Development of the Vertical Comet Assay - Nevis Laboratories · Development of the Vertical Comet...

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.


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


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!


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


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


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.


Confocal Microscope Imaging


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


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


Grayscaling and Background Filtering


Gaussian Filtering

Convolution Gaussian Filter

.0751 .0751.1238

Original Image Normalized Gaussian convolution filter: σ = 2.0

.0751 .0751

.1238

.1238

.1238.2042


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


Original Picture: SYBL Green Fluorescently Labeled Cells


Grayscaled and Gaussian Filtered (σ=2.0)


Background Filtered


Sobel Edge Detection


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


Hysteresis Low Maximum Threshold Hysteresis High Maximum Threshold


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)


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!


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!


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


Radiological Research Accelerator Facility

Development of the Vertical Comet Assay - Nevis Laboratories · Development of the Vertical Comet...

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