We’ve all heard the phrases ‘small and agile’, ‘first mover advantage’, ‘big and beautiful’, and ‘large and slow’. They’re particularly noteworthy when new product development is being discussed, especially for small, agile companies seeking the first mover advantage but challenged to resource future upgrades while supporting the existing product range. How tempting the added resources of big and beautiful sound, as long as they don’t lead to large and slow!

HT-1 to HT-2

Tomocube launched the HT-1 holotomography microscope to the delight of cell scientists, since it delivered quantitative nanoscale, real-time, dynamic images of individual living cells quickly and simply without any sample preparation. However, many researchers also wanted some confirmation of specificity, and Tomocube’s development team responded immediately with a plan to create a second light path in the microscope for fluorescence – a relatively simple upgrade for the fully-motorised HT-1. Then, growing user feedback suggested a more ambitious program to embrace a major advantage. So, ‘simply add fluorescence’ became ‘add the ability to do fluorescence in correlation with the 3D Holotomogram so that the relative positions of the RI structures could be determined against the backdrop of fluorescence in 3D space’. In less than 18 months the HT-2 arrived in 2018.

Both the HT-1 and HT-2 microscopes offer the advantages of high resolution and high measurement precision afforded by a dual beam interferometric microscope. For those working with individual samples or cells in suspension, for example, their speed, power, and precision are indispensable. But, for others, the limitation of one sample at a time is problematic and illustrates perfectly that there is no ‘one size fits all’ solution. For instance, when you intend to work with multi well plates there is the issue of using dry lenses as well as a longer working distance on the condenser unit to accommodate larger vessels with both affecting resolution. Another issue is fluorescence efficiency with lower NA lenses, since lower NA equals less light delivery and light collection and results in longer exposure times. Multi well plates also mean a larger stage movement on a larger and heavier stage.

HT-1 and HT-2 to HT-?

Tomocube has been continually discussing the complex imaging requirements of researchers working with live cells since the HT-1 was conceived. However, almost immediately after the introduction of the HT-2, the development focus intensified as a result of an approach by ETH Zurich, Switzerland’s foremost research university. Ranked 4th in the world and 2nd in Europe for engineering and technology, 1st in the world for earth and marine science, and 6th overall by QS World University Rankings in 2021, ETH had already seen and tried HT-2 and expressed significant interest in future developments for the HT-1 and HT-2 products. Over many months and hundreds of hours of debate, a ‘shopping list’ of requirements was hammered out. However, the shopping list was so extensive that it became obvious that further development of the HT-2 was not going to give an optimum solution. An entirely new microscope platform with a new imaging system was required.

An initial test rig was set-up to confirm the feasibility of the proposed new optical system. In parallel, Tomocube’s engineers began describing and designing the components needed to build a commercial version of what was being tested on the optical bench. Soon after, software design was also started in parallel with the overriding aim of making the system simple and intuitive but flexible and powerful. In this, they adopted the key philosophy of keeping the software GUI as close to that of other microscopes as possible, so that it was easy to adopt with minimum training requirements. The result of this intensive effort is now available.

HT-? becomes HT-X1

The HT-X1 is the first of a completely new generation of holotomography microscopes overcoming many limitations scientists and researchers face, including work with confluent cell sheets. Utilizing incoherent light to generate holographic images of unlabelled live cells, the new microscope is ideally suited to higher-throughput and automated screening applications thanks to its ability to image multi-well plate formats, large field-of-view, laser autofocus system, and very high performance 0.95NA objective. Core imaging facilities will also value its integrated gassed incubator for long-term studies and its software-driven approach, which allows multiple users to access its formidable performance simply and quickly through its intuitive GUI, TomoStudio X.

Dr. Gabor Csúcs from ETH Zurich heads the first research team to use the revolutionary microscope, pre-ordering the HT-X1 before its official launch date. He says, “We tested HT-X1 for our studies on both animal cells and microorganisms. The advantage of observing cells and organelles in their intact and live state with little requirement for preparative steps makes the HT-X1 a powerful tool for long-term, comparative experiments. The new software, TomoStudio X, is easy to use and intuitive, which enables us to increase our productivity. I believe HT-X1 is a very powerful research tool for label-free imaging and see it as a good choice for imaging centres around the world.”

The technology underpinning the HT-X1

The new microscope builds on the optical diffraction tomography (ODT) of Tomocube’s award-winning HT-1 and HT-2 microscopes. Like them it presents morphological, physical, and chemical properties of individual living cells through the 3D refractive index (RI) tomograms quickly and simply without any sample preparation, and molecular specificity information through fluorescence imaging. However, the new HT-X1 uses a conventional single-beam LED instead of a laser light source. Instead of rotating the laser beam to illuminate the sample at various incidence angles it illuminates the sample with various beam patterns specifically designed to retrieve the refractive index and captures a sequence of holograms from different positions.

This unique single-beam technique simplifies the imaging process by eliminating the need for background calibration and allowing imaging in confluent samples without an increase in light intensity. As well as being easily combined with complementary imaging modalities, it is mechanically more stable and less sensitive to speckle noise for high contrast imaging.

The HT-X1 incorporates a 450 nm LED light source for transmitted light together with a customisable four-channel fluorescence filter engine, with any 3 of the channels being captured for overlaying. The large, motorized stage will easily accommodate 24-, 12- and 6-well plates and standard 35 mm imaging dishes in the integrated incubator, which is equipped with a sealing lid to allow gassing and combines with a motorized laser-powered autofocus module for accurate and repeatable monitoring of locations during long-term studies. A 40x 0.95 NA air objective lens allows rapid imaging of multiple locations with a lateral resolution of up to 156 nm. The extremely high numerical aperture also allows more illumination for fluorescence imaging while the large 160 x 160 micron field-of-view allows rapid and easy imaging of multiple locations.

HT-X1 and HT-2 at ETH Zurich

ETH’s HT-X1 offers users the unique capability to perform multiparameter, multi-well assays with the highest possible resolution. Meanwhile, their existing HT-2 continues to enable rapid, high-resolution imaging in 3D alongside high-speed 2D for membrane fluctuation analysis. Though single sample, the HT-2 remains the highest resolution system available in the market and the only one to have the capability and speed to image free floating cells and bacteria.