The classic mask producing a quasi donut shape for the stimulating wavelength is mandatory for confocal-STED microscopy. To preserve the co-aligment of the excitation beam (355 nm) and the Stimulating beam (532 nm) the mask has to be dichroic. This device is based on chromatic waveplates (λ/2 for 532 nm and λ for 355 nm) : four segments of such a waveplate are assembled in a star-like configuration, so that, with circularly polarized incident light, the polarization of the output beam is a combination of radial and azimuthal polarizations. It is difficult to assemble perfectly the 4 quadrants and in conseqence the "zero" intensity of the donut is not not exactly zero as shown in the figure below. This explains why the intensity of the STED light sheet is reduced when the power of the stimulating beam increases.
For STED-SPIM, the stimulating light sheet can be obtained in a different and simpler way by using a mask that produces directly a zero intensity light sheet instead of a zero intensity central spot. The required phase mask is then simpler since it can be made up of only two quadrants able to divide an incident beam into two sub beams with opposite phases.
We have adapted this idea to our purpose by adding dichroïc properties since we need an activity for the stimulating wavelength (532 nm) and a complete passivity for the pump beam at 355 nm. This can be done by two different approaches.
- Type 1 Mask by using an assembly of two identical dual wavelength quartz waveplates (half wave at 532 nm and wave at 355 nm), their axis being crossed and the incident beam well centered at the interface between the two plates.
Type 2 by using a single thin optical window with its straight edge at the middle of the incident beam, its thickness and its refractive index dispersion chosen to give an optical thickness equal to a multiple of wavelength at 355 nm and simultaneously an odd multiple of half wavelength at 532 nm.
These dichroïc masks, specially type 2, are really advantageous compared to the classic 4 quadrant mask because they limit the number of quadrants to connect, which can be a difficult task. In addition they can benefit from low coast commercially available components.
We have checked these two masks. The type 1 mask was obtained from a single quartz waveplate (half wave, 7th order at 532nm and wave, 12th order at 355nm) cut into two equal parts. The type 2 mask is a simple microscope coverslip (High performance” SCHOTT NEXTERION® ). A thickness tolerance of 170 +/- 5 µm has allow us to find several coincidences (for example half wave, 474th order at 532 nm and wave, 722th order at 355 nm) among a lot of 100 coverslips. In fact, and this is a net advantage of this kind of mask, the coincidence does not to be perfect because it is possible to tilt slightly the coverslip to compensate a small mismatch.
The figure shows the light intensity distribution obtained for a well collimated Gaussian beam after passing through these masks. As expected a dark sheet is created for the stimulating beam (532 nm) for Type 1 and type 2 as well.
For type 2 mask, a small tilt angle on the plate gives a perfectly uniform light intensity distribution at 355 nm while preserving the dark sheet at 532 nm.
For type 1 mask we observe a slight attenuation at the sheet position for 355nm which is not possible to compensate. This behavior is normal since the design of the quartz waveplate is optimized for half wave at 532 nm and a small mismatch for wave at 355 nm is tolerated. It is certainly possible to find plates with higher order having better coincidences at 532 nm and 355 nm. Nevertheless type 1 design is also interesting since its low sensitivity to tilt allows slightly diverging incident beams while for type 2 a perfect collimation is mandatory.
We have check the ability of these masks to produce thin sheets by STED depletion in a Coumarin dye solution. To measure the sheet thicknesses care has been taken to precisely orient the sheets perpendicular to the plane of observation by mounting the masks in a rotating mount. The performances of the two masks are identical. The STED light sheets have nearly the same thicknesses of those obtained with the 4 quadrant mask (see figure just above). We measure a small attenuation of the STED fluorescent beam compare to the fluorescent beam without stimulation. The attenuation obtained with the 4 quadrant mask was much more important, we attribute this effect to the difficulty to realise a perfect mask made up of numerous quadrants.