Extracting the Meaning: Painless processing and analysis of image data with Fiji and Ruby

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painless processing and analysis of image data with Fiji and Ruby extracting the meaning Based on the experience from the summer project by Gregory Goltsov melville f o u n d a t i o n Supervisor Dr Anne Savage

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who am i?

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who am i? 4th year Computer Games Technology

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who am i? 4th year Computer Games Technology Ruby fan

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who am i? 4th year Computer Games Technology Ruby fan (basically) a programmer

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the project

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Data

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Breakthrough Breast Cancer Research Unit and Division of Pathology, University of Edinburgh

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Breakthrough Breast Cancer Research Unit and Division of Pathology, University of Edinburgh Breast cancer spheroid

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Breakthrough Breast Cancer Research Unit and Division of Pathology, University of Edinburgh Breast cancer spheroid Drug 396 403 S4 ...

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Breakthrough Breast Cancer Research Unit and Division of Pathology, University of Edinburgh Breast cancer spheroid Drug 396 403 S4 ... ?

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Breakthrough Breast Cancer Research Unit and Division of Pathology, University of Edinburgh Aim To develop a metric to objectively measure the eﬀectiveness of said drugs on the spheroids Breast cancer spheroid Drug 396 403 S4 ... ?

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Data

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Data

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pre-processing • processing • analysis Data

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pre-processing • processing • analysis Data Results

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pre-processing • processing • analysis Data Results ──────

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pre-processing • processing • analysis Data Results ────── Scalable

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pre-processing • processing • analysis Data Results ────── Scalable Robust

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pre-processing • processing • analysis Data Results ────── Scalable Robust Fully automatic

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pre-processing • processing • analysis Data Results ────── Scalable Robust Fully automatic ──────

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pre-processing • processing • analysis

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pre-processing • processing • analysis Filtering invalid data

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pre-processing • processing • analysis Filtering invalid data Renaming

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396 3um well1 time0 403Contwell1time0 409BCont4time0 616inact 30um well2 time0 403 Cont 1 24h S4 1uM 1 24h 616 (inact) 1uM 1 time 48h S4 3 uM 4a time 48h

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396 3um well1 time0 403Contwell1time0 409BCont4time0 616inact 30um well2 time0 403 Cont 1 24h S4 1uM 1 24h 616 (inact) 1uM 1 time 48h S4 3 uM 4a time 48h

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396 3um well1 time0 403Contwell1time0 409BCont4time0 616inact 30um well2 time0 403 Cont 1 24h S4 1uM 1 24h 616 (inact) 1uM 1 time 48h S4 3 uM 4a time 48h How to retrieve a particular image?

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396 3um well1 time0 403Contwell1time0 409BCont4time0 616inact 30um well2 time0 403 Cont 1 24h S4 1uM 1 24h 616 (inact) 1uM 1 time 48h S4 3 uM 4a time 48h How to retrieve a particular image? W hat is the id?

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So I implemented the RENAMER

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RENAMER

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RENAMER

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RENAMER 396 3um well1 time0 403Contwell1time0 409BCont4time0 616inact 30um well2 time0 403 Cont 1 24h S4 1uM 1 24h 616 (inact) 1uM 1 time 48h S4 3 uM 4a time 48h

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RENAMER 396 3um well1 time0 403Contwell1time0 409BCont4time0 616inact 30um well2 time0 403 Cont 1 24h S4 1uM 1 24h 616 (inact) 1uM 1 time 48h S4 3 uM 4a time 48h 396 3uM 1 0h 403 (cont) 1 0h 409 (cont) 4 0h 616 (inact) 30uM 2 0h 403 (cont) 1 24h S4 1uM 1 24h 616 (inact) 1uM 1 48h S4 3uM 4 48h

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But why bother with RENAMER ?

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But why bother with RENAMER ? Consistent

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But why bother with RENAMER ? Consistent Scalable

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But why bother with RENAMER ? Consistent Scalable DB-like querying

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But why bother with RENAMER ? Consistent Time-series analysis Scalable DB-like querying

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But why bother with RENAMER ? Consistent Time-series analysis Robust Scalable DB-like querying

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pre-processing • processing • analysis

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pre-processing • processing • analysis Brightness/contrast correction

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pre-processing • processing • analysis Brightness/contrast correction Pseudo Flat-Field correction

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pre-processing • processing • analysis Brightness/contrast correction Pseudo Flat-Field correction Background subtraction

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pre-processing • processing • analysis Brightness/contrast correction Pseudo Flat-Field correction Background subtraction Thresholding

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Unprocessed data varies a lot 0 h 24 h 48 h

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Unprocessed data varies a lot, making it impossible to threshold 48 h 0 h 24 h

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Original

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Brightness/ contrast correction # Code: Computations.enhance_contrast imp, la

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Pseudo Flat-Field correction # Code: # This is the kernel size for Gaussian blur. # Smaller kernel removes more background kernel_size = 100 pffc = Computations.pffc imp, kernel_size, true

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Background subtraction # Code: IJUtils.run 'Subtract Background...', :rolling => 100, :light_disable => ''

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Otsu thresholding # Code: processor.set_auto_threshold AutoThresholder::Method::Otsu,false IJUtils.run 'Convert to Mask' IJUtils.run 'Despeckle'

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Original Brightness/contrast adjustment + PFFC Subtract background Otsu thresholding

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pre-processing • processing • analysis

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pre-processing • processing • analysis Klonowski landscapes

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pre-processing • processing • analysis Klonowski landscapes Higuchi fractal dimension

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pre-processing • processing • analysis Klonowski landscapes Higuchi fractal dimension Area and perimeter

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klonowski landscapes

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klonowski landscapes 2D image ➝ 1D “signal”

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Vertical landscape Horizontal landscape

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Klonowski et al. Nonlinear Biomedical Physics 2010, 4:7 http://www.nonlinearbiomedphys.com/content/4/1/7 Vertical landscape Horizontal landscape

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Vertical landscape Horizontal landscape

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higuchi fractal dimension

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higuchi fractal dimension Complexity, D, of a “signal”/curve

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No content

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“Jagged, uneven, rough edge” “Smooth, gradual, round” D = 1.84 D = 1.07

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“Jagged, uneven, rough edge” “Smooth, gradual, round” D = 1.84 D = 1.07

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No content

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Untreated

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Untreated Treated (1μM 396)

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D ≃ 1 Untreated Treated (1μM 396)

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D ≃ 1 Untreated Treated (1μM 396) D ≃ 1.3

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D ≃ 1 Untreated Treated (1μM 396) D ≃ 1.3 D ≃ 1.32

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D ≃ 1 Untreated Treated (1μM 396) D ≃ 1.3 D ≃ 1.32 D ≃ 1

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D ≃ 1 Untreated Treated (1μM 396) D ≃ 1.3 D ≃ 1.32 D ≃ 1 D ≃ 1.15

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D ≃ 1 Untreated Treated (1μM 396) D ≃ 1.3 D ≃ 1.32 D ≃ 1 D ≃ 1.15 D ≃ 1.28

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results

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resultsalpha

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Lower % is better

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Lower % is better

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what next?

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what next? ──────

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what next? ────── Open-source Higuchi fractal dimension code

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what next? ────── Open-source Higuchi fractal dimension code More analysis

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what next? ────── Open-source Higuchi fractal dimension code More analysis Paper

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what next? ────── Open-source Higuchi fractal dimension code More analysis Paper ──────

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thank you! gregory@goltsov.info www.gregory.goltsov.info Questions? melville f o u n d a t i o n Big thanks to: School of CONTEMPORARY SCIENCES