Additional information about slide performance
Broad spectral profile
The laser-written features fluoresce across the visible spectrum, having a broad excitation/emission profile peaking around 488nm / 550nm. The above profiles indicate the fluorescence intensity for a range of different fabrication powers. The broadband profile is particularly useful for checking image alignment across multiple colour channels.
Feature size vs Fabrication power
When the fabrication optics is corrected for aberrations, the lateral size of the fluorescent feature varies linearly with the pulse energy of the fabrication laser. Images of the features shown above were taken by Dr Mike Shaw at the National Physical Laboratories in Teddington, UK. Dr Shaw used a structured illumination microscope with a lateral resolution of 160 nm. Validation of the measured feature size can also be achieved by using other super-resolution methods or by comparison to fluorescent beads of known size.
The PSFcheck fluorescent features are compatible with 2D and 3D structured illumination microscopy and single molecular localisation (SML) microscopy. These techniques have been used (see above) to measure the absolute size of the fluorescent features in the large and small 3D point arrays. The large features in the 3D array pattern have been measured to be 750 nm laterally and the small features in the 3D array measured to be 300 nm laterally (10% tolerance).
The refractive index of the polymer substrate in PSFcheck varies relatively slowly across the visible range. At the peak excitation value of 488 nm the refractive index is 1.57. As the laser written features are located just below the protective coverslip (< 30 um) any index mismatch with the immersion oil will not have a significant effect on the PSF measurement.
We are constantly looking to revise and improve our calibration products. In particular we are currently investigating solutions for water immersion lenses, so watch this space!