Spring 2012:Vincent Lee Lab 2

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Objectives

  • 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.


Materials

Microscope

  • Epi-fluorescence Microscope[1] with parts from Thorlabs

Drivers

Figure 1. The Pololu A4983 Microstepper with voltage regulators.
  • Pololu A4983 Stepper Motor Driver Carrier with Voltage Regulators[2]
  • Thorlabs 8 mm Travel Stepper Motor Actuator[3]

Processing

  • Matlab
  • Matlab Image Acquisition Toolbox[4]
  • Matlab Allied Vision Technologies Adaptor[5]
  • National Instruments USB Data Acquisition[6]
  • ImageJ[7]
  • ImageJ plugins: Deconvolution Lab[8] and Diffraction PSF 3D[9]. Manually save in the plugins folder in ImageJ.

Subject

  • Submicron beads, small enough to be considered point sources

Microscope

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.

Safety

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.

Laser Alignment

  • The laser could be aligned using two 2-degrees tunable mirrors, allowing both angle and position of the beam to be controlled.

Data Acquisition

Actual PSF

  • Open PSF GUI
  • Enable Sleep
  • Manually focus on fluorescent beads
  • Disable Sleep
  • Run PSF Scan from PSF GUI
  • Open in imageJ with 'deconvolution' plugin

Theoretical PSF

  • 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
  • Visualize

Results

Figure 3. A 3D view of our PSF found by looking at the light recorded by the microscope by a single fluorescent 190 nm bead.
Figure 4. An image stack of our PSF.
Figure 5. A 3D view of a calculated theoretical PSF.
Figure 6. An image stack of a calculated theoretical PSF.


Code

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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