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.
Fluorescence stability - Widefield
Like all fluorophores, the fluorescence intensity of the laser-written features does decrease over time. The rate at which this occurs depends very much on the intensity of the excitation light and the size of the feature being images (i.e. how much fluorescent material is present initially). The above graph shows the change in fluorescence brightness of a laser-written 'F' shape under 5mW of continuous widefield illumination (CW, 532 nm) through a 0.5 NA objective. A single frame from the sequence (250 ms exposure) is shown inset. Extrapolating from an exponential fir to this curve indicates that fluorescence intensity would halve after ~ 3,500 exposures.
Fluorescence stability - Confocal
The excitation intensity at the sample is much higher in confocal than in widefield. The extremely low background signal from the PSFcheck slides allows high signal to noise ratios to be achieved at relatively low excitation intensities. Performing the same exponential extrapolation of the confocal fluorescence indicates that even with medium to high excitation powers, over 120 exposures can be acquired before the fluorescence intensity is reduced to 40% of the original intensity. At lower excitation powers (10%) there is no appreciable decay over time. Assuming regular use, we have conservatively estimated a 6-12 month lifetime.
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). PSFs can then be extracted from images of the features using freeware such as the ImageJ plugin PSFj.
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!