Expansion microscopy (ExM) is a powerful technology to resolve biological structures below the optical diffraction limit. Molecules of interest are anchored to the gel polymers, which then uniformly expand to open up space between the crowded structural features (Chang et al., 2017, Chen et al., 2015, Chen et al., 2016, Tillberg et al., 2016, Zhao et al., 2017). Researchers could then use easily attainable, uncomplicated imaging devices, such as confocal or epifluorescence microscopes to observe and perform super-resolution imaging. ExM has dramatically reduced the dependence on high-end super-resolution imaging systems. However, premature photobleaching of fluorescence signals at the out-of-focus region becomes a severe problem in ExM due to relatively sparse fluorescence-labelled proteins within the expanded samples and the need for higher intensity laser illumination to compensate for low labelling density. To overcome such a problem, it is desired to reduce the illumination in the out-of-focus area where lattice lightsheet microscopy (LLSM), which offers excellent optical section capability, seems to be the best choice (Chen et al., 2014). In LLSM, only the on-focus sample plane of a high numerical aperture objective lens (NA=1.1) is illuminated by the lightsheet, and the out-of-focus photobleaching is minimized.

As the sample gets larger, a longer lattice lightsheet (LLS) is required to cover the length of the sample. However, the use of a longer LLS for excitation leads to a technical challenge where the energy confinement of the LLS reduces as the length of the LLS gets extended (Gao, 2015b). There are two consequences for imaging as the confined energy in LLS reduces. First, the optical sectioning ability of the LLS is deteriorated due to the increase in the thickness of the LLS (though it remains a controversial issue as to how the thickness of the lightsheet is defined). The reduced optical sectioning capability results in a poorer axial resolution in the reconstructed images. Second, less energy confinement implies that more illumination is distributed at the out-of-focus region, which would introduce unnecessary photobleaching and create noise in the reconstructed images. The tradeoff between the image quality and the imaging volume, therefore, becomes an obvious issue for larger samples. Although LLSM has been applied to mm-scale expanded sample, the above issues are the obstacles that hinder the use of the full capability of LLSM on an expanded sample (Gao et al., (2019)). To be specified, a very long LLS (~160 µm) is used this study to suppress the total imaging time at a reasonable level. The drawback from this approach is that the excitation confinement of LLS reduces and the axial resolution obtained is lowered as well.

One way to overcome the problem of reduced optical section capability is to replace the long lightsheet by tiling many short lightsheets along the direction of light propagation. One could move the sample relative to the excitation objective (EO) to spatially translate the lightsheet within the sample. For example, a 4X expanded cell would have a dimension up to hundreds of micrometers, and an LLS that covers a 100 µm length would have less than 40% energy within the central component. If we have, instead, ten LLSs to cover the same length, each LLS would have more than 70% of energy within the central component (Gao, 2015b). However, we may encounter another problem: stitching artifact. Due to the technical difficulty in precisely spatially translating the lightsheet, after the subvolumes are acquired, it is necessary to eliminate the image stitching artifact during subvolume fusion, which introduces a computational burden (Chalfoun et al., 2017, Hörl et al., 2019). If this is not conducted correctly, the artifact might affect the subsequent analysis. To avoid such stitching artifact, we have developed an alternative approach, the tiling lattice lightsheet microscopy (tLLSM), as a solution to this problem (Gao et al., 2019, Tsai et al., 2020). Instead of mechanically moving the sample or the excitation objective, a phase modulation is used at the excitation path to translate the lightsheet in space. With this method, we can image the expanded and/ or clarified cells, tissues, or organisms with ease (Chen et al., 2020, Feng et al., 2021, Fu et al., 2016, Gao, 2015a, Wang et al., 2019).

In this review we will first briefly explain the working principle of tLLSM, then we will discuss the advantages of our approach over other techniques, and the future outlook of the field will be provided at the end.