YALE JOURNAL OF BIOLOGY AND MEDICINE 91 (2018), pp.267-277. Review Combining Three-Dimensional Quantitative Phase Imaging and Fluorescence Microscopy for the Study of Cell Pathophysiology Young Seo Kima,b,c, SangYun Leec,d, JaeHwang Jungc,d, Seungwoo Shinc,d, He-Gwon Choie, Guang-Ho Chae, Weisun Parkb,c,d, Sumin Leeb, and YongKeun Parkb,c,d,* aDepartment of Chemical and Biomolecular Engineering, Korea Advanced Institute of Science and Technology (KAIST), Daejeon, Republic of Korea; bTomocube Inc., Daejeon, Republic of Korea; cKAIST Institute of Health Science and Technology, KAIST, Daejeon, Republic of Korea; dDepartment of Physics, KAIST, Daejeon, Republic of Korea; eDepartment of Medical Science, Chungnam National University, Daejeon, Republic of Korea 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 luorescence microscopic techniques have been recently developed for superior spatiotemporal resolution as well as high molecular speciicity. In this review, we briely summarize recent advances in three-dimensional QPI combined with luorescence techniques for the correlative study of cell pathophysiology. Through this review, biologists and clinicians can be provided with insights on this rapidly growing ield of research and may ind broader applications to investigate unrevealed nature in cell physiology and related diseases. INTRODUCTION techniques have been developed to achieve better imag- ing capabilities. In particular, luorescence microscopy is Optical imaging of biological cells and tissues has one of the important advances that opened a new era in been utilized as an indispensable technique providing in- molecular biology and molecular diagnosis. Via speciic valuable information on the pathophysiology of diseases. labeling of target molecules with luorescence probes, un- Since the observation of cork cells using a microscope precedented molecular speciicity and imaging contrast th by Robert Hooke in the 17 century, various microscopic could be achieved. However, the signals from luores- *To whom all correspondence should be addressed: YongKeun Park, Department of Physics, KAIST, Daejeon, Republic of Korea; Email: [email protected]. †Abbreviations: QPI, quantitative phase imaging; RI, refractive index; 2D, two-dimensional; 3D, three-dimensional; ODT, optical difraction tomography; RBCs, red blood cells; CT, computerized tomography; RS, Raman Spectroscopy; RAFTOR, RI and luores- cence tomography with optoluidic rotation; EM, electron microscopy; Pf, Plasmodium falciparum; Bm, Babesia microti; BMDM, bone marrow-derived macrophages; GNPs, gold nanoparticle particles; AI, artiicial intelligence. Keywords: quantitative phase imaging, luorescence imaging, label-free imaging, holotomography, correlative imaging, microscopy Disclosures: Mr. Y.S. Kim, Mr. S. Shin, Dr. S. Lee, Dr. W. Park, and Prof. Park have inancial interests in Tomocube Inc., a company that commercializes optical difraction tomography and quantitative phase imaging instruments. Copyright © 2018 267 268 Kim et al.: Combining 3D quantitative phase imaging and luorescence microscopy cence probes are qualitative due to varying permeability ity. Although the exogenous labeling agents are required, of luorescence dyes. Also, repeated measurements are synergetic advantages between QPI and luorescence limited due to photobleaching and phototoxicity. More microscopy suggested new applications. importantly, they require the use of exogenous labeling Here, we review the recent advances in the correla- agents which may prevent from live cell imaging of intact tive imaging techniques combining 3D QPI with various cells, and labeled cells are very limited to in vivo appli- luorescence microscopic techniques. First, we introduce cations. the principle of QPI and ODT. Then, we summarize Quantitative phase imaging (QPI) is an interferomet- important demonstrations of the correlative imaging for ric microscopy technique, which measures the optical various biological and medical studies. Prospective ap- phase delay induced by refractive index (RI) diferenceplications and futures of the correlative imaging will also between a sample and medium [1,2]. Because RI is an be discussed. intrinsic optical property of a material, no exogenous la- beling agent is required to generate an imaging contrast PRINCIPLE OF QUANTITATIVE PHASE in QPI. Furthermore, from the measured phase delay, the IMAGING morphological and chemical properties of a sample can be quantitatively retrieved. These advantages make QPI By exploiting the interference nature of light, QPI increasingly attractive in studying various biological sam- techniques enable us to retrieve not only the amplitude ples, including blood cells [3-5], bacteria [6-9], neurons but also the phase information of scattered light from [10,11], parasites [12,13], plant cells [14,15], cancer cells a sample. Interference between the scattered light and [16-19], inlamed tissues [20], and tissue slices [21,22]. well-deined reference light produces an interference Optical difraction tomography (ODT), one of the pattern, called a hologram or an interferogram [47,48] three-dimensional (3D) QPI methods, reconstructs the (Figure 1A). Several ield retrieval algorithms [49,50], 3D RI distribution of a sample from the measurements utilizing temporally or spatially modulated reference of multiple two-dimensional (2D) holograms via inverse light, have been developed to extract the optical ield scattering principle [23]. Multiple 2D holograms of a information, i.e., both the amplitude and phase, from a sample can be obtained by utilizing illumination angle measured hologram. scanning [24-28] or sample rotation [29-32]. RI distribu- In an aspect of imaging, the phase information of tion of a sample serves as an intrinsic optical imaging the light scattered by an object is the optical phase delay contrast, which provides physical and chemical infor- map, which is related to the light refraction. Although mation including protein concentration and cellular dry most biological cells and tissues are transparent under mass in a quantitative manner [33,34]. In particular, the optical wavelengths, light passing through these biolog- applicability of ODT to various research area also have ical samples exhibits optical phase delays depending on been demonstrated, such as the physiology of various bi- the morphology and distribution of RI values of each ological samples including blood cells [35-37], immune sample. In principle, the optical phase delay is calcu- cells [30,38], embryos [39], bacteria, and various eukary- lated as the integration of RI values along the trajectory otic cells [40-42]. of light passing through a sample subtracted inside the QPI approaches have provided a new methodology integration of those passing through surrounding media. for investigating the pathophysiology of live cells and Even microscopic cells usually produce signiicant op- tissues via label-free and quantitative imaging. Although tical phase delays, which can be precisely measured by label-free and high-speed 3D imaging capability of QPIQPI techniques without using an exogenous agent. On provides the advantage for live cell imaging, the limited the contrary, these biological cells are mostly transparent molecular speciicity strongly restricts broader applica- in visible light ranges and thus do not produce enough tions in cell biology and biochemistry. imaging contrast to be imaged in bright-ield microscopy. To overcome the limited molecular speciicity in QPI This aspect makes QPI an especially useful imaging tool while maintaining the advantages of the method, several for observing live cell morphology and dynamics without multimodal approaches have been recently demonstrated. disturbing its physiological condition. For example, ODT integrated with multi-spectral light In 2D QPI, the measured optical phase delay is sim- sources [43], Raman spectroscopy [44], and structured ply a coupled value between the thickness of a sample illumination microscopy [45,46] have demonstrated the and the mean RI value of the sample. To retrieve one val- potential for combining molecular speciic information ue, the other value should be known. Although 2D QPI and morphological information. In particular, correlative can be efectively applied to several applications such as imaging approaches combining luorescence microscopy imaging red blood cells (RBCs) or bacteria, the detailed and QPI take the advantages of quantitative imaging, su- 3D morphology of eukaryotic cells or their internal struc- perior spatiotemporal resolution, and molecular speciic- tures cannot be directly investigated. 3D QPI enables the Kim et al.: Combining 3D quantitative phase imaging and luorescence microscopy 269 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. reconstruction of 3D RI distributions [23,51,52] (Figure ODT can be understood as an optical analogous 1B). to X-ray computerized tomography (CT). Whereas 2D The principle of ODT is the inverse solving of wave X-ray only provides the integrated projection images equation (Helmholtz equation for monochromatic wave); (Figure 1C), X-ray CT reconstructs 3D X-ray absorptiv- from the multiply scattered waves obtained with various ity tomograms of a human body (Figure 1D). Similarity,