olympus Hoya CIE Standard Observer < x ( f ), y ( f ), z ( f ) > Tungsten Lamp L m ( f ) Daylight Filter F( f ) < X, Y, Z> CIE Tristimulus Values W y Normalization (Y max = 1) T( f ) Stain Transmission Spectra
5 Histological Stain Spectra T(f) Cukierski, et al. (9) McNamara (3) Garini, et al. (6) Van der Laak (1)
6 Construction of Color Transform Stain Spectra T(f) Neutral Gray Hematoxylin Eosin DAB Methyl Green Scanner Model Microscope Model < R i, G i, B i > < X i, Y i, Z i > Target Spectra [T k (f)] γ x[t l (f)] α Interpolator 3D LUT < X, Y, Z > =LUT(< R, G, B >)
7 Viewing of Digital Slides Color Management ICC Profile Workflow Digital Slide ICC Profile Use create_clut_profile application (ICC website) Chromatic Adaptation to D50 connection space Microscope White Point = (0.9984, , ) Monitor ICC Profile srgb mode for monitor Use generic srgb profile Viewing Software Aperio ImageScope LCMS library Microscopic Viewing Nikon Eclipse E with Hoya 80A filter
8 Viewing of Digital Slides Raw RGB srbg (Using ICC Profile) Raw RGB srbg (Using ICC Profile)
9 Viewing of Digital Slides Raw RGB srbg (Using ICC Profile) Raw RGB srbg (Using ICC Profile)
10 Viewing of Digital Slides Raw RGB srbg (Using ICC Profile) Raw RGB srbg (Using ICC Profile)
11 Validation of Spectral Models IT8.7 Ektachrome Film Target (Wolf Faust) Calibration File XYZ (D50) Spectral Transmittance nm (10nm) Scanner Model Validation Scan/Measure Target RGB values Calculate Model-Predicted RGB values Compare Measured vs Predicted values Microscope Model Validation Change Lamp to D50 (no filter) Calculate Model XYZ values Compare with calibration XYZ values
12 Validation of Spectral Models Scanner Model Standard Error 4-5 counts (shown above) All Model data based upon manufacturer data sheets Microscope Model D50 values agree to 10-4 (precision of spectral data) Obviously manufacturer calculated these too
13 Measuring Scanner Spectral Response JDS Uniphase Corp. Linear Variable Filter JDS Uniphase Corp. 1. Scan along length of filter 2. Spectral response R(f), G(f), B(f) 3. Compare to model camera/light response JDS Uniphase Corp.
14 Measuring Scanner Spectral Response Model / LVF Comparison Scan direction Semrock Quad Band Filter: FF01-440/521/607/700 used for referencing the scan axis to nm.
15 Slide-Specific Color Profiling Observer Metamerism R 1 G 1 B 1 R 2 G 2 B 2 T 1 (f) T 2 (f) X Y Z R G B Device Metamerism T 1 (f) T 2 (f) X 1 Y 1 Z 1 X 2 Y 2 Z 2 This problem can be avoided altogether for histology. Histology slides mostly have two or three stains, designed to not be observer metameric likely not device metameric either. A color transform can be calculated for each slide, based upon the specific stains and their spectral properties.
16 Conclusions / Questions Spectral models for scanner and microscope were combined to generate a color transform based upon manufacturer data specs and stain transmittance spectra from published literature. Significantly, the color transform was calculated without actually scanning a target slide. The models were then validated using an IT8.7 film target, having known spectral transmittance. Calibration of the scanner s transfer function was also performed using a Linear Variable Filter (LVF) and compared favorably to the generic model. This approach suggests the possibility of generating slidespecific profiles for each digital slide, based upon precalibrated spectral properties of the actual stains.
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