mesoSPIM_laser_selection

Laser selection for a mesoSPIM

The excitation path consists of a laser combiner unit coupled to two single-mode fibers, one for each excitation path. Some of the existing mesoSPIM instruments have Omicron SOLE-6 laser with 405, 488, 515, 561, 594 and 647 nm excitation lines and a 50:50 split between fibers. Other mesoSPIMs have two Toptica MLE laser combiners with 405, 488, 561, 640 nm lines, one for each excitation path. Recently, Benchtop mesoSPIMs were fitted with Oxxius L4Cc combiners (405, 488, 561, 638 or more red-shifted range: 488, 561, 638, 785 nm), with dual-fiber switching module, which is more budget-friendly.

In general, it is recommended to use laser combiners with the highest available output powers, each line should have at least 30 mW on the sample to properly illuminate the full FOV (which can be up to 22 mm).

The laser selection should take the following considerations into account:

• Relatively high laser powers (50-100 mW out of the fiber) are required for imaging endogenous labels in samples cleared with methods with loss of fluorescent proteins (in our experience especially passive and active CLARITY).
• In contrast, well-stained samples – for example, after amplification with secondary antibodies such as in iDISCO – generally leads to high signal levels and thus can be imaged at lower laser intensities.
• If it can be anticipated that only very little imaging will be done in samples requiring FOVs of more than 5 mm, lower laser power specifications will be sufficient as the available power will be concentrated onto a smaller light-sheet.
• We do not recommend having a 405-nm laser line unless it is absolutely needed – in general, the penetration depth even in cleared samples at this wavelength is very low and it is recommended to use red labels instead of UV-excitable dyes.
• White-light laser sources are not recommended (though not tested) as it can be expected that the axial chromatic aberration of the mesoSPIM excitation path will lead to worse effective light-sheet thickness because ETL sweep parameters depend on the dispersion of the immersion medium.

Most existing mesoSPIMs are part of either research labs or imaging facilities with a wide range of clearing methods available. Having sufficient laser powers and a wide selection of laser lines available therefore is beneficial to support all kinds of imaging needs. Nonetheless, high-power laser combiners with 4 or 6 wavelengths are a major cost factor and account for 30-50% of the total cost of a mesoSPIM setup. If a mesoSPIM has to be built on a lower budget, we recommend starting with a single laser line and a single excitation path. If multiple lines are required, building an open-source laser combiner might be an option.