Multimodal Optical Microscopy Methods Reveal Polyp Tissue Morphology and Structure in Caribbean Reef Building Corals

Abstract

An integrated suite of imaging techniques has been applied to determine the three-dimensional (3D) morphology and cellular structure of polyp tissues comprising the Caribbean reef building corals Montastraeaannularis and M. faveolata. These approaches include fluorescence microscopy (FM), serial block face imaging (SBFI), and two-photon confocal laser scanning microscopy (TPLSM). SBFI provides deep tissue imaging after physical sectioning; it details the tissue surface texture and 3D visualization to tissue depths of more than 2 mm. Complementary FM and TPLSM yield ultra-high resolution images of tissue cellular structure. Results have: (1) identified previously unreported lobate tissue morphologies on the outer wall of individual coral polyps and (2) created the first surface maps of the 3D distribution and tissue density of chromatophores and algae-like dinoflagellate zooxanthellae endosymbionts. Spectral absorption peaks of 500 nm and 675 nm, respectively, suggest that M. annularis and M. faveolata contain similar types of chlorophyll and chromatophores. However, M. annularis and M. faveolata exhibit significant differences in the tissue density and 3D distribution of these key cellular components. This study focusing on imaging methods indicates that SBFI is extremely useful for analysis of large mm-scale samples of decalcified coral tissues. Complimentary FM and TPLSM reveal subtle submillimeter scale changes in cellular distribution and density in nondecalcified coral tissue samples. The TPLSM technique affords: (1) minimally invasive sample preparation, (2) superior optical sectioning ability, and (3) minimal light absorption and scattering, while still permitting deep tissue imaging. Video LinkThe video component of this article can be found at

Visualising the Cardiovascular System of Embryos of Biomedical Model Organisms with High Resolution Episcopic Microscopy (HREM)

“…For coping with the drawbacks of conventional histological sections and section-based imaging methods [8,9,10], the last few decades have seen the development of large numbers of innovative 3D imaging technologies covering the meso- and microscopic range. The most promising are micro computed tomography (µCT)—even termed as virtual histology [11,12,13,14,15,16,17,18,19], micro-magnetic resonance imaging (µMRI) [20,21,22,23,24,25,26,27,28,29,30], optical projection tomography (OPT) [31,32,33,34,35,36,37], optical coherence tomography (OCT) [38,39,40,41], photoacoustic tomography (PAT) [40,42], serial block face microscopy [43,44], ultrasound microscopy [45,46,47,48,49], Episcopic 3D microscopy (Epi3D) [50], Episcopic Fluorescence Image Capturing (EFIC) [51,52] and High Resolution Episcopic Microscopy (HREM) [53,54,55,56]. A similar, episcopic approach is used for serial block-face electron microscopy (SBFSEM), which offers ultrastructural 3D information [57,58,59].…”

In vivoimaging of coral tissue and skeleton with optical coherence tomography

“…Descriptions of coral tissue structure have largely been based on light microscopy (Brown et al, 1995;Hayes and Bush, 1990), histological sectioning, and electron microscopy, which requires extensive tissue sample preparation that can introduce artefacts such as tissue shrinkage and dehydration. Microscopic imaging of live intact corals has also been realized in the lab (e.g., Shapiro et al, 2016;Sivaguru et al, 2014) and in situ (Mullen et al, 2016) but information about internal tissue organization is limited to early developmental stages, tissue explants or corals with thin tissue layers without pronounced calcified structures. Depth resolution and contrast is severely hampered by the absorption and scattering of visible light in the tissue-skeleton matrix, and the same effects contrive the depth resolution of confocal laser microscopy on coral tissue to the outermost 100-200 µm using high numerical aperture objectives with a small field of view (Salih, 2012).…”

Cardiac Muscle Organization Revealed in 3-D by Imaging Whole-Mount Mouse Hearts using Two-Photon Fluorescence and Confocal Microscopy

“…Whole-hear t and ventricular volumes for normal and t/t hearts (see Supplementary Material) were traced (every 4050 micron step through the z-axis) using the Surpass module (Contour Tracing Algorithm) in the Imaris Suite (25) software (version 8.0; Bitplane, Inc., Zurich, Switzerland). The whole-heart volume was created using the Isosurface module in the same Imaris program after applying background subtraction, smoothing and a threshold for the muscle based on the intensity of muscle volume.…”