Kerstens A et al. (2019), A Label-free Multicolor Optical Surface Tomogra...

XB-ART-55612

BMC Biol 2019 Jan 07;171:1. doi: 10.1186/s12915-018-0614-4.

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A Label-free Multicolor Optical Surface Tomography (ALMOST) imaging method for nontransparent 3D samples.

Kerstens A , Corthout N , Pavie B , Huang Z , Vernaillen F , Vande Velde G , Munck S .

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BACKGROUND: Current mesoscale 3D imaging techniques are limited to transparent or cleared samples or require the use of X-rays. This is a severe limitation for many research areas, as the 3D color surface morphology of opaque samples-for example, intact adult Drosophila, Xenopus embryos, and other non-transparent samples-cannot be assessed. We have developed "ALMOST," a novel optical method for 3D surface imaging of reflective opaque objects utilizing an optical projection tomography device in combination with oblique illumination and optical filters. RESULTS: As well as demonstrating image formation, we provide background information and explain the reconstruction-and consequent rendering-using a standard filtered back projection algorithm and 3D software. We expanded our approach to fluorescence and multi-channel spectral imaging, validating our results with micro-computed tomography. Different biological and inorganic test samples were used to highlight the versatility of our approach. To further demonstrate the applicability of ALMOST, we explored the muscle-induced form change of the Drosophila larva, imaged adult Drosophila, dynamically visualized the closure of neural folds during neurulation of live Xenopus embryos, and showed the complementarity of our approach by comparison with transmitted light and fluorescence OPT imaging of a Xenopus tadpole. CONCLUSION: Thus, our new modality for spectral/color, macro/mesoscopic 3D imaging can be applied to a variety of model organisms and enables the longitudinal surface dynamics during development to be revealed.

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	Fig. 1. OPT and ALMOST imaging modalities. a Diagram of the imaging light path for a sample (green orb) with back illumination. b Theory of image formation in a tomographic system like OPT. The sample object resides at the center of a coordinate system. Parallel beams spaced by r pass through the sample to form a projected image (Pθ). The theory of this imaging process is based on the Radon transform (see also Additional file 1: Text S1 for more detailed background; r is used in the CT imaging and is less relevant for light-based approaches). c Diagram of the oblique illumination light path to create reflected light images of opaque samples, while standard OPT works with transparent samples and uses fluorescence or back illumination. It is also possible to add color filters in the reflected light path to collect spectral information. d Theory of image formation when reflected light interacts with an opaque sample that contains surface topography information. e The oblique illumination/imaging chamber for reflected light imaging is depicted. It is crucial to use a reflective chamber, for example, lined with white paper, to promote diffuse illumination. f Depiction of diffuse reflection compared to specular reflection. g, h Flowcharts of the imaging, reconstruction, and visualization process. The filtered back projection algorithm is abbreviated as filtered BPA. It is of note that if NRecon is used for reconstruction that the images are converted and need to be inverted back (see Additional file 1: Text S1 for more information)
	Fig. 2. Color and shape in test samples. Color code on a resistor. a Photograph of the resistor. b 3D reconstruction of the blue channel. c 3D reconstruction of the green channel. d 3D reconstruction of the red channel. e Relative intensity profile of the color channels as indicated in b–d. f 3D RGB color reconstruction. Small artifacts like the flare on the thread above the actual resistor are a consequence of extended reflexes of the reflective metal part connecting the resistor. The brown, green, red, and gold color rings (from top to bottom) imprinted on the resistor, part of the four color code used to describe its properties, can be revealed. The color balance for the three colors was adapted manually. Seed cone sample (Metasequoia glyptostroboides) with complex surface structure. g Photograph of the seed cone. h Individual image from the ALMOST device (blue channel). i Reconstructed sagittal section through a central plane of the seed cone (blue channel). j Intensity profile along the line indicated in i to compare inside and outside of the complex shape. k, l 3D semitransparent volume rendering of the seed cone in three colors (colors inverted compared to j, for realistic color display). The color balance for the three colors was adapted manually. Images show different angles, including a view from below the cone, showing that complex samples can be imaged with ALMOST. Scale bars = 2 mm. Imaging conditions are summarized in Additional file 20: Table S1
	Fig. 3. Micro-CT and ALMOST imaging of the same samples. Samples of the same shape but with different color patterning are imaged, namely Lego figurines. a, b Photograph of a Lego figurine with a beard dubbed “Dalton” and of a Lego figurine with glasses and happy face dubbed the “Workman.” c A reconstructed sagittal section from the Dalton obtained with micro-CT. d Dalton maximum intensity 3D reconstruction of the micro-CT data made similarly as the ALMOST data (with Arivis). e Dalton volumetric 3D reconstruction of the micro-CT data. f A reconstructed sagittal section from the Dalton obtained with ALMOST, blue channel. g Dalton maximum intensity 3D reconstruction of the ALMOST data, made with Arivis. h The Workman maximum intensity 3D reconstruction of the ALMOST data, made with Arivis. i Using ALMOST, the surface can be revealed similar to the depicted micro-CT surface (cfr e) using, for example, one color channel (here blue). j Overlay of the surfaces from ALMOST (green) and micro-CT imaging (red). The ALMOST surface matches well the CT surface, thereby showing that the retrieved surfaces can be quantitative. Both characters are revealed in ALMOST imaging and can be discriminated, whereas in micro-CT, the figurines look similar. Optical imaging conditions are summarized in Additional file 20: Table S1. Scale bar = 2 mm
	Fig. 4. Combining ALMOST and fluorescence OPT on adult Drosophila. A mutant fruit fly expressing GFP in the eyes is imaged. a shows the reflective image acquired by ALMOST in the blue channel. b Section through reconstruction of the fly in reflective mode (front view, blue channel). c 3D rendering of the fly imaged in reflective mode. d Combination of the 3D rendering of the reflective and the fluorescence modes. e Single three-color (raw) ALMOST image before reconstruction and rendering of a red-eyed wild-type Drosophila head. f ALMOST 3D reconstruction of a sequence of rotational images as in e. g Single three-color ALMOST image (before reconstruction and rendering) of a glace-eyed mutant Drosophila head with narrowed eyes of reduced size. h ALMOST 3D reconstruction of a sequence of rotational images as in g. Scale bars = 500 μm. Imaging conditions are summarized in Additional file 20: Table S1
	Fig. 5. Live imaging of a Xenopus tropicalis embryo; complementarity of the ALMOST approach. Different stages of the same Xenopus tropicalis embryo are shown during neurulation. a Top view from dorsal to ventral side of a stage 12 embryo. b Lateral side view of a. c Same embryo as in a and b after ~ 1.5 h. Stage 14.5 is shown. d Lateral side view of the embryo in c. e Same embryo as in a–d after ~ 2.8 h (relative to a, b). Stage 19 is shown. f Lateral side view of e. g The GFP fluorescence signal of the same embryo is shown after fixation and imaged with a spinning disk. Gray information is the transmitted light signal from the spinning disk. The embryo is a crest3- gfp transgenic line, labeling a subset of neuronal precursor cells. h Zoomed view, comparing e and g. Horizontal section view of the embryo from anterior to posterior and cut open at the line indicated in e and g. As the animals get older, they become more transparent. To demonstrate the complementarity of our approach, we imaged a semitransparent tadpole of stage ~ 50 with ALMOST, with fluorescence OPT, and with transmitted light OPT. i Side view using ALMOST displayed in purple, with more brightness in purple indicating less reflection. Insert is showing a raw reflection image. j Side view using autofluorescence displayed in cyan. Brighter signals indicate stronger autofluorescence. k Side view using transmitted light displayed in green. Brighter signals indicate lower transmission. l Merged view showing the tadpole from the top. m–p Virtual sections through the animal as indicated in l. q Merged side view of i–k, section as indicated in l. The different approaches reveal different aspects of the tadpole. Part of the difference between transmitted light and ALMOST is due to scattering inside the sample. Scale bar = 500 μm in all images. Imaging conditions are summarized in Additional file 20: Table S1

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