clinton jung [email protected] advisor: bir bhanu center for research in intelligent systems august 20,...
TRANSCRIPT
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Clinton [email protected]
Advisor: Bir BhanuCenter for Research in Intelligent Systems
August 20, 2009
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Overview
Background Project Objectives Technical Methods
Otsu’s MethodConnected Components
Algorithm
Results and Analysis Conclusions
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Background What are stem cells? Two lines
Embryonic stem cellsAdult stem cells
Five statesAttached to substrateUnattachedDancing, or pre-attachment behaviorDeath, or apoptosisMitosis
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Background cont. Stem cell culture conditions
37 degrees CelsiusTreated with cigarette smoke—traditional and
harm reductionMouse embryonic stem cells are used because
they are easier to obtain and manipulateMatragel substrate
Video CaptureUses Biostation HardwareTime-lapse style3.5 hours, 106 still images
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Background cont. Stem cells alternate
between five statesDancing always comes
before a cell attachesMitosis only occurs when
cells are unattachedApoptosis is often confused
with dancing behavior
Cell ArrangementsSingle CellColony
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Project ObjectivesCrop images for the cells undergoing
dancing and play as video for each cellDevelop image segmentation techniquesFind connected components and compute
featuresQuantify the dancing phenomenon and
change in shape
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Example of Video
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Video Cropped for Dancing
Dancing Cells
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Observations Cells undergoing pre-
attachment behavior (dancing)
• Often have several legs or appendages when dancing. These bulbs are roughly one third or less of the original size of the cell before dancing
• A cell may undergo several cycles of detachment, dancing, attachment
• May affect the state of surrounding cells and influence them in some manner
• Dancing often occurs after mitosis but not necessarily
Cells undergoing pre-attachment behavior (dancing)
• Often have several legs or appendages when dancing. These bulbs are roughly one third or less of the original size of the cell before dancing
• A cell may undergo several cycles of detachment, dancing, attachment
• May affect the state of surrounding cells and influence them in some manner
• Dancing often occurs after mitosis but not necessarily
Cells attached to substrate
• Can be identified by an increase in surface area and exhibit a darker inner intensity value
• When cells attach, they lose their circular shape and instead become noncircular. There were cells that were semi-attached
Cells attached to substrate
• Can be identified by an increase in surface area and exhibit a darker inner intensity value
• When cells attach, they lose their circular shape and instead become noncircular. There were cells that were semi-attached
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Technical Methods
Image Segmentation- Process of dividing an image into different segments (sets of pixels). This technique can be used to locate objects and boundaries based on lines, curves, contrasts
BenefitsAutomatically reduce image to simpler one to analyzeIdentify different components and features of an image
Simpler, processed images can then be analyzed
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Otsu’s Method Step 1: Input Original Image and convert to grayscale Step 2: Find threshold automatically using histogram. Two groups of
pixels created such that the intra-class variance is minimal and inter-class variance maximal
Step 3: Split image into two classes (binary) based on the threshold value. This final image will have pixel values of either 0 or 255.
Grayscale Value
Num
ber
of
Pix
els
Calculated Threshold Value: 138
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Otsu’s Method–Video application
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Connected Components Algorithm Step 1: Input Original Image which must be a binary image Step 2: For each pixel, examine surrounding 8 pixels
If the pixel is neighboring, assign label 1 If pixel is not neighboring, assign label 0
Step 3: Continue to check each pixel line by line until entire image is checked, resulting in a matrix of 1’s and 0’s
Step 4: Convert image back to rgb in order to display components in colors.
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Connected Components Algorithm—Video Application
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Analysis Expand connected components program to count the
number of pixels in a cell undergoing pre-attachment behavior and plot this value over time
Total Number of Pixels vs. Time
Time (Video Frame Number)
Tot
al N
umbe
r of
Pix
els
in
conn
ecte
d co
mpo
nent
fr
ame
Frame 41
Frame 85
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Analysis cont. Expand program to compute average grayscale value of a
dancing cell over time and examine the averages and standard deviations of these values over time per frame.
Time (Video Frame Number)
Ave
rage
Gra
ysca
le V
alue
Average Grayscale Value vs. Time
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Analysis cont.
Time (Video Frame Number)
Gra
ysca
le S
tand
ard
Dev
iatio
n
Grayscale Standard Deviation vs. Time
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Conclusions A preliminary calculation of the changes in pixel count for a cell
undergoing pre-attachment behavior displays no periodic behavior. Pixel count data is also consistent with our hypothesis of a high pixel count for cells in dancing and a low count for a cell in attachment.
The graphical analysis shows that the cells in the dancing, attached, and unattached states have very distinct average grayscale values per frame over the course of the video and can potentially be automatically differentiated using this property
However, the standard deviations of these values for the different stem cell states have a more inconsistent pattern which results in interference with each other
The average and standard deviation values can be combined using pattern recognition techniques
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Acknowledgements
I would like to extend much gratitude toDr. Bir Bhanu for his guidance and adviceBenjamin Guan for his willingness to teach Jun Wang for the opportunity this summer to do
researchTalbot Lab—Dr. Prue Talbot and Sabrina Lin for
growing and filming the stem cells Shubham Debna and Lindsay Kulkin for their
collaborationOther members of the BRITE program and C.R.I.S.