Computational multifocus fluorescence microscopy for three-dimensional visualization of multicellular tumor spheroids

Abstract

Significance: Three-dimensional (3D) visualization of multicellular tumor spheroids (MCTS) in fluorescence microscopy can rapidly provide qualitative morphological information about the architecture of these cellular aggregates, which can recapitulate key aspects of their in vivo counterpart.

Aim: The present work is aimed at overcoming the shallow depth-of-field (DoF) limitation in fluorescence microscopy while achieving 3D visualization of thick biological samples under study.

Approach: A custom-built fluorescence microscope with an electrically focus-tunable lens was developed to optically sweep in-depth the structure of MCTS. Acquired multifocus stacks were combined by means of postprocessing algorithms performed in the Fourier domain.

Results: Images with relevant characteristics as extended DoF, stereoscopic pairs as well as reconstructed viewpoints of MCTS were obtained without segmentation of the focused regions or estimation of the depth map. The reconstructed images allowed us to observe the 3D morphology of cell aggregates.

Conclusions: Computational multifocus fluorescence microscopy can provide 3D visualization in MCTS. This tool is a promising development in assessing the morphological structure of different cellular aggregates while preserving a robust yet simple optical setup.

Keywords: computational optical imaging; fluorescence microscopy; stereoscopic pairs; three-dimensional visualization.

Fig. 1

Scheme of the multifocus custom built fluorescence microscope. LED: Light emission diode as excitation source with center wavelength at 385 nm, L: illumination lens system, DM: dichroic mirror, MO: microscope objective, EFTL: electrically focus-tunable lens. MCTS: multicellular tumor spheroid stained with DAPI (emission peak centered at 461 nm).

Fig. 2

(a)–(o) 15 multifocus images acquired ($z$-stack) for currents in the EFTL between 265 and 125 mA in steps of $-10\mathrm{mA}$. See Video 1 for a visualization of the stack (Video 1, MP4, 0.2 MB [URL: https://doi.org/10.1117/1.JBO.27.6.066501.1]).

Fig. 3

Extended DoF for a virtual centered pinhole view (${\beta }_x=0$, ${\beta }_y=0$); see Video 2 for a complete set of novel viewpoints corresponding to $-0.25\le {\beta }_x\le 0.25$ and $-0.25\le {\beta }_y\le 0.25$ (Video 2, MP4, 1.0 MB [URL: https://doi.org/10.1117/1.JBO.27.6.066501.2]).).

Fig. 4

Stereoscopic pair for cross eyed visualization. (a) Reconstructed and (b) perspective images [images corresponding to pinhole virtually displaced to the right and to the left, respectively].

Fig. 5

Synthetic stack of fluorescent beads and reconstruction of viewpoints. (a) 3D scene. (${\mathrm{b}}_{1-3}$) Images of the stack corresponding to $R_0\approx 12.7{\mathrm{mA}}^{-1}$ and the system focusing for currents through the EFTL 50, 33.3, 0 mA, respectively. (${\mathrm{c}}_{1-3}$) Ground-truth for fractional displacements ${\beta }_x=-0.5,0,0.5$, respectively [vertical dashed guideline passing through a bead in the central viewpoint (${\mathrm{b}}_2$) added to visualize more clearly the displacements]. (${\mathrm{d}}_{1-3}$) Reconstructed viewpoints for fractional displacements ${\beta }_x=-0.5,0,0.5$, respectively.