Before acquiring images for deconvolution, ideal sampling density has to be found. The XY Calibration and Z Step calculations are different for confocal and widefield microscopy.
Ideal value for a widefield microscope is calculated as follows:
Figure 593. XY Calibration
Figure 594. Z Step
where λem is the sample emission wavelength, n is the refractive index of the objective lens, and α is the objective half-aperture angle which is calculated as follows:
Figure 595.
where NA is the numerical aperture of the objective calculated as follows:
Figure 596.
Note
Calculated values for XY calibration and Z step are ideal. Use this value or the closest possible value. A range from zero to 3x the ideal value (for XY calibration) or 2x the ideal value (for Z step) can be used. A warning message is shown for values higher than these limits, notifying that the deconvolution effect may be weak or ineffective.
Ideal value for a confocal microscope is calculated as follows:
Figure 597. XY Calibration
Figure 598. Z Step
where λex is the excitation wavelength of the light source, n is the refractive index of the objective lens, and α is the objective half-aperture angle.
Note
Calculated values for XY calibration and Z step are ideal. Use this value or the closest possible value. A range from zero to 3x the ideal value (for XY calibration) or 2x the ideal value (for Z step) can be used. A warning message is shown for values higher than these limits, notifying that the deconvolution effect may be weak or ineffective.