Difference between revisions of "Locating objects in a fluorescent microscopic image"
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− | == | + | ==Overview== |
− | + | ||
+ | ===Goals=== | ||
+ | * Develop software to find locations of fluorescent microspheres in a digital image, σ<sub>x,n</sub> and σ<sub>y,n</sub>. | ||
+ | * Investigate the effect of optical noise sources on centroid accuracy and precision. | ||
+ | |||
+ | ==Procedure== | ||
===Image 1 micron fluorescent microspheres=== | ===Image 1 micron fluorescent microspheres=== | ||
# Use imaqtool to record an image of 1 micron fluorescent microspheres at 40X magnification. | # Use imaqtool to record an image of 1 micron fluorescent microspheres at 40X magnification. |
Revision as of 04:59, 2 January 2013
Overview
Goals
- Develop software to find locations of fluorescent microspheres in a digital image, σx,n and σy,n.
- Investigate the effect of optical noise sources on centroid accuracy and precision.
Procedure
Image 1 micron fluorescent microspheres
- Use imaqtool to record an image of 1 micron fluorescent microspheres at 40X magnification.
- Minimize quantization noise by selecting 16-bit monochrome mode.
- Adjust the camera settings to achieve good contrast with minimal noise.
- Plot a histogram of the pixel values to verify optimal exposure
- Use a log axis for the pixel count.
- Take another image if the contrast is low or some pixels are over-saturated.
- Write a function in Matlab or another languageto compute the centroid of each microsphere
- The function should return an N row by 2 column matrix, where N is the number microspheres in the image. The first column should contain the X coordinate and the second column should contain Y.
Relevant Matlab commands
imaqtool imhist semilogy im2bw graythresh regionprops
Precision and accuracy of centroids of stationary objects
- Image fluorescent microspheres as before, but change the settings in imaqtool to capture 5 sequential frames instead of 1.
- Run your centroid finding code on each image.
- Compute the standard deviation of each particle's centroid, σx and σy.
Error sources
Discuss the effect of the following error sources on the centroid values.
- Shot noise
- Readout noise
- Dark current noise
- Ambient light
- Mechanical vibration
<-- Todo: image representations, histogram and how to expose image page imaqtool/camera settings page -->