Date post: | 15-Jan-2016 |
Category: |
Documents |
View: | 223 times |
Download: | 0 times |
Im Rahmen des K plus Programms gefördert durch:
Analysis of Cell Arrays: A Tool for Evaluation of High Density
Micro Colony Chips in Fluorescence Assays
Martina Uray & Horst Bischof Institute for Computer Graphics and Vision
Jochen Gerlach & Ingo Klimant Institute of Analytical Chemistry and Radiochemistry
Brigitte PohnResearch Centre Applied Biocatalysis
Helmut Schwab Institute for Molecular Biotechnology
2PPGT 2005 Martina Uray
Overview
• Motivation
• System Setup
• Enzymatic Reaction
• Automatic Analysis
• Results
• Conclusion and Future Work
3PPGT 2005 Martina Uray
Motivation
• Need– Generation of highly active, specific and enantioselective
biocatalysts High throughput screening methods, suitable for desired enzyme reaction
• New development– Screening of whole cell arrays without decomposition of cells
and further cleaning steps – High throughput due to a semi-automatic software tool
4PPGT 2005 Martina Uray
• Two strains with significant difference in esterase activity• Spotting of the array (~5000 colonies) with Microgrid II robot
(Biorobotics) – Regular sub-blocks, strains are randomly distributed– Reference spots for activity comparison– Cells grow at 30° over night and form micro colonies
• Replication of arrays
– Studies on different substrates or reaction conditions
• Preparation of the sensor– 100 µm thick– Includes the indicator, substrate and reaction buffer
System Setup I
5PPGT 2005 Martina Uray
System Setup II
• Start of the reaction by putting the array onto the sensor– Substrate diffuses into the micro colonies– Reaction leads to a flux of protons into the sensor– Usage of fluorescent pH indicator which fluorescence
intensity is dependent on proton concentration– Excitation with a blue LED – Signal detection with a CCD camera– Measurement in constant time intervals
Image stack which shows the sensory response over time
6PPGT 2005 Martina Uray
Enzymatic Reaction I
120 s 220 s
320 s
7PPGT 2005 Martina Uray
Enzymatic Reaction II
8PPGT 2005 Martina Uray
Automatic Analysis I
• Given– Composition of the array
(number of blocks, number of spots per block, fix spot-diameter)• Automatically …
– …find the position of the spots Corresponding block Situation inside the block
– …find the centre of the colonies Spot-content from centre and diameter
– …calculate the kinetic properties of every spot in the array Intensity development over time (intensity curve) Gradient (regression line) reflects the turnover rate
– …rate colonies Mapping to high and low activity
9PPGT 2005 Martina Uray
Automatic Analysis I
• Software tool with graphical user interface• Automatically find the centre and the position of the spots
(fixed diameter)• Manually select an image and delineate the array with a
rectangle
• Normalize image for comparison
10PPGT 2005 Martina Uray
Automatic Analysis II
1. Gaussian filter Noise reduction
2. Cross correlation Binary image
3. Summation over stack Grey scale image
4. Radon Transformation Estimation of rotation and starting point of grid Rough grid alignment
5. Fast Mean Shift (adaptive gradient ascent method) points towards the direction of maximum increase in density Exact spot centres
11PPGT 2005 Martina Uray
Results I
12PPGT 2005 Martina Uray
Results II
Spot positions are …• … clearly identified as long as there is a
signal• … approximated when there is no signal• … missed if the real centre is too far from
the first grid approximation
13PPGT 2005 Martina Uray
Results III
• Comparison between standard and unknown spots Distinction of high (NJ70) and low (N27) activity esterase mutants spotted randomly over the array
14PPGT 2005 Martina Uray
• Developed a semi-automatic system for fast screening of a huge amount of datasets
• Demonstrated robust performance
• Next Steps:– Calculate grid from reflected light image
Evaluate the exact size of the spots (Fast Mean Shift Algorithm) Calculation of spot intensity in comparison to its size
– Handle bright spots on dark background– Evaluate spot intensities in different parts of the array over
different time intervals
Conclusion & Future Work
15PPGT 2005 Martina Uray
Thank you for your attention!