You need to be able to locate specific stars in the sky. Note: Using a Star Analyser grating requires that you already be somewhat proficient at capturing images of stars. In a hurry or want some help getting started? Submit your equipment details in our Equipment form and we'll send you a short report that shows you the best way to use a Star Analyser to capture spectra. If you plan to use a standalone DSLR, please read this: link. Need support? Please contact us if you find any bug or have any suggestion.Are you using a DSLR? Please be sure to watch this video: link. You can use the PSF calculator option in the form to see the expected size of the PSF of your imaging setup and accordingly, of a distilled Experimental PSF from bead images. as 2D images) and do 2D deconvolution in the Huygens Software planewise. Hint: If you have a data stack that is dramatically undersampled in Z (not fulfilling the Nyquist Criterion by a large factor) it is better to interpret the different planes as independent images (i.e. In case of low Numerical Apertures, like 0.4, we recommend not to undersample in the axial direction. Widefield microscopy data is more sensitive to undersampling, so it is better to stay below a factor of 1.5. When large pinholes are used, up to 2 times larger even. For Confocal Microscope images, sampling distances may be up to 1.7 times the Nyquist ones. In these cases larger sampling distances may be used and a good job can still be done when deconvolving these images. While sampling at the Nyquist rate is a very good idea, it is in many practical situations hard to attain. The correct Nyquist rate is defined in terms of the system Bandwidth (in the frequency domain) which is determined by the Point Spread Function. The pixelation corresponding to the Nyquist rate will be clearly seen.Ī common rule of thumb defines the ideal sampling in terms of spatial resolution ("sample with half of the resolution") but this is not exactly correct, and in some cases will lead to undersampling. The images are shown as Maximum Intensity Projections along Z and Y, and upscaled for a better display. The size of the PSF image will be given in µm. You have also the option to generate an image of the Point Spread Function (PSF) which only takes a few seconds more. The data will be returned in nanometers (nm). (To see what equations are used in this calculator and some theory behind the scenes read the Nyquist rate background article). With the form on this page you can calculate the Ideal Sampling from your optical conditions to acquire a well sampled image. See the examples on anti aliasing and aliasing artifacts, and some consequences in Quality Vs Sampling. Frequently, the Nyquist rate is considered as being two times lower than the spatial resolution limit of a microscope, which is incorrect and will also lead to undersampling. Images obtained with sampling distances (voxel dimensions) larger than those established by this rate will suffer from undersampling. This Nyquist rate is equal to twice the highest frequency (bandwidth) of the signal. It is recommended to sample the image at a rate close to the ideal Nyquist Rate. The ideal Sampling Density (or inversely, Voxel Size) for image acquisition depends on the optics of the microscope. For further details, see below.įor installing our Nyquist app for Android devices, please visit this page. To compute these backprojected values, see the Backprojected Pinhole Calculator. divided by the total magnification of the system. ![]() ![]() The Pinhole Radius and Pinhole Distances parameters are not physical sizes, but rather BackProjected sizes, i.e. The Huygens Theoretical PSF page contains more information and interactive examples on the impact of RI mismatch on the PSF shape. ![]() ![]() Note that the pinhole size doesn't alter the bandwidth of the detection system. To determine the ideal sampling density you need to image at the Nyquist rate Additional parameters appear if you check the option to calculate the Theoretical PSF. Microscopy Nyquist rate and PSF calculatorīefore you're performing a microscopy experiment, calculate the correct pixel size so that you image enough detail of your object.
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