Imaging tissue-mimic with light sheet microscopy: A comparative guideline

Abstract

Tissue mimics (TMs) on the scale of several hundred microns provide a beneficial cell culture configuration for in vitro engineered tissue and are currently under the spotlight in tissue engineering and regenerative medicine. Due to the cell density and size, TMs are fairly inaccessible to optical observation and imaging within these samples remains challenging. Light Sheet Fluorescence Microscopy (LSFM)- an emerging and attractive technique for 3D optical sectioning of large samples- appears to be a particularly well-suited approach to deal with them. In this work, we compared the effectiveness of different light sheet illumination modalities reported in the literature to improve resolution and/or light exposure for complex 3D samples. In order to provide an acute and fair comparative assessment, we also developed a systematic, computerized benchmarking method. The outcomes of our experiment provide meaningful information for valid comparisons and arises the main differences between the modalities when imaging different types of TMs.

Light-sheet microscopy: a tutorial

“…This is a remarkable result taking into account that this BB enables twice the FOV as the reference LSFM, but gives better axial resolution. DSLM with BBs has been successfully used for imaging a variety of biological samples, from cells [63] and small invertebrates [64] to tissue-mimic constructs [70]. This modality can be further combined with the confocal line detection that may provide an additional increase of contrast and optical sectioning [121].…”

“…One of the most critical points, however, is the high light dose received by the sample, which could produce phototoxic effects in developing systems. For example, it has been reported that after 1 h of imaging, the division in tumor spheroids stops, while in linear excitation it can be maintained for more than 24 h [70].…”

“…This fact has been exploited by using ultrashort pulsed lasers in LSFM to generate two-photon excited fluorescence (TPEF) of standard visible fluorescent markers [56,64]. Two-photon LSFM (2P-LSFM) has, thus, been shown to have excellent performance for in vivo cellular-resolution 3D imaging of large biological samples such as the fruit fly [67], zebrafish embryos [68,69], and multicellular spheroids [51,70]. 2P-LSFM, in combination with structured illumination, have also been tested on zebrafish samples [8,71], increasing signal contrast and imaging depth.…”

“…(4) allows us to compute the number of photons obtained per pixel in the SPIM configuration. Considering the added benefits of nonlinear excitation, TPEF has therefore been implemented and investigated in several LS applications [51,56,63,64,[67][68][69][70]108,109]. One of the most critical points, however, is the high light dose received by the sample, which could produce phototoxic effects in developing systems.…”

Large Scale Imaging by Fine Spatial Alignment of Multi-Scanning Data with Gel Cube Device

“…Sectioning of biological samples following paraffin embedding or freezing can yield better images for thick samples, but samples may deteriorate during slicing, and it is difficult to see correlations between large scale of tissue form and molecular expression. Light sheet microscopy [11][12][13] can overcome these problems and image tissue-sized samples, but these samples must be transferred to a special container for imaging, a process that may damage the sample. In addition, this system is quite expensive and not accessible to all researchers.…”