Nyquist calculator

Nyquist rate and PSF calculator Nyquist calculator

The Numerical Apperature must be lower than the Lens immersion refractive index.

Excitation wavelength should be shorter than emission wavelength.

Conflicting combination of wavelengths and excitation photon count.

Microscope type

Numerical aperture

Excitation wavelength

Emission wavelength

Number of excitation photons

Lens immersion refractive index

Backprojected pinhole radius

STED Depletion wavelength

STED 3x percentage

STED Saturation factor

SPIM excitation mode

Width of Gaussian light sheet

µm

Light sheet NA

Light sheet fill factor

Array Detector model

Calculate a Point Spread Function

Specimen medium refractive index

Acquisition depth

µm

Backprojected pinhole radius

B.P. distance between pinholes

µm

Objective Quality

A true Nyquist rate does not exist for STED. Instead, we calculate a sampling rate that is both practical and high enough to capture all information realistically available.

Acquisition depth is always 0 µm for 4Pi.

Results

This is the parameter list used in this calculation:

Parameter	Value
Microscope type
Numerical aperture
Excitation wavelength
Emission wavelength
Number of excitation photons
Lens immersion refractive index
Backprojected pinhole radius
STED Depletion wavelength
STED 3x percentage
STED Saturation factor
Specimen medium refractive index
Specimen medium refractive index
SPIM excitation mode
Width of Gaussian light sheet
Light sheet NA
Light sheet fill factor
Array Detector model
Backprojected pinhole radius
B.P. distance between pinholes

The optical axis lays along z. Your Nyquist sampling is:

x: nm
y: nm
z: nm

Set your zooms and scanning steps so that each pixel covers a x-y area of nm × nm (or smaller)

Calibrate and set your z-stepper so that it takes steps of nm when acquiring a 3D stack (or smaller)

For Confocal Microscope images recorded with pinholes of around 1 Airy disk diameter, lateral sampling distances may be up to 1.6 times of the recommended Nyquist values without significantly compromising image quality. When small pinholes are used (< 0.5 Airy disk), up to 1.3 times larger; with very large pinholes (> 4 Airy disks), up to 2 times.

Images captured with Airyscan Fast Mode, have a 4 times larger y-sampling distance, because 4 rows are sampled simultaneously to increase acquisition speed.

For Array Detector images that will be deconvolved with Huygens super-sampling mode 'superXY', lateral sampling distances may be up to 1.7 times the recommended Nyquist values, without significantly compromising image quality.

PSF: XY MIP grayscale — xy MIP grayscale

PSF: XY MIP false color — xy MIP false color

PSF: XZ MIP grayscale — xz MIP grayscale

PSF: XZ MIP false color — xz MIP false color

These are xy and xz MIP projections of the 3D point spread function.
The 3D image dimensions (x × y × z) are × × µm.
It was calculated using the Nyquist sample sizes specified above.
The number of samples (x × y × z) to cover this volume at the Nyquist rate is × × .
To enhance dimmer regions the gamma is corrected by 1.7 in the false color image.

For Array Detectors a PSF of the center detector is shown.

For Airyscan Fast Mode, a combined PSF is shown.