Difference between revisions of "Locating objects in a fluorescent microscopic image"
From Course Wiki
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(→Precision and accuracy of centroids of stationary objects) |
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regionprops | regionprops | ||
| − | == | + | ==Centroid precision and accuracy== |
# Image fluorescent microspheres as before, but change the settings in imaqtool to capture 5 sequential frames instead of 1. | # Image fluorescent microspheres as before, but change the settings in imaqtool to capture 5 sequential frames instead of 1. | ||
| + | #* Ensure that the camera gain is set to 0. | ||
# Run your centroid finding code on each image. | # Run your centroid finding code on each image. | ||
# Compute the standard deviation of each particle's centroid, σ<sub>x</sub> and σ<sub>y</sub>. | # Compute the standard deviation of each particle's centroid, σ<sub>x</sub> and σ<sub>y</sub>. | ||
| + | # Turn the gain up to 5; re-adjust the exposure setting, and acquire another set of 5 images | ||
| + | # Turn the gain up to 10; re-adjust the exposure setting, and acquire another set of 5 images | ||
| + | # Run your centroid finder on the gain=5 and gain=10 image sets and compute the standard deviation. | ||
| + | |||
| + | ==Report== | ||
| + | # Compare the accuracy an precision of intensity-weighted centroids at different gain settings. | ||
| + | #* What is the trend in the uncertainty? | ||
| + | #* What is the likely explanation for the trend? | ||
===Error sources=== | ===Error sources=== | ||
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image representations, histogram and how to expose image page | image representations, histogram and how to expose image page | ||
imaqtool/camera settings page | imaqtool/camera settings page | ||
| + | Determine gain value to use | ||
--> | --> | ||
{{Template:20.309 bottom}} | {{Template:20.309 bottom}} | ||
Revision as of 05:05, 2 January 2013
Overview
In this exercise, you will figure out where stuff is in an image.
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
Centroid precision and accuracy
- Image fluorescent microspheres as before, but change the settings in imaqtool to capture 5 sequential frames instead of 1.
- Ensure that the camera gain is set to 0.
- Run your centroid finding code on each image.
- Compute the standard deviation of each particle's centroid, σx and σy.
- Turn the gain up to 5; re-adjust the exposure setting, and acquire another set of 5 images
- Turn the gain up to 10; re-adjust the exposure setting, and acquire another set of 5 images
- Run your centroid finder on the gain=5 and gain=10 image sets and compute the standard deviation.
Report
- Compare the accuracy an precision of intensity-weighted centroids at different gain settings.
- What is the trend in the uncertainty?
- What is the likely explanation for the trend?
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 Determine gain value to use -->
