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.

3D visualization of multicellular tumor spheroids in fluorescence microscopy

“…Once a multifocus stack is acquired, post processing algorithms enable the synthesis of an EDOF image. 4,7 Let i k be the intensity distribution of the k-th image of a stack of N images. (For color images in…”

“…where (u, v) are spatial frequencies, ⃗ I , ⃗ F are N -element column vectors and H is a N × N symmetric matrix (see 7 and references therein).…”

“…[4][5][6] In the present work a custom built fluorescence microscope with an electrically focus-tunable lens (EFTL) was employed. 7 The EFTL allows a non mechanical scanning. Since the optical system is not telecentric, once a multifocus z-stack is acquired, image registration is performed to match the different fields of view.…”

All-in-focus reconstruction of a dynamic scene from multiple-multifocus captures

“…Once a multifocus stack is acquired, post-capture processing algorithms enable the synthesis of an all-in-focus reconstructed image. 4,7 Let i k be the intensity distribution of the k-th image of a stack of N images. (For color images in…”

“…[1][2][3] Alternatively our strategy is based on acquiring a set of differently focused images along the z-axis, i.e., a set of multifocus images or z-stack, where the structure of the volumetric sample is optically swept in depth. [4][5][6][7] In the present work a custom built fluorescence microscope with an electrically focus-tunable lens (EFTL) was employed. The EFTL allows a non mechanical scanning avoiding lateral mechanical displacements between acquired images.…”