How to get 3D Tomography
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
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.
Cells are composed of mostly proteins, and cellular body can be understood as a bag of a protein solution. The RI of a protein solution is linearly proportional to the concentration of the solution. Thus, the simultaneous measurement of the volume and the mean RI value of a cell can provide a non-aqueous mass or dry mass of the cell, because the concentration is the ratio of a mass to a volume. For further technical information, please refer to the publication section.
A biological cell is neither a solid or a fluid; it is in-between, or a vixcoelastic material. How stiff or soft a cell is strongly related to its physiological or pathological conditions. TomoCube provides an unique means to measure cellular elasticity of soft cells, such as red blood cells (RBCs), by precisely measuring dynamic fluctuations in cell membrane. Because RBCs are so soft and elastic, they exhibit vibrations in the cell membrane in the order of tens of nanometers, from which cellular elasticity can be assessed. For further technical information, please refer to the publication section.
Technical specs of Tomocube product
Unlike HT-1, the new HT-2 model can capture fluorescence images. HT-2 model includes three channels of LED source (385nm, 470nm, 570nm), which can be used to acquire 2D or 3D fluorescence images. Previous HT-1 model only captures holotomography and therefore lacked specificity. On the other hand, the new HT-2 model allows simultaneous acquisition of both holotomography and fluorescence images.
The difference between HT-S and HT-H model is the lens. HT-S model uses an air lens, which is used in dry conditions to observe the sample. However, the HT-H model uses a water-immersion lens that can be immersed in water or other liquids. Therefore, HT-H model does not require coverslip on top of the sample and can be directly immersed into the media. Also, HT-H model’s water-immersion lens has higher resolution (Refer to HT series technical specifications).
TomoDish is mainly used to obtain 3D holographic images by HT series, but it can also be used to general light microscope images. It is very effective when taking DIC images or using only a small amount of media if you are treating high cost reagent. Details are uploaed onto Tomocube website: Support – Supporting Material – TomoDish