Live Cell Imaging with Holotomography and Fluorescence

Aubrey Lambert Tomocube Inc., 2nd floor, KHE Bldg., 48 Yuseong-daero 1184beon-gil, Yuseong-gu, Daejeon 34109, South Korea

Abstract: Despite its vital role in advancing live cell imaging (LCI) and unraveling the complex processes that control cellular function, fluorescence microscopy presents challenges for the researcher, including labeling agents that can interfere with normal molecular activities, and limitations on repeat measurements and long-term studies from photobleaching and phototoxicity phenomena. Holoto- mography microscopy delivers nanoscale, label-free, real-time LCI and can combine this quantitative phase imaging (QPI) with fluo- rescence for state-of-the-art spatiotemporal

resolution as well as

high molecular specificity. This article introduces QPI, examines its advantages for LCI, and reviews correlative microscopy studies in cell pathophysiology in combination with fluorescence.

Keywords: Holotomography, quantitative phase imaging (QPI), label- free imaging, fluorescence, live cell imaging (LCI)

Introduction Fluorescence microscopy has played an important role in

the recent advancements in live cell imaging (LCI), helping to unravel the complex processes that control cellular func- tion. Alongside various sophisticated light microscopes in the world’s biomedical facilities, a toolbox of specific fluorescent probes, laser light sources, digital camera systems, image anal- ysis soſtware, and inexpensive computing power combine to make advances almost routine. However, LCI still poses a number of challenges for the

researcher. Fluorescence microscopy techniques require the use of labeling agents to obtain high-contrast molecular information, but these agents can interfere with normal molecular activities. Labeling is also a time-consuming procedure, and the phenom- ena of photobleaching and phototoxicity limit repeat measure- ments. Cells that need to be analyzed and then reinserted into the body for in vivo studies, such as stem cells or immune cells, pose an especially difficult challenge. Tat same availability of laser-illuminated imaging systems,

computing power, and sophisticated soſtware has contributed to a new 3D imaging technology that relies on a fundamental prop- erty of light employed by Frits Zernike in 1934 [1]. His phase- contrast microscope measured variations in light as it passed through transparent specimens, revealing details of cellular structures without fixing and staining. Now, quantitative phase imaging (QPI) technology, pioneered by Tomocube, is breaking new ground in LCI, providing a label-free, real-time means to image and measure live cells and tissues in three dimensions. Te holotomography (HT) microscope delivers nanoscale, real- time, dynamic images without any sample preparation. Te latest Tomocube HT microscope combines QPI technology with fluo- rescence for state-of-the-art spatiotemporal resolution as well as high molecular specificity. Tis article introduces the technol- ogy behind HT, examines its advantages for LCI, and reviews potential applications for 3D QPI combined with fluorescence techniques for correlative studies of cell pathophysiology.

18 doi:10.1017/S1551929519001032

Materials and Methods How holotomography works. Refractive index (RI) is a

fundamental optical parameter describing the speed of light passing through an object. As light traverses the object, the specimen material scatters the light and changes the phase of the light. If a living cell is viewed, then the various constitu- ents of the cell scatter the light differently according to their RI. Phase contrast microscopy, where light emerging from the specimen constructively interferes with a separate beam of light that did not pass through the specimen (reference beam), produces small but observable brightness differences caused by the degree of the phase shiſt. Tese brightness variations result in contrast that reveal the features of an object. In this way, the dynamics of biological processes may be observed and recorded in fine detail. Te Tomocube HT microscope expands the concept of

phase contrast to reveal details of the sample in a 3D tomo- gram. Te sample is held on a stage between the objective and condenser lens. A 532 nm laser beam is split into two optical paths: an imaging path to illuminate the sample and a refer- ence path (Figure 1). Light in the imaging path is expanded

Figure 1: Schematic light path. The sample is located on the stage between the objective and a condenser lens. A laser beam is split into specimen and reference beams. The specimen and reference beams generate a 2D hologram, which is recorded by a digital camera. Under the control of the digital micromirror device (DMD), the laser illuminates the specimen by rotating the incident beam about the optic axis at an incident angle of 53°. A 3D RI tomogram of the sample is reconstructed from the measured multiple holograms acquired at various illumination angles. • 2020 January

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