Combining Three-Dimensional Quantitative Phase Imaging and Fluorescence Microscopy for the Study of Cell Pathophysiology

Abstract

Quantitative phase imaging (QPI) has emerged as one of the powerful imaging tools for the study of live cells in a non-invasive manner. In particular, multimodal approaches combining QPI and fluorescence microscopic techniques have been recently developed for superior spatiotemporal resolution as well as high molecular specificity. In this review, we briefly summarize recent advances in three-dimensional QPI combined with fluorescence techniques for the correlative study of cell pathophysiology. Through this review, biologists and clinicians can be provided with insights on this rapidly growing field of research and may find broader applications to investigate unrevealed nature in cell physiology and related diseases.

Keywords: correlative imaging; fluorescence imaging; holotomography; label-free imaging; microscopy; quantitative phase imaging.

Figure 1

Overview of 2D and 3D imaging. The schematic of (A) 2D quantitative phase imaging, (B) 3D quantitative phase imaging, (C) 2D X-ray imaging, and (D) 3D X-ray computerized tomography.

Figure 2

2D QPI with other correlative imaging techniques. (A) Through this imaging technique, the mitosis of a kidney cell was accurately visualized both through quantitative phase image, fluorescence image, and their overlaid image. (B) Through this technique, axially averaged refractive index of cells was determined. This was used for the calculation of protein concentrations. (C) Through this multimodal imaging system, normal and P. falciparum-infected red blood cells (RBCs) were analyzed to have different morphology and hemoglobin distribution of the RBCs, which were determined by QPM and confocal Raman microscopy, respectively. (A-C) are modified from refs. [68,69], and [44], respectively, with permissions.

Figure 3

3D QPI with other correlative imaging techniques. (A) ODT visualizes and quantifies lipids in algae, which are validated through 2D FL Nile red dye imaging. (B) Through this multimodal optical system, volume of nucleus and cytoplasm of HeLa and NIH-3T3 cells were determined by quantitative analysis. (C) Due to improvements in lateral resolution and depth localization, ODT and 3D SIM visualize clearer sub-diffraction structures of A549 cell, outlined in yellow box, compared to the conventional widefield (WF) imaging system. (D) Through multi-plane phase and SOFI imaging, clear subcellular structures of HeLa cell were imaged, making this system an accurate 4D imaging modality. (A-D) are modified from refs. [76,77,45], and [80], respectively, with permissions.

Figure 4

Demonstration of applicability of ODT and multimodal approach combining ODT and 3D fluorescence. (A) Study of Toxoplasma gondii infecting ARPE-19 through timelapse QPI. (B) NIH-3T3 cell research through ODT and 3D FL (GFP-Mito and mCherry-Golgi). Both images are obtained by commercialized ODT setup (HT-2H, Tomocube, Inc., South Korea).