• Insert a size-standard (a nano-bead) instead of your specimen. Size-standards are available commercially. The size of the bead should be as small as possible - i.e. ideally match the size of one subpixel (smaller than a pixel) in the resulting image.

  Note

  The same nano-bead can be used with different objectives (20x, 40x, etc.) as long as the sub-pixel condition is satisfied.

• Capture an image (or a Z-stack) of a single nano-bead. If it is difficult to set just one bead into the field of view, you can crop the image after acquisition. Make sure the background of your image is black (0). It is likely that the image of the bead will be blurry - this is the actual PSF of your microscope. If you plan to deconvolve multichannel images, you should capture a nano-bead with multi emission wavelength.

• Save the image to a file. For 3D deconvolution, the image must be a Z-stack ND2 file, for 2D deconvolution, the image must be in TIFF format.

• Use these images within the deconvolution dialog windows after pressing the Import PSF button.

  Caution

  It is important that ALL(!!!) capturing settings used during PSF acquisition (for example Z-step, objective, scan zoom settings for confocal, etc...) match the settings used for capturing the specimen. Deconvolution results where a non-matching PSF was used can not be satisfactory!

The PSF can be mathematically calculated from the following parameters:

• Numerical aperture of the objective

• Refractive Index of the immersion medium used

• Microscope modality

• Pinhole size (confocal microscopes)

• Objective magnification (confocal microscopes)

• Objective calibration

• Z-step

• Spherical aberration

• Excitation wavelength (confocal microscopes)

• Emission wavelength

• Refractive index of the sample