Paul Matsudaira
WI/MIT BioImaging Center, Dept Biology and Div Biological Engineering, MIT
Cellular machinery, biomechanics, and bioinformatics
IC-21 macrophage cells transfected with GFP, imaged in 3D, and rendered as a projected solid (Imaris)
•bioengineering models describe biological processes•complex movements via cellular machines•mechanics + chemistry•structures capture states of movement•informatics/computing resources
ManipulateMolecular GeneticsChemical GeneticsCell Engineering
MeasureBiochemistry
ImagingProteomics
Bioelectronics
Systematic Experiments
ModelDifferential Eq. Matrices
Bayesian NetworksBoolean Networks
MineBioinformatics
GenomicsData Semantics
Quantitative Models
Quantitative Biology Paradigm
courtesy of CSBi
Cell biologists study large, dynamic structures
G. Borisy and S. Tatiana
1 nm 1 µm 1 mm
cellsorganelles
molecules protein machines
NMR, x-rays
cryo electron microscopy
single particle det. dynamic 4D light microscopy
resolution and size scale
Overlapped structural and cell biology approaches
zone of adhesion and extenstion
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Forces generate movements, machines generate forces
fimbrin-EGFPtimelapse 30 sec
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Changes in structure-assembly, force, chemistry
mother adhesiondaughter adhesions
top side
Formation of cell adhesions at the leading edge of a macrophage cell(J. Evans et al J. Cell Biol. in press)
•multispectral fluorescence data acquisition•video rate data acquisition•cellular tomography and single particle cryoEM imaging•high content screening•whole proteome studies
Cell Biology Appetite for More Information
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High content: Tracking all
cell adhesions
(J. Evans et al J. Cell Biol. in press)
Dynamics of structure modulated by microtubules
10 µm paclitaxel
10 µm demecolcine
(J. Evans et al J. Cell Biol. in press)
Imaging storage/processing requirements
object file sizegenome sequence 12 MBprotein structures1 2 GB2D localization2 6 GB3D localization3 300 GB4D localization4 3.6 TB152kDa (ave. MW) x 110 MW/residues x 10 atoms2512x512 two channel, 16 bit TIFF350 image slices/stack412 images/series
The BioImaging Pipeline
acquisition
management
processinganalysis modelling
Podosomes split, merge, and appear de novo
polar assembly
simple
dendroid
genomics imagingtraces counts
ATGC voxelsequence image
function function
Imaging is an informatics science
WI/MIT BioImaging Net
terminal
terminal
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Origin3400
4 TB
confocalmicroscopy
Origin2400
4 TB
IBM Power4 6550
Cryo-EM
2-photon microscopy
Tape
RAID30TB
imaging modes
Expansion and management of imaging data
channel 0 channel 1 channel 2
multi-spectral (channels) 12 or 16-bit acquisition
256 x 256 1 130 KB3 910 KB
1024x 1024 1 2 MB3 14 MB
image size (pixels) channels file size
1+2+3
(x,y,z,t,ch) file size 256 x 256 x 1 x 3 400 KB 256 x 256 x 50 x 3 20 MB 256 x 256 x 50 x 20 x 3 400 MB 1024 x 1024 x 50 x 3 800 MB 1024 x 1024 x 50 x 20 x 3 16 GB
3D/4D image data (stacks)
35KB to 8.2 MB per channel/stack (X:Y:Z or X:Y:T)
NMR
x-raycryoEM
light
microscopy
raw data12 or 16-bit
deconvolved data
32-bit
intermediates32-bit
raw data32-bit
40 MB 80 MB 80 MB 80 MB 80 MB
40 MB 120 MB 200 MB 280 MB 360 MB
accumulated file size
Image processing (deconvolution) RAM 256 x 256 x 50 x 3 (x,y,z,ch)
3.2 GB
ch1 vs. ch2ch1 vs. ch3 ch2 vs. ch3ch1 vs ch2 vs. ch3
colocalization
Total data accumulation
rendering
1 GB
movies
object analyses
1 GB
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5 5 53 5 5 53 5 5 53 5 5 53 5 5 5345 43 53 45 43 53 45 43 53 45 43 53 45 43 53
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5 5 53 5 5 53 5 5 53 5 5 53 5 5 5345 43 53 45 43 53 45 43 53 45 43 53 45 43 53
545578 8787 8786 545578 8787 8786 545578 8787 8786 545578 8787 8786 545578 8787 878645 5 335 45 5 335 45 5 335 45 5 335 45 5 335
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1 GB ?
raw 0.4 GBpost-deconvol 3.6 GBanalysis 7.2 GB
total 11.2 GB
terminal
terminal
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SGI Origin3400
32 GB RAM4 TB
Zeiss510 META ftp
data acquisition data storageimage restorationimage analysisbatch rendering
database management remote visualizationrenderingbatch design
Current WI (local) imaging net
stack (512 x 512 x 50 x 12 x 6)4.8 GB
Microtubules at podosomes
tubulinactinfimbrin
QuickTime™ and aVideo decompressorare needed to see this picture.QuickTime™ and aVideo decompressorare needed to see this picture.
Dynamic structures from light microscopy
fimbrin-EGFPtimelapse 15 sec
side/bottom view top view
‘finger’
‘palm’‘finger’
‘finger-tip’‘palm’
assembly of cell adhesions in a live cell
Dynamic Cell Adhesions - Static
