Since Robert Hooke's microscopic observations of cork cells in the 17th century, optical imaging has proven an indispensable technique in elucidating the pathophysiology of diseases. Perhaps the real transformation occurred last century with Zernike's invention of the phase-contrast microscope, measuring the variations in light as it passes through transparent specimens to reveal details of the cellular structures without fixing and staining. At a stroke, these detailed 2D images kickstarted the science of cell biology and provided the foundations for the explosive advancements in live cell imaging (LCI) in recent years.

Fluorescence microscopy has played an important role in these advances, helping to unravel the complex processes that control every aspect of cellular function. Cellular studies are routine in today's biomedical facilities given the widespread availability of laser scanning microscopes, cheap computing power, sophisticated image analysis software, megapixel digital camera systems, and a plethora of highly specific fluorescent probes. However, LCI still poses a number of challenges, given that fluorescence microscopy techniques require the use of labelling agents and a time-consuming labelling procedure, in order to render high-contrast molecular information. These agents can also interfere with normal molecular activities. For example, photobleaching and phototoxicity limit repeat measurements.

Cells that need to be analyzed and then reinserted into the body for in vivo studies — such as stem or immune cells — pose an especially difficult challenge.

Availability of laser illuminated imaging systems, computing power, and sophisticated software have also contributed to a new 3D imaging technology that relies on the same fundamental property of light as traditional phase contrast. Holotomography (HT) provides a quantitative, label-free means to image live cells and tissues in three dimensions to deliver nanoscale, real-time, dynamic images quickly and simply, without any sample preparation. A new microscope from Tomocube (Daejeon, South Korea) combines quantitative phase imaging (QPI) technology with fluorescence, for superior spatiotemporal resolution as well as high molecular specificity.

In this article, we briefly introduce HT, examine its advantages for live cell imaging and review potential applications for 3D QPI combined with fluorescence techniques for the correlative study of cell pathophysiology.

How Holotomography Works

The refractive index (RI) is an intrinsic optical parameter describing the speed of light passing through any object, such as a cell. When light passes through a cell, diffraction occurs according to the object's RI, changing some properties of the light, including wavelength and phase shift. In phase contrast microscopy, the emergent light is viewed together with the original light, in order to observe brightness changes, which are dependent on the degree of phase shift.

Figure 1. Hepatocyte detail captured by Tomocube’s HT-1 holographic microscope.

In an HT microscope, a laser beam is used to illuminate the sample and multiple two-dimensional RI measurements are stitched together to display a 3D tomogram image (Figure 1). This is similar to the way CT scanners use x-rays — a rotating energy source captures 2D image slices that can be assembled into a 3D tomogram. Because the RI values are a quantitative measurement, HT microscopy has the significant advantage of providing quantitative data about the sample, including morphological and chemical information.

The light path schematic is shown in Figure 2, with the sample held on a stage between the objective and condenser lenses. The laser beam is split into specimen and reference arms to generate a 2D hologram recorded by a digital image sensor. The laser illuminates the sample with an incident angle of 53° (Tomocube HT-1S) or 63° (Tomocube HT-1H), which rotates 360° with respect to the optical axis. The 3D RI tomogram of the sample is reconstructed from the 2D holograms with various illumination angles.

Figure 2. Schematic light path. A sample is located on the stage between the objective and a condenser lens. A laser is split into a specimen and reference arm, generating a 2-D hologram to be recorded by a digital camera. The digital micromirror device (DMD) controls the laser to illuminate the rotating sample.

At the heart of the light path is a complex digital micromirror device (DMD) optical light shaper, consisting of several hundred thousand micromirrors arranged in a rectangular array. Each individual mirror can be rapidly tilted plus or minus 12 degrees. Tomocube's patented electronic control of the light path through the DMD eliminates moving parts for better stability and improved optical resolution.

Holotomography - Advantages for Live Cell Imaging

In exploiting the intrinsic optical properties of all materials and directly measuring the optical phase delay introduced by RI differences between samples and their medium, HT microscopy eliminates the need for stains or other labelling agents to quantitatively and non-invasively investigate biological cells and thin tissues. Furthermore, reconstructing the 3D RI distributions of biological material, resolves at high resolution, structural and chemical information, including dry mass, cell volume, shapes of sub-cellular organelles, cytoplasmic density, surface area, and deformability. And, this only requires a low power laser system, which eliminates the potential for photobleaching and phototoxicity.

Figure 3. Cellular images captured by the HT-1 holographic microscope.

The effectiveness of holotomography in capturing three-dimensional images of live cells without exogenous labelling agents is shown in Figure 3. Cell membranes and organelles can be imaged at a resolution of 110 nm, with no photo-bleaching or phototoxicity-induced damage to cells or interference with normal molecular activities. Holotomography has been successfully applied to cytopathology, single-cell analysis, drug or toxicity reactions, cytology, and hematology.

Holotomographic Quantitative Phase Imaging and Fluorescence

Quantitative phase imaging provides a new methodology for investigating the pathophysiology of live cells and tissues via label-free and quantitative imaging. However, although its label-free and high-speed 3D imaging attributes are highly advantageous for live cell imaging, limited molecular specificity restricts broader applications in cell biology and biochemistry. What is needed is a correlative imaging “double act” combining 3D QPI with fluorescence.

Figure 4. The Tomocube HT-2 holotomography and fluorescence microscope.

This is the approach taken by Tomocube with the launch of its second microscope, the HT-2, which adds 3D fluorescence imaging capability to the existing holotomography set-up (Figure 4). The HT-2 delivers holotomography/fluorescence correlative analysis in 2D, 3D, and 4D. The highly detailed fluorescence images show the position of specific target organelles or structures in living cells, while simultaneous measurement of time-lapse 3D RI tomography enables the monitoring of cells and their structures with minimal stress.

Potential applications for Correlative Imaging with QPI

A review of work combining 3D QPI and various fluorescence microscopy techniques for the study of cell pathophysiology has recently been published in the Yale Journal of Biology and Medicine.1 This recognizes the demand for more detailed morphological imaging of biological samples and the possibility of superior spatiotemporal resolution, quantitative imaging, and high molecular-specific analysis of live cells through the combination of holotomography and other microscopic techniques.

Among the 2D work reviewed, was the mitosis of a kidney cell accurately visualized through quantitative phase imaging, fluorescence imaging, and their overlaid image.2 In another paper, the axially averaged refractive index of cells was determined and was used for the calculation of protein concentrations.3 Among the 3D work reviewed was holotomography combined with 3D fluorescence images used correlatively to image live cells for quantitative and precise spatial molecular specificity.4 Simultaneous 3D QPI and 3D fluorescence imaging are optimal for cellular dynamics imaging, especially targeting the organelles, as phototoxicity and photobleaching can be minimized through QPI. Recently, optical diffusion tomography (ODT) and 3D fluorescence correlative imaging have been used in RI and fluorescence tomography with optofluidic rotation (RAFTOR) to quantitatively analyze a suspended cell with molecular specificity.5 Also, 3D QPI, and 3D confocal fluorescence imaging were used together for the study of yeast cells.6

Figure 5. A shows time-lapse 3D images of Toxoplasma gondii infecting an ARPE-19 cell, which undergoes death as it is penetrated. These images can provide important insights on the mechanism of infection and were measured using a Tomocube HT-1 holotomography microscope. Figure 5B shows NIH-3T3 cell research using QPI and 3D fluorescence (GFP-Mito and mCherry-Golgi). Both images were obtained using a Tomocube HT-2 holotomography microscope. (Images first published in Kim YS, Lee S, Jung J, Shin S, Choi H-G, Cha G-H, Park W, Lee S, and Park Y. Yale J Biol Med. 2018 Sep; 91(3): 267–277).

The power of QPI and of combining it with fluorescence in a single instrument, is shown in Figure 5. The high-resolution time-lapse 3D images of Toxoplasma gondii infecting an ARPE-19 cell (Figure 5A) provide important insights into the mechanism of infection and were achieved using the Tomocube HT-1 microscope. Figure 5B shows NIH-3T3 cell research using both QPI and 3D fluorescence (GFP-Mito and mCherry-Golgi) using a Tomocube HT-2 holotomography microscope.

The future outlook

Holotomography and other QPI techniques have much to offer researchers in their efforts to understand the complex interactions within living cells. With the emergence of correlative imaging, as seen in Tomocube's HT-2 microscope, which combines the speed and quantitative features of QPI with the molecular specificity of fluorescence, these innovative approaches could play an important role in enhancing our understanding of the physiology and pathology of cells and tissues and make a critical impact on the diagnosis and treatment of various diseases.

This article was written by Aubrey Lambert, Chief Marketing Officer, Tomocube Inc. (Daejeon, South Korea). For more information, visit here .


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