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.
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.
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.
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.