Development of hydrogel-based standards and phantoms for non-linear imaging at depth

Abstract

Significance: Rapid advances in medical imaging technology, particularly the development of optical systems with non-linear imaging modalities, are boosting deep tissue imaging. The development of reliable standards and phantoms is critical for validation and optimization of these cutting-edge imaging techniques.

Aim: We aim to design and fabricate flexible, multi-layered hydrogel-based optical standards and evaluate advanced optical imaging techniques at depth.

Approach: Standards were made using a robust double-network hydrogel matrix consisting of agarose and polyacrylamide. The materials generated ranged from single layers to more complex constructs consisting of up to seven layers, with modality-specific markers embedded between the layers.

Results: These standards proved useful in the determination of the axial scaling factor for light microscopy and allowed for depth evaluation for different imaging modalities (conventional one-photon excitation fluorescence imaging, two-photon excitation fluorescence imaging, second harmonic generation imaging, and coherent anti-Stokes Raman scattering) achieving actual depths of 1550, 1550, 1240, and $1240\mu m$, respectively. Once fabricated, the phantoms were found to be stable for many months.

Conclusions: The ability to image at depth, the phantom's robustness and flexible layered structure, and the ready incorporation of "optical markers" make these ideal depth standards for the validation of a variety of imaging modalities.

Keywords: axial scaling; depth imaging; hydrogel; non-linear imaging; phantoms; standards.

Fig. 1

(a) One pot method for the fabrication of the double network hydrogels. Agarose, acrylamide, N, N′-methylene-bis-acrylamide, and the ultraviolet (UV) activated initiator were mixed in water, heated, cooled, and UV cured to obtain the double network hydrogel. (b) Casting mold assembly comprising two glass plates (treated with 1H,1H,2H,2H-Perfluorooctyldimethylchlorosilane) using a variety of spacers of differing thicknesses; (c) $490\mu \mathrm{m}$ double network hydrogel mounted on a glass plate ; (d) the 4 stages involved in the fabrication of the depth phantoms, i.e., slicing, coating with modality specific markers, layering, and sealing.

Fig. 2

Measurement of the thickness of a gel layer. (a) Gel sample used for thickness analysis with a lower layer of $310\mu \mathrm{m}$ (test layer) capped with a 2 mm support layer. (b) Imaging a single gel layer ($310\mu \mathrm{m}$) coated with FS beads on both surfaces and then capped with a support layer of gel (2 mm). (c) Microscopy images of the marker layers on the top and bottom of the $310\mu \mathrm{m}$ gel. The distance traversed by the microscope stage between the image planes of the beads gives the apparent thickness of the gel. The scale bar represents $50\mu \mathrm{m}$.

Fig. 3

Images of well plates containing the depth standards. (a), (b) Side views and (c) bottom view of the fabricated standards. Well plate outer dimensions ($w\times l$) $25.5\mathrm{mm}\times 75.5\mathrm{mm}$.

Fig. 4

Measurements of the thickness of the gel layers in the multi-layered phantoms showing the expected thickness from the fabrication process. (a)–(c) The design of the depth standards for the testing of optical systems. Depth standards (a) and (b) were made of 6 layers of signaling markers sandwiching 5 layers of hydrogels of defined thicknesses giving overall heights of 950 and $1550\mu \mathrm{m}$. Depth standard (c) was made of 5 layers of signaling markers sandwiching 4 layers of hydrogels of defined thicknesses giving overall heights of $1520\mu \mathrm{m}$. (d)–(f) Images of signaling markers in each sample, generated via 1PEF with the separation between each layer in $\mu \mathrm{m}$, are given in red. The scale bar represents $50\mu \mathrm{m}$.

Fig. 5

Analysis of depth standard design 1 on multimodal imaging systems. (a)–(c) Illustration of depth standards incorporating “modality markers” BaTiO3 (red), FS beads (green), and PS beads (blue) and imaged via SHG, 2PEF, and coherent anti-Stokes Raman scattering, respectively. Each depth standard had 6 layers of signaling markers sandwiching 5 layers of hydrogels of defined thicknesses ($190\mu \mathrm{m}$) giving an overall height of $950\mu \mathrm{m}$. (d)–(f) SHG, 2PEF, and CARS imaging up to depths of 696, 620, and $649\mu \mathrm{m}$, respectively. The microscopy images represent the marker layers, and the distances between each layer are given in red. The scale bar represents $50\mu \mathrm{m}$.

Fig. 6

Analysis of depth standard design 2 on multimodal imaging systems. (a)–(c) Depth standards incorporating BaTiO3 (red), FS beads (green), and PS beads (blue) imaged via SHG, 2PEF, and coherent anti-Stokes Raman scattering, respectively. Each depth standard had 6 layers of signaling markers (BaTiO3/FS beads/PS beads) sandwiching 5 layers of hydrogels of defined thicknesses ($310\mu \mathrm{m}$) giving an overall height of $1550\mu \mathrm{m}$. (d)–(f) SHG, 2PEF, and CARS imaging up to depths of 835, 1046, and $814\mu \mathrm{m}$, respectively. The microscopy images represent the marker layers, and the distances between each layer are given in red. The scale bar represents $50\mu \mathrm{m}$.