the world’s most versatile 4D Live Cell Analysis (LCA) system.


HT delivers nanoscale, real-time results based on quantitative phase imaging (QPI) with high precision of measurement and reproducibility, optionally combined with fluorescence for molecular specificity.

The Principle

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 constituents 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 shift. These 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.

Example images of microscopes

This is the principle utilized in Phase Contrast and Differential Interference Contrast microscopy, techniques that are widely used to image unstained biological samples. However, these methods image in two dimensions while biological samples are actually three dimensional. The Tomocube HT solves this problem. When we combine the image data from the beam passing through the sample with the reference beam we create a hologram image. By rotating the imaging beam through 360° we can capture a sequence of holograms from different angles. The resulting 2D hologram images of a sample obtained with various illumination angles are reconstructed into a 3D RI tomogram. Cellular Holotomography is analogous to X-ray computed tomography (CT) and magnetic resonance imaging (MRI). Unlike phase contrast microscopy, HT provides quantitative (RI-based) and 3D tomographic reconstruction capability.

CT vs HT

How is this achieved?

Just like in a conventional microscope, the sample is located on a stage between an objective lens and a condenser lens. A light source is split to follow two paths, the specimen beam and the reference beam (the principle behind a Mach-Zehnder interferometer). The sample and the reference arms when combined generate a 2-D hologram, which is recorded by a digital image sensor (s-CMOS). This reflects a QPI measurement principle of the phase shift relative to the reference beam.
Tomocube_Label-free 3D Live Cell Imaging_Holotomography_Schematic light path
Tomocube_Label-free 3D Live Cell Imaging_Holotomography_Technology animation
The imaging beam illuminates the sample with an incident angle of 53°(S models) or 63°(H models). The beam is rotated through 360° with respect to the optical axis. From the 48 overlapping captured holograms a 3-D RI tomogram of the sample is then reconstructed.

Patented beam rotation technology

The Tomocube system uses a Digital Micro-mirror Device (DMD) to enable the illumination beam rotation We developed our proprietary technology (patent ##s) [E1] to precisely control the intensity and angle of the beam reflected from a DMD. The patented technology behind the beam rotation provides unique advantages over other methods. Highly stable, fast and reliable electronic control of the light path through the DMD eliminates moving parts for better stability and improved image resolution. Being solid state, the rotation can be achieved rapidly. Combined with the high speed of the detector, the full 3D data set can be captured in less than half a second. Even free-floating objects can be captured with the system without displaying artifacts resulting from Brownian motion.
Caption: DMD consists of several hundred thousand micromirrors arranged in a rectangular array. Each individualal mirror can be rapidly tilted electronically to create a mirror pattern which can rotate the beam through 360° around the optic axis at a desired angle.

A Rubik’s Cube of RI voxels

As the holographic data is quantitative, what we can calculate is a virtual Rubik’s Cube of voxels of known RI. Using Tomostudio software, we can also selectively pseudo-colour bands of RI to highlight structures within the captured volume and render them as a 3D image or animation. Effectively, we can visualize in 3D, colour-coded structures that were previously undetectable without staining.

More than just an image - Quantitative data

By highlighting the structure of the cell and its subcellular components based up their differing refractive indices, quantitative data can be extracted without invasive labelling. Volume, surface area, sphericity and more can be calculated from the cells of interest. But more than that, the RI directly relates to the quantity of material present in the voxel. That means that the system can measure mean RI, dry mass, and concentrations of known substances like aggregated proteins or lipid droplets. See it and measure it.

Quantitative Bioimaging Meets Kinetics,
from 3D to 4D

With full automation Tomocube HT affords the opportunity for safe, long-term study of live cells on a large scale, and real time live cell analysis. This is possible because of the minimal specimen damage, instrument’s motorized stage, environmental controls, and custom-designed stitching software developed in cooperation with the Korea Advanced Institute of Science and Technology (KAIST).
Tomocube_Label-free 3D Live Cell Imaging_Holotomography_TomoAnalysis

Holotomographic microscopy delivers nanoscale, label-free, real-time LCA and can combine quantitative phase imaging (QPI) with fluorescence for state-of-the-art spatiotemporal resolution as well as high molecular specificity.