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Measuring dOPM optical performance
Before any of these experiments ensure system is aligned.
For these experiments you'll need fluorescent beads embedded in agarose.
Beads need to be on the scale of the point spread function (PSF) of the microscope.
Image a sparse bead sample with dOPM. For spatial resolution 100 nm TetraSpeck beads are suitable balance between brightness and size.
Use code to extract line profiles from isolated bead volumes. Compare measured line profiles with theoretical values.
Image a sparse bead sample with dOPM using epi-illumination rather than light-sheet illumination an empty filter position in the dOPM filter wheel.
Image a target bead with dOPM camera then image the same bead with the wide-field camera under the same epi-illumination setting i.e. same illumination power, same exposure time, camera binning, filter cube in microscope lower turret.
For minimal bleaching 200 nm TetraSpeck beads are suitable balance between brightness and size.
Use code to measure the integrated signal from isolated bead volumes. Compare integrated signal between left and right hand cameras per bead.
Theoretical FWHM:
- NA
- wavelength
- refractive index
Theoretical collection efficiency: