Initialisation

Micron
ND images
Octave

Image IO
GNU Octave for
GraphicsMagick
Bio-Formats
Microscope Image Processing
Threshold

Epigenetic
markers
Carnë Draug (carandraug)
FRAP

Future work
Octave Forge Image package maintainer
Credits

March 20, 2017

Initialisation
Micron
ND images
Octave GNU Octave for
Image IO
GraphicsMagick
Microscope Image Processing
Bio-Formats

Threshold

Epigenetic
markers
David Miguel Susano Pinto
FRAP
Micron Oxford — Advanced BioImaging Unit
Future work
Department of Biochemistry
Credits University of Oxford

March 20, 2017

 Micron
Initialisation
Micron
ND images
Octave

Image IO
GraphicsMagick
Bio-Formats

Threshold
Develop and apply advanced microscopy methods for cell and
Epigenetic developmental biology at the University of Oxford.
markers

FRAP
development Building new microscopes
Future work facility Support researchers
Credits
 Micron
Initialisation
Micron
ND images
Octave

Image IO
GraphicsMagick
Bio-Formats

Threshold

Epigenetic
markers

FRAP

Future work

Credits
 Typical microscope images
Initialisation
14000
Micron
ND images
12000
Octave

Image IO 10000

GraphicsMagick
8000
Bio-Formats

Threshold 6000

Epigenetic 4000

markers
2000
0 100 200 300 400 500
FRAP 14000

Future work 12000

Credits 10000

8000

6000

4000

2000
0 100 200 300 400 500
 Typical microscope images
Initialisation
Micron
ND images
Octave

Image IO
GraphicsMagick
Bio-Formats

Threshold

Epigenetic
markers

FRAP

Future work

Credits
 Typical microscope images
Initialisation
Micron
ND images
Octave

Image IO
GraphicsMagick
Bio-Formats

Threshold

Epigenetic
markers

FRAP

Future work

Credits
 Typical microscope images
Initialisation
Micron
ND images
Octave

Image IO
GraphicsMagick
Bio-Formats • 512 * 512 pixels
Threshold
• 1024 * 1024 pixels
• 1 colour
Epigenetic • 2 colours
markers • 1 focal plane
FRAP • 21 Z stack
• 2500 time frames
Future work • 16 bit
Credits
• 8 bit
• total of 44MB
• total of 655MB
 Images as ND Arrays
Initialisation
Micron
ND images
Octave

Image IO
GraphicsMagick
• x and y
Bio-Formats
• time
Threshold

Epigenetic • z (volume)
markers
• wavelength
FRAP

Future work • phase

Credits • angle
• lifetime
Think “data”, not “picture”
 Absolutely barbaric
Initialisation
Micron
ND images
Octave

Image IO
GraphicsMagick
Bio-Formats • binary_dilation
Threshold
• grey_dilation
Epigenetic
markers • conv2
FRAP
• convn
Future work

Credits
• bwlabel2
• bwlabeln
 Image functions
Initialisation
Micron
ND images
Octave

Image IO
GraphicsMagick
• When colour matters
Bio-Formats
• imread
Threshold
• imwrite
Epigenetic
markers
• rgb2hsv
• lab2xyz
FRAP

Future work • Image related algorithms

Credits • imdilate
• watershed
• bwlabeln
 Typical problems
Initialisation
Micron
ND images
Octave

Image IO
GraphicsMagick
Bio-Formats
Concentration
Threshold Protein expression, number of complexes in a
Epigenetic cellular compartment.
markers

FRAP
Co-localization
Future work Do two signals overlap and correlate?
Credits Dynamics
How fast does it move?
 Why Octave
Initialisation
Micron
ND images
Octave

Image IO
GraphicsMagick • Array programming language
Bio-Formats

Threshold
• Logical indexing
Epigenetic • REPL
markers

FRAP • Community
Future work • Image package
Credits
• Octave Forge
• Free software
 Analysis steps
Initialisation
Micron
ND images
Octave

Image IO
GraphicsMagick
Bio-Formats

Threshold

Epigenetic
1 Open image file
markers
2 Identify regions of interest
FRAP

Future work
3 Perform measurements
Credits
 GraphicsMagick
Initialisation
Micron
ND images
Octave

Image IO
GraphicsMagick
Bio-Formats Not really suitable for science
Threshold

Epigenetic
• No real image values
markers
• No reliable bitdepth
FRAP

Future work
• No reliable image type
Credits
 GraphicsMagick
Initialisation
Micron
ND images
Octave

Image IO
GraphicsMagick
Bio-Formats Not really suitable for science
Threshold

Epigenetic
• No real image values
markers
• No reliable bitdepth
FRAP

Future work
• No reliable image type
Credits
Surprisingly good enough.
 GraphicsMagick
Initialisation
Micron
ND images
Octave

Image IO
GraphicsMagick
Bio-Formats
• TIFF is de facto standard in biological imaging
Threshold

Epigenetic • TIFF is a multi-page

markers

FRAP
img4d = imread (fpath, "Frames", "all");
Future work
imwrite (img4d, fpath);
Credits
 Bio-Formats
Initialisation
Micron
ND images
Octave

Image IO • Java library

GraphicsMagick
Bio-Formats
• Reading of microscope images
Threshold

Epigenetic
• Focus on metadata
markers
• Free software under GPL
FRAP

Future work
• Matlab toolbox
Credits

http://www.openmicroscopy.org/site/products/bio-formats
 Outcome
Initialisation
Micron
ND images Changes in Octave:
Octave

Image IO • conversion between Octave and Java data types

GraphicsMagick
Bio-Formats • conversion of Java arrays
Threshold
• fieldnames and isa
Epigenetic
markers • get and set static class properties
FRAP

Future work

Credits
Changes in Bio-Formats:
• use of javaObject and javaMethod
• 2 cases of isoctave
• release of an Octave package in their build system
 Manual threshold
Initialisation
Micron
ND images
Octave

Image IO
GraphicsMagick
Bio-Formats

Threshold

Epigenetic
markers

FRAP

Future work

Credits
 Automatic threshold
Initialisation
Micron
ND images
Octave
HistThresh by Antti Niemistö:
Image IO
GraphicsMagick • MaxEntropy
Bio-Formats

Threshold • MaxLikelihood
Epigenetic • MinError
markers

FRAP • Otsu
Future work • concavity
Credits
• intermeans
• intermodes
• moments
 Automatic threshold
Initialisation
Micron
ND images
Octave

Image IO
GraphicsMagick
Bio-Formats

Threshold
thresh = graythresh (img, "moments");
Epigenetic
markers bw = im2bw (img, thresh);
FRAP bw = im2bw (img, "moments");
Future work thresh = graythresh (hist, ...);
Credits
 The problem
Initialisation
Micron
ND images
Octave

Image IO
GraphicsMagick
Bio-Formats

Threshold

Epigenetic
markers

FRAP

Future work

Credits
 The solution
Initialisation
Micron
ND images
Octave

Image IO
GraphicsMagick
Bio-Formats

Threshold

Epigenetic
markers

FRAP

Future work

Credits
 Nuclei ROI
Initialisation
Micron
ND images
Octave

Image IO
GraphicsMagick
Bio-Formats

Threshold

Epigenetic
markers
1 convn + fspecial for blur
FRAP 2 graythresh for threshold
Future work 3 imfill for filling holes
Credits
Initialisation
Micron
ND images
Octave

Image IO
GraphicsMagick
Bio-Formats

Threshold im2bw (foci, graythresh (foci(nuclei_mask)));

Epigenetic
markers

FRAP

Future work

Credits
 Watershed
Initialisation
Micron
ND images
Octave

Image IO
GraphicsMagick
Bio-Formats

Threshold

Epigenetic
markers

FRAP

Future work

Credits
 Watershed
Initialisation
Micron
ND images
Octave

Image IO
GraphicsMagick
Bio-Formats

Threshold

Epigenetic
markers

FRAP

Future work

Credits
 Watershed
Initialisation
Micron
ND images
Octave

Image IO
GraphicsMagick
Bio-Formats

Threshold

Epigenetic
markers

FRAP

Future work

Credits
 regionprops
Initialisation
Micron
ND images
Octave

Image IO
GraphicsMagick
Bio-Formats
regionprops (bw, img, props);
Threshold

Epigenetic
markers
• Eccentricity
FRAP • Image
Future work
• Perimeter
Credits
• Area
• WeightedCentroid
 DAPI
Initialisation
Micron
ND images
Octave

Image IO
GraphicsMagick
Bio-Formats

Threshold

Epigenetic
markers

FRAP

Future work

Credits
 Distance to IC
Initialisation
Micron
ND images
Octave

Image IO
GraphicsMagick
Bio-Formats

Threshold

Epigenetic
markers

FRAP

Future work

Credits
 Outcomes
Initialisation
Micron
ND images
Octave

Image IO
GraphicsMagick
Bio-Formats

Threshold • regionprops
Epigenetic
markers • bwconncomp
FRAP • imregionalmin
Future work
• imreconstruct
Credits
 Outcomes
Initialisation
Micron
ND images
Octave

Image IO
GraphicsMagick
Bio-Formats
• tif log file
Threshold

Epigenetic
• CSV with center coordinates and nearest neighbour.
markers x y z NN dist NN idx Voxels
5642.53 9789.85 484.85 1012.14 31 23
FRAP
6135.53 10192.63 441.54 518.02 30 14
Future work ...
Credits
• boxplot of distances
 Outcomes
Initialisation
Micron
ND images
Octave

Image IO
GraphicsMagick
Bio-Formats

Threshold

Epigenetic
markers

FRAP

Future work

Credits
 Fluorescence Recovery After
Initialisation
Photobleaching (FRAP)
Micron
ND images
Octave

Image IO
GraphicsMagick
Bio-Formats

Threshold pre bleach bleaching recovery

Epigenetic
markers
Fluorescence Intensity

FRAP

Future work
Immobile fraction
Credits

Time
 frapinator
Initialisation
Micron
ND images
Octave

Image IO
GraphicsMagick
Bio-Formats

Threshold

Epigenetic
markers

FRAP

Future work

Credits
 frapinator
Initialisation
Micron
ND images
Octave

Image IO
GraphicsMagick
Bio-Formats

Threshold

Epigenetic
markers

FRAP

Future work

Credits
 Don’t port from Matlab
Initialisation
Micron
ND images
Octave

Image IO
GraphicsMagick
Bio-Formats

Threshold • More people win

Epigenetic
markers • No need to backport upstream changes
FRAP • If you wait long enough, you don’t even need to do
Future work anything
Credits
 Future work
Initialisation
Micron
ND images
Octave

Image IO
GraphicsMagick
Bio-Formats • more java for OMERO
Threshold • Java multi-dimensional arrays
Epigenetic • Java import statement and dot notation
markers

FRAP

Future work

Credits
 Future work
Initialisation
Micron
ND images
Octave

Image IO
GraphicsMagick
Bio-Formats • more java for OMERO
Threshold • Java multi-dimensional arrays
Epigenetic • Java import statement and dot notation
markers

FRAP • libtiff interface

Future work

Credits
 Future work
Initialisation
Micron
ND images
Octave

Image IO
GraphicsMagick
Bio-Formats • more java for OMERO
Threshold • Java multi-dimensional arrays
Epigenetic • Java import statement and dot notation
markers

FRAP • libtiff interface

Future work • More threshold algorithms
Credits
 Future work
Initialisation
Micron
ND images
Octave

Image IO
GraphicsMagick
Bio-Formats • more java for OMERO
Threshold • Java multi-dimensional arrays
Epigenetic • Java import statement and dot notation
markers

FRAP • libtiff interface

Future work • More threshold algorithms
Credits
• ND image viewer
 Acknowledgements
Initialisation
Micron
ND images
Octave

Image IO
GraphicsMagick
Bio-Formats

Threshold

Epigenetic
markers

FRAP

Future work

Credits
 Acknowledgements
Initialisation
Micron
ND images
Octave

Image IO
GraphicsMagick
Bio-Formats

Threshold • Ezequiel Micron, University of Oxford

Epigenetic
markers
• CassandraVictoria Innocent, University of Oxford
FRAP • Davide Mazza, National Institutes of Health
Future work
• Antti Niemistö, Tampere University of Technology
Credits
 Acknowledgements
Initialisation
Micron
ND images
Octave

Image IO
GraphicsMagick
Bio-Formats

Threshold

Epigenetic
• Søren Hauberg
markers
• Pantxo Diribarne
FRAP

Future work
• Jordi Hermoso
Credits
 Acknowledgements
Initialisation
Micron
ND images
Octave

Image IO
GraphicsMagick
Bio-Formats

Threshold
• Søren Hauberg
Epigenetic
markers
• Avinoam Kalma
• Pantxo Diribarne
FRAP • Harmut Gimpel
Future work
• Jordi Hermoso
Credits