PSFcheck slides

How PSFcheck slides work

Each PSFcheck slide contains microscopic laser-written fluorescent patterns. These patterns provide a highly accurate 'ground truth' against which you can compare the image of the pattern you capture on your optical microscope. By quantifying the differences between your image and the pattern on the slide, it is possible to correct your science images.

Check more than just the PSF

The PSFcheck slides enable you to measure more than the Point Spread Function (PSF) of your system. The four patterns provided on each PSFcheck slides allow you to measure multiple imaging performance parameters. See below for a description of each of the patterns.

1. 3D point array

The first pattern is an array of fluorescent features. These features are on a 10 micrometre pitch in X, Y and Z dimensions. The pattern covers a range of 70 x 70 micrometres laterally (XY) and 20 micrometres axially (Z). Each slide has two versions of the 3D point array; one with larger features (approx 650 nm lateral) which are easier to see and another with smaller features (approx 350 nm lateral) which are better suited to PSF measurement when used in conjunction with the PSFj ImageJ plugin. 

2. 2D Grid

The second pattern is a large area grid. The grid lines are spaced by 10 micrometres in X and Y and cover an area of 500 x 500 micrometres in total. The axial extent (thickness) of the grid is < 600 nm. Using the grid pattern it is possible to quantify absolute magnification and the degree of distortion (barrel, pincushion) present in individual objective lenses. By low-pass filtering the 2D Grid image, a measure of the field flatness can also be obtained.

3. Large area power series

The large area power series is a series of bright strips of variable fluorescence intensity. Each strip has an area of 20 x 250 micrometres, with a 40 micrometre gap between adjacent strips. As the fluorescence brightness of the strips have a known ratio, it is possible to estimate the linearity of your fluorescence detector.

4. Single point power series

The last pattern is a series of single features fabricated with variable laser power. Increasing the laser power used to fabricate the feature increases the width of the donut-shaped feature. The ability to identify the central intensity dip in these features can provide a quick check of the resolving power of your microscope. Alternatively, by imaging the smallest sub-diffraction features in the point array it is possible to estimate the PSF directly.