By capturing the intrinsic refractive index (RI) of cells using a low level of light intensity, Holotomography has emerged as a unique solution for live cell imaging that surpasses the compromise between obtaining high image quality and maintaining healthy cells.
The latest generation of Holotomography – Tomocube HT-X1 – utilizes a low-coherence light source. This groundbreaking advancement unlocks label-free 3D and 4D quantitative live cell imaging on diverse multi-well plates with improved resolution and free of laser-induced speckle noise.
The new system works well with a wide range of biological samples, including confluent cell sheets and thick specimens. It provides not only high-content 3D visualization but also quantitative data for individual cells and their intracellular structures, such as the volume, surface area, and dry mass.
Observations of cellular morphology and activity are typically based on labeling of target molecules. These methods are invasive, affect the nature of the target molecules and potentially interfere with their biological relevance. In addition, these forms of label are not effective in providing quantitative information. The tools used to visualize labels are generally damaging to cells as often laser-based illumination is used to excite fluorochromes which in turn causes phototoxic damage to the cells.
The “holy grail” of imaging is to visualize cells without labeling to ensure the cells behave and grow normally. Holotomography (HT) does this by capturing the intrinsic light scattering properties of cellular materials using very low levels of light intensity, just enough to allow the light to pass through the cell. In doing so, the refractive index (RI) information of structures within the cell can be collected and selectively pseudo-colored to reveal the cell and its organelles.
In capturing the RI distribution, the Tomocube HT-X1 Holotomography can also provide quantitative data in 3D such as the volume, surface area, and dry mass of cells and their intracellular structures.
Holotomography and Fluorescence |
Fluorescence |
| Objective Lens |
|
|---|---|
| Condenser Lens |
|
| Lateral Resolution (Dish/6/12/24 well) |
1069/1211/1387/1695 nm |
| Axial Resolution
(Dish/6/12/24 well) |
|
| Holotomography Light Source |
450 nm LED |
| Fluorescence Light Source |
LEDs 3 channels (R/G/B) + 1 channel (R or G or B) |
| Basic |
|
| Power | 100-240 V, 50/60 Hz, 5-3 A, 400 W |
| Temperature / Humidity | 20-28 °C / 35-85 %, non-condensing |
As the control software for the HT-X1, TomoStudio X is designed to operate the system as you design the experiment. TomoStudio X offers flexible workflows for multi-dimensional image acquisition of holotomography and fluorescence in multi-well imaging conditions. All the essential control and status of the system are displayed all at one glance.
HT-X1 uses an LED illumination module integrated with a Digital Micro-mirror Device (DMD). When HT-X1 captures a 3D image, the illumination module is rapidly cycling through a set of optimized illumination patterns programmed in the DMD to encode specific spatial features of the specimen. By synthesizing these images acquired from multiple angles, HT can overcome the optical resolution limit, achieving higher synthetic NA compared to the physical NA value of objective lens. Therefore, HT-X1 imaging provides high-content, high-resolution 3D imaging at exceptional stability
The Holotomography approach in live cell imaging does not require fixation or labeling, which reduces unwanted artificial intervention and the need for long exposure to light. By minimizing the risks of phototoxicity and photobleaching, the specimen remains at its nature state for observation. With a rapid capture speed of 1 second per frame, HT enables real-time monitoring of dynamic cellular processes with exceptional temporal resolution. Additionally, its integrated stage-top feature facilitates long-term observations, empowering researchers to explore cellular phenomena over extended periods with stability and precision.
Especially designed to maximize user flexibility, the HT-X1 employs a unique adaptive illumination module that is tailored for multi-well plates. The combination of high NA, a long working distance condenser, DMD, and a motorized illumination unit delivers an efficient illumination pattern for a diverse range of vessel types, from 35-mm dishes to 96-well plates. With the HT-X1, researchers will have more freedom to design their experiments on different sample types using any imaging vessel of their choice.
TomoStudio X works in concert with the HT-X1 platform to visualize and analyze RI tomograms. This intuitive, easy-to-use software empowers users with complete system control, allowing them to handle even the most complex experiments through simple mouse clicks. Researchers can easily set up complex time-lapse sequences, perform large-area tile imaging and stitching tasks, and image multiple points with ease using this software.
