Spring 2012:Vincent Lee Lab 2
- Building a fluorescence microscope, tuning
- Learning how optical components like those from ThorLabs come together and work.
- Learning about stepper control in a lab setting
- Learning about 3-dimensional deconvolution and looking at the differences in image reconstruction for the actual and theoretical PSF.
- Epi-fluorescence Microscope with parts from Thorlabs
- Pololu A4983 Stepper Motor Driver Carrier with Voltage Regulators
- Thorlabs 8 mm Travel Stepper Motor Actuator
- Matlab Image Acquisition Toolbox
- Matlab Allied Vision Technologies Adaptor
- National Instruments USB Data Acquisition
- ImageJ plugins: Deconvolution Lab and Diffraction PSF 3D. Manually save in the plugins folder in ImageJ.
- Submicron beads, small enough to be considered point sources
The microscope built was an epi-fluorescence microscope which used a green laser for fluorescence. The stage was designed to be controlled by microstepper motors such that the 3D PSF could be obtained through scanning the z-axis of a sample.
Since a moderately high-power laser was being used, safety precautions were taken:
- An ND filter was used during laser alignment and while the apparatus was being built
- When laser was turned on for actual measurements, the ND filter was removed and 532nm protective laser goggles were worn by all in the vicinity.
- The laser could be aligned using two 2-degrees tunable mirrors, allowing both angle and position of the beam to be controlled.
- Open PSF GUI
- Enable Sleep
- Manually focus on fluorescent beads
- Disable Sleep
- Run PSF Scan from PSF GUI
- Open in imageJ with 'deconvolution' plugin
- Use imageJ plugin PSF 3D.
- Use parameters
- Index of Refraction of the media = 1.398
- Numerical Apeture n*sin(theta) = 0.65
- Wavelength = Nile Red (525-605) – 590 because the cut off of the barrier filter is 590nm
- Longditudinal Spherical Aberration = 0
- Image pixel spacing = 7.4um/40x = 185nm
- Slice spacing = 100nm
- Width = 20px
- Height =20px
- Depth = 84px
- Set settings for an 8 bit image with correct contrast and intensity
Leanna wrote the code, I defer to what she has said about it.
There were two matlab files used, PSFconstruct and PSFmicrometer.
PSFconstruct (stack,bounds) -> (sizedtif)
PSFmicrometer (zdistance, stepdistance) -> Talks to stepper controller at Dev1, signals it to step zdistance in stepdistance intervals.
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