RI is an intrinsic parameter of any material, describing the speed of light when light passes through the material. The use of RI can be utilized for bioimaging contrast, due to its label-free and quantitative properties. For further information, please see
https://www.biorxiv.org/content/early/2017/02/06/106328

Different parts of cellular structures such as cell membrane, cytoplasm, nucleus, and sub-cellular organelles have distinct values of RIs. Thus, the measurements of 3-D RI tomogram of a cell provides a means to visualize 3-D structures of live cells without using staining or labeling-agents.

The principle of the 3-D RI tomogram, or holotomography, is the optical version of X-ray CT. In X-ray CT, X-ray beams with various illumination angles passes through a human body, and the corresponding multiple 2-D X-ray images are obtained, from which 3-D absorption tomogram is obtained via a reconstruction algorithm. In holotomography, laser beams with various illumination angles passes through a live cell, and the corresponding multiple 2-D holographic images are obtained, from which 3-D RI tomogram is obtained via a reconstruction algorithm. Due to the same physical principle, both the X-ray CT and holotomography shares the physical governing equations, cores of reconstruction algorithms, etc.

Phase contrast or Differential interference contrast (DIC) microscopy provides visualization of unstained live cells with high contrast. However, both phase contrast and DIC microscopy only generate 2-D and qualitative imaging. Whereas, Holotomography measures quantitative 3-D tomograms.

The fluorescence microscopy has the disadvantage of causing stress to the cells, however, its advantage is that the target can be labelled with specificity. On the other hand, holotomography minimizes the stress on the cells, but the downside is that the target organelles cannot be distinguished with certainty with RI alone. Therefore, if we use holotomography together with the fluorescence microscopy, we can achieve both the specificity and 3D spatial resolution.

Holotomographic images can be analyzed using Tomostudio. Through analysis, you can acquire three parameters: morphological, chemical and mechanical. The morphological parameters include volume, surface area, projection area and sphericity. The chemical parameter measures dry mass and concentration, and in the case of red blood cell, Hb concentration can also be measured. The mechanical parameter measures cell stiffness.

Conventional fluorescence microscopies require use of fluorescent proteins or dyes, which cause stress and damage to the cells. Furthermore, fluorescent signal accuracy decreases due to photobleaching in a long-term imaging. However, use of fluorescent dyes is unnecessary with holotomography. Stress and damage on the cells can be significantly reduced, and photobleaching does not occur. Holotomography therefore allows long-term observation, as well as acquisition of 3D time-lapse images.