Spring 2011:3D PSF lab
Objectives
- Measure the point spread function of the 20.309 fluorescence microscope
- Examine the effect of NA, magnification, and optical corrections on PSF
- Generate a dataset for deconvolution problem set
Materials and methods
Exercise left to the reader
Microscope at Station 5
Feb 11 2011 The microscope currently at station 5 is not finalized. Bright field images reveal the sample along with a ghost image of the sample due to some reflection. One possible reason is that the 200mm imaging lens is too far from the objective.
Feb 12 2011 The ghost reflections disappeared when decreasing the bright field illumination. The microscope exhibits some astigmatism apparent when entering and leaving a focal plane. It is most likely due to a dirty lens in the imaging path.
Microscope at Station 3
Feb 14 2011 This microscope (formerly at station 8) has a complete optical construction, and was utilized to obtain images down to the 100X objective without major issues.
The computer controlled stage is being attempted with Matlab software, but it is really easy to just switch to Python control if preferred. The only caveat would be that the computer at station 3 doesn't have Python yet (ask (Steve)^2)
The camera adapter for Matlab has been successfully installed, but again it would be straightforward to switch to Python control based on the code developed by other class members.
ImageJ Tutorial: Deconvolution Lab
PSF: ImageJ 3D Deconvolution tutorial
Introduction:
ImageJ is free open source image analysis program. Download and install imageJ through the website below.
Loading a plug in:
Most pluggins will come with detailed instructions on how to load and run them. For the few that don't, here are some instructions.
1. Save pluggin '.java' file into C:\Program Files\ImageJ\plugins\'PlugginCategory'
Deconvolution Tutorial:
1.
Cited from the site below. <http://bigwww.epfl.ch/algorithms/deconvolutionlab/>
Links
Fluorescence Microscopy in 3D (Paper)