development of the vertical comet assay - nevis laboratories · development of the vertical comet...
TRANSCRIPT
Development of the Vertical Comet Assay and Associated BioComet
SoftwareSoftware
J E R E M I A H W A L A
R A D I O L O G I C A L R E S E A R C H A C C E L E R A T O R F A C I L I T YC O L U M B I A R E U 2 0 0 7
Original 2D Comet Assay
“Microelectrophoreteic Study of Radiation-Induced DNA Damages in Indi idual Mammalian Cells” 1983 Ostling and JohansonIndividual Mammalian Cells” – 1983, Ostling and Johanson
i iDNA Migration
Jeremiah Wala - Columbia 2007 REU: RARAF
Vertical Comet Assay
“Miniaturizing the C A Wi h D
comet lysed cellconducting substrate+
Comet Assay With 3D Vertical Comets” –2002, Baert and O ld
- x
zy
OostveldFirst and only vertical comet assay paper publishedCells placed loosely in a gel
Vertical comet assay for studying dense cell monolayers. Confocal images are taken in the z-plane
Cells placed loosely in a gelDeveloped to demonstrate superiority over 2D comet assay (more information
id d h h i iprovided through imaging.Used confocal microscope to image cells at different slices in the z-direction.
Jeremiah Wala - Columbia 2007 REU: RARAF
Conducting Surface
Sustain cell attachment and growth
Thin enough to pass 6 MeV alpha particle radiation with minimal energy loss and scattering
Inert to cell buffer
Sturdy enough to sustain being transported and handled Electrode and
substrate
Initial Tests…2 cm
substrate
1 cm diameter hole
Jeremiah Wala - Columbia 2007 REU: RARAF
Materials: SABM, 0.05% Trypsin, microdishes, large plastic dishes,hemocytometer, glass coverslips, centrifuge capsule (and counterbalance)
Cell Platingcapsule (and counterbalance).
Procedure: 1. Sterilize aspirator and aspirate off medium. 2. Use 5 mL Hank's Solution to remove serum
Add L % T i ( % dil d i 3. Add .5 mL 0.05% Trypsin (2.5% diluted in Hank's Solution)4. Shake lightly, place in incubator for a few minutes. Check that cells are removed.5. Add 4 mL SABM buffer to inactivate trypsin. 5 4 yp6. Place small amount (2 uL or so) of cell mixture into hemocytometer7. Count cells (only those reflecting light). One big square signals 10^4 cells per mL.8 Place cells centrifuge capsule Place capsule in
Small Airway Epithelial Cells (SAEC) l t d t l i i d 8. Place cells centrifuge capsule. Place capsule in
centrifuge with counterweight.9. Centrifuge at 1500 rpm for 5 min (Program 3)10. Aspirate off excess media, leaving cell pellet.11. Add enough SABM to make 10^6 cells per mL
(SAEC) plated onto aluminized Mylar.
· Successful creation of Microbeam dish wells· Cells plated to metal surface
12. Place 2 uL on each microdish. Immediately cover with glass coverslip.13. Place in incubator (20-25 min for fibroblasts, 45-50 min for EC). 14 Remove glass coverslips and add 2 mL SABM
· Cells plated to metal surface· Required conductance and thickness· Successful imaging of Hoechst 33342 nuclear stain 14. Remove glass coverslips and add 2 mL SABM
per microdish. 15. Replace in incubator
Aluminum Reacted!
Jeremiah Wala - Columbia 2007 REU: RARAF
Gel Electrophoresis
1) Place 100 µl LMP Agar in cell well.
2) Fill with 90 ml 1x TBE electrophoresis buffer and insert top electrode.
3) Apply voltage to create high current (50 250 mA)current (50 – 250 mA)
4) Open system and remove buffer
Heating Concerns
The electrophoresis produces The electrophoresis produces heat from the high current. At 150 mA, total power transferred to buffer by heat: 6.3 W
Jeremiah Wala - Columbia 2007 REU: RARAF
Radiation Considerations
Must pass 6 MeV alpha particles with minimum energy loss
Solve Bethe-Bloch equation for local energy loss behaviorq gy6 MeV incident on gold: 421 KeV/µmTotal Energy Loss in 9.8 µm
l d f f ldMylar and 10s of nm of gold~1 MeV
Jeremiah Wala - Columbia 2007 REU: RARAF
Monte Carlo Simulations
Transport of Ions in Matter Transverse scattering is Transport of Ions in Matter simulation for 9.8 µm Mylar
Transverse scattering is minimal. However, a thinner substrate is always desired.
Jeremiah Wala - Columbia 2007 REU: RARAF
Confocal Microscope Imaging
Jeremiah Wala - Columbia 2007 REU: RARAF
http://staff.science.uva.nl/~zoon/sms/SMS.html
Image Analysis: BioComet
User-friendly image handling software designed for y g g guse with vertical comet assay
Multiple algorithms optimize image data for reducing noise and obtaining comet statistics
Currently available comet assay programs unsuited f ith D t lli f i h for use with 3D comets, calling for in-house development of BioComet
Java based for ease of GUI development crossJava based for ease of GUI development, cross-platform compatibility, and availability of JAI API
Jeremiah Wala - Columbia 2007 REU: RARAF
Operation Sequence
I Fil F ldImage File Folder
Image: a0 Image: a2Image: a1
PNG or JPEG File
Grayscale Gaussian Background Sobel Edge Thinning and
General Image Processing Algorithms
Grayscale Gaussian Filter
Background Filter
Sobel Edge Detection Thresholding
Comet Radius
Comet Center
Comet Tail
Comet Head
Comet Parameter Quantification
DAT FileComet Moment
Jeremiah Wala - Columbia 2007 REU: RARAF
Gaussian Filtering
Convolution Gaussian Filter
.0751 .0751.1238
Original Image Normalized Gaussian convolution filter: σ = 2.0
.0751 .0751
.1238
.1238
.1238.2042
Jeremiah Wala - Columbia 2007 REU: RARAF
Sobel Edge Detection
Approximates the derivatives in x and ypp y
Computes gradient of image
1 0 1 00 0 1 0 2 0 1 01.0
2.0
-1.0
-2.0
0.0
0.0
-1.0 -2.0 -1.0
0.0 0.0 0.0
1.0 -1.00.0
X Direction
1.0 1.02.0
Y Direction X Direction Kernel
Y Direction Kernel
Jeremiah Wala - Columbia 2007 REU: RARAF
Edge Thinning and Hysteresis Thresholding
“A Computational Approach to Edge Detection” –p pp g1986 Canny, J.F
Non Maximum Suppression Hysteresis Thresholdingo S pp o y o d g
Follows edges and assumes line continuity!g y
Jeremiah Wala - Columbia 2007 REU: RARAF
Hysteresis Low Maximum Threshold Hysteresis High Maximum Threshold
Jeremiah Wala - Columbia 2007 REU: RARAF
Implementing the Algorithmsp g g
Comet Location Detection: Use edge detection and thinning (Figure 2)
Comet Head Radius: Use Sobel edge detection, but no thinning or thresholding( i )(Figure 3)
Jeremiah Wala - Columbia 2007 REU: RARAF
Maintaining the Code: Javadocg
Current Javadoc: http://www.people.cornell.edu/pages/jaw226
l d f l d d f h hProgram is compiled into JAR file, and defines the CLASS-PATH to the JAI API. Any user with basic JRE (free) can run BioComet!
Jeremiah Wala - Columbia 2007 REU: RARAF
Conclusions
Both experimental and theoretical tests setup the project well but more materials are needed including a cooler well, but more materials are needed, including a cooler and a more stable gold substrate.Canny edge detection method and homemade algorithms
d i d b t i l i f l fl reduce noise and boost signal in confocal fluorescence imaging.Comet parameterization should be performed at specific
i t i i h dli l ith points in image handling algorithms. The BioComet software is designed for vertical comets, but can easily be adapted to provide a backbone for any
D i i 3D imaging. I learned a lot of biology, radiation physics, computer science, and particle physics in just 10 weeks!
Jeremiah Wala - Columbia 2007 REU: RARAF
Acknowledgements
Dr. Brian Ponnaiya – Biologyy gy
Mr. Steve Marino, Dr. Guy Garty, Dr. Alan Bigelow, Dr. Gerhardt Randers-Pehrson – Physics
Dr. David Brenner – Radiation Oncology
Dr. John Parsons for supporting this REU.
RARAF for funding my stay here at Columbia
di l i l h l ili
Jeremiah Wala - Columbia 2007 REU: RARAF
Radiological Research Accelerator Facility