Here at Tomocube we are always interested to see the variety of ways the HT series of holotomography microscopes is being used to make new discoveries and advance human understanding of the world around us. Here are just some of those stories, and you can find many more at our website – Publications.
 

HT-1 to HT-2
 

Balamuthia mandrillaris trophozoites ingest human neuronal cells via a trogocytosis-independent mechanism

A team from Thailand’s Mahidol University led by Kasem Kulkeaw and Worakamol Pengsart, recently reported in Parasites & Vectors on a study that may lead to the development of new drugs to combat a fatal brain disease.
 

 

Their work involves Balamuthia mandrillaris, a natural nonsymbiotic amoeba that lives freely in soil and fresh water worldwide. Despite being an environmental microorganism, B. mandrillaris is pathogenic to humans and can infect the brain, causing haemorrhagic necrosis known as granulomatous amoebic encephalitis (GAE). Even with current drug treatments, the mortality rate is exceptionally high at approx. 98% and the mechanism by which the amoebae utilize human neuron-related cells as an energy source is uncertain.

The team used a Tomocube holotomography microscope to observe cell-to-cell interactions B. mandrillaris trophozoites and human neuroblastoma SH-SY5Y cells in 3D. The SH-SY5Y cells were cultured in a Tomocube cell culture dish in a centred quadrilateral well and the trophozoites were then added. Fluorescent probes specific to lipids, proteins and nucleic acids were used to image the ingestion of human cells in real-time.
 

Figure1. A) Schematic diagram of 3D imaging of cell-to-cell interactions using a holotomographic microscope. B) The 3D images of pseudo-coloured cells along the X–Y, X–Z and Y–Z planes are shown in the panels from left to right. Arrowheads, lipid droplet-like dots. C) X–Y view of the B. mandrillaris trophozoites interacting with human neuroblastoma SH-SY5Y cells (left panel), and the cytoplasmic bridge (arrows in the middle and right panel) in the Y–Z plane. D) The snapshot image of a rotating trophozoite shows anchoring sites (arrows in the left, middle and right panels). Abbreviations: B.M., B. mandrillaris; SH-SY5Y, human neuroblastoma cell line.


Label-Free Morpho-Molecular Imaging for Studying the Differential Interaction of Black Phosphorus with Tumor Cells
 

The June issue of Nanomaterials carried a paper (Valentina Mussi et al.) from an international group of researchers based in Italy and the USA on the use of optical diffraction tomography (ODT) in the exploration of the chemotherapeutic use black phosphorous.

The authors explain that ‘designer nanomaterials’ that can selectively probe and destroy cells in the human body is rapidly opening the door to the development of promising approaches for early disease diagnosis and personalized therapies. Among these, black phosphorus nanosheets (2D BP) are emerging as very promising, highly selective chemotherapeutic agents due to their fast degradation in the intracellular matrix of cancer cells, including human cervical cancer cells, alveolar adenocarcinoma, lung carcinoma, hepatoma, and prostate cancer cells.

Mussi et al. used Tomocube’s HT microscope together with Raman spectroscopy to achieve integrated, label-free insights into the processes that accompany the introduction of black phosphorus to both carcinomic and normal human prostate cells. According to the paper, the ODT experiments provided “unambiguous visualization of the 2D BP internalization in cancer cells and the morphological modifications of those cells in the apoptotic phase. The cellular internalization and damaging occurred, respectively, 18 h and 36–48 h after the 2D BP administration. Changes in the chemical properties of the internalized 2D BP flakes were monitored by Raman spectroscopy. Interestingly, a fast oxidation process of the 2D BP flakes was activated in the intracellular matrix of the cancer cells after 24 h of incubation. This was in sharp contrast to the low 2D BP uptake and minimal chemical changes observed in the normal cells.”
 

Figure2. Phase, maximum intensity projection, and 3D RI images of a PC-3 cell after 18 h (A) and a group of PC-3 cells after 48 h (B) of incubation with 2D BP flakes. In the phase and RI images, the BP clusters are visible as red dots (maximum RI: 1.38) in the cytoplasm marked in yellow (maximum RI: 1.34). Scale bar: 10 μm.
 

As well as elucidating the fate of black phosphorous flakes in cancer cells, these scientists suggest that their ODT-based label-free morpho-molecular approach “offers a powerful, rapid tool to study the pharmacokinetic properties of engineered nanomaterials in preclinical research.”

Label-Free Vibrational and Quantitative Phase Microscopy Reveals Remarkable Pathogen-Induced Morphomolecular Divergence in Tumor-Derived Cells.
 

Cancer-derived cells and label-free ODT and Raman spectroscopy were also the focus of a recent paper from a group at John Hopkins University, USA, in ACS Sens. Zhenhui Liu, Sheetal Parida, Shaoguang Wu, Cynthia L. Sears, Dipali Sharma and Ishan Barman used a model system of human breast cancer cells to demonstrate that quantitative phase microscopy can detect biomolecular and morphological changes in single cells exposed to toxin.

The background to the study is the understanding that defining the morphological and molecular changes in cancer cells subject to extracellular stimuli is crucial for identifying factors that promote tumour progression. In this regard, the label-free optical imaging of the Tomocube holotomography microscope retrieves valuable single-cell information through the detailed visualization of the morphology and quantitative changes in biomolecular composition.

The authors state that the potential of ODT microscopy in the analysis of microbiota–cancer cell interactions is “surprisingly underappreciated, despite the growing evidence of the critical role of dysbiosis in malignant transformations.” Using their model system, they imaged the biomolecular and morphological changes in single human breast cancer cells exposed to Bacteroides fragilis toxin (BFT), a toxin secreted by enterotoxigenic B. fragilis. What’s more, the team went on to use machine learning to elucidate subtle, but consistent, morphomolecular differences between BFT-exposed and control breast cancer cells, which were accentuated after in vivo passage. This corroborates their findings that a short-term BFT exposure imparts a long-term effect on cancer cells and promotes a more invasive phenotype.
 

Figure3. Overview of the study. (Left) MCF7 breast cancer cells were treated with BFT and orthotopically implanted into mice. Tumors were harvested, and the dissociated tumor cells were subjected to in vivo limiting dilution. (Middle) The cells before and after tumor derivation were characterized by Raman microspectroscopy, quantitative phase microscopy, and fluorescence microscopy. (Right) Captured images and spectra were analyzed to elucidate the BFT-induced changes in cell molecular composition and morphology.
 

The team suggest that this label-free approach delivers measurements representative of the overall cellular phenotype and offers a global detection approach that may pave the way for further investigations into the multifaceted interactions between the cancer cell and the microbiota.



Morphological alterations in primary hepatocytes upon nanomaterial incubation assessed by digital holographic microscopy and holotomography

The impact of nanomaterials on the morphology of cells was also the focus of a paper by the German team of Kai Eder, Anne Marzi, Martin Wiemann, Ursula Rauen, Björn Kemper, and Jürgen Schnekenburger.

Writing in the proceedings of SPIE BiOS 2022 in San Francisco, USA, they explain that the effects of nanoparticles on cells can range from a reduction in viability to alterations in morphology and intracellular structures. They also underline the establishment of holotomography microscopy and digital holographic microscopy (DHM) as tools for the label-free imaging of cell morphology and the assessment of the 3D distribution of intracellular structures through significant improvements in quantitative phase imaging (QPI) technologies.

The team go on to present the use of DHM and holotomography microscopy applied consecutively on the same samples to observe and quantify the impact of nanomaterials on the morphology of primary hepatocytes, which make up 90% of the cells in the liver. The hepatocytes were isolated from collagenase-perfused rat livers and seeded into HT-compatible cell culture dishes. After cultivation and incubation with different types of nanoparticles (CeO2, Ag, Au) for 24 hours the cells were fixed with a mixture of glutaraldehyde and paraformaldehyde to preserve cell morphology and structure. An automated, modular DHM setup was applied for large-area QPI screening of the entire hepatocyte populations while a Tomocube HT-2H was utilized to observe selected tiny 3-dimensional intracellular changes of interest via refractive index tomograms.

Their results demonstrate that QPI with DHM is capable for efficient large-area 2D screening and to reveal of nanomaterial-related alterations in the entire hepatocyte populations while HT provides high performance complementary 3D insights and the localization of tiny intracellular damage.
 

Figure4. Representative HT measurements of fixed primary rat hepatocytes incubated with nanoparticles and imaged in PBS buffer. In each part of the figure an x, y, z cross-sections within refractive index range from 1.320 to 1.400 is shown. The larger, right side image (yellow box) shows the refractive index tomograms of the hepatocytes in a false color representation. High refractive index volumes are shown in red color, while lower refractive index volumes are shown in green color. Each FOV is 240 μm x 240 μm. Nanoparticles and controls were incubated for 24 hours on the primary rat hepatocytes. A) Vehicle control 2.5% BSA in cell culture medium incubated showed normal morphology. Bile canaliculi are visible as tubes with a low refractive index. Green arrows indicate lipid droplets with high refractive index. B) CeO2 incubated primary rat hepatocytes show morphology similar to the vehicle control and intracellular nanoparticle agglomerates are visible as smaller red volumes indicated with yellow arrows. C) AgPVP nanoparticles incubated on primary hepatocytes. Cell damage is visible by detachment and rounding of cells from the substrate. Two cells in different stages of cell death are shown with red arrows. Cell content condenses, visible in higher refractive index for most volumes of the cells. (D) Primary rat hepatocytes incubated with AuPVP nanoparticles. Cell morphology is similar to the cell culture vehicle control and no increased detachment of cells from the substrate is visible. Green arrows in A) and D) show lipid droplets.
 

The team suggest that this label-free approach delivers measurements representative of the overall cellular phenotype and offers a global detection approach that may pave the way for further investigations into the multifaceted interactions between the cancer cell and the microbiota.

Localization analysis of intercellular materials of living diatom cells studied by tomographic phase microscopy
 

Although the observation of living cells using holotomography microscopy provides unique information on the optical properties of cellular components, the interpretation of the data from living cells requires further research.

A 2022 paper from Ryo Hamano, Shigeki Mayama and Kazuo Umemura in Appl. Phys. Lett. provides further evidence of this. They point out that information on the refractive index of cells provides a 3D dataset and that rapid progress in computer technology has enabled the generation of large datasets using holotomography. They also make the point that while one pixel of a holotomography image is approx. 100–200 nm in size, more than several millions of pixels are contained in the several tens of cubic microns of space of a typical observation area.

The team used the Tomocube HT microscope to quantitatively analyse localization of cell components of living diatom cells from X–Y cross sections, and the distribution of RI values at the cell surfaces and inside the cells quantified. The results showed that the RI values were slightly lower at the cell centre than the cell boundary and that RI values also fluctuated according to the depth below the cell surface. Furthermore, statistical analysis by root mean square and Moran’s I methods revealed unique localization of RI values for several cells among the 25 individuals. In addition, the volumes of the cells estimated using TPM data corresponded to the cell volumes obtained via scanning electron microscopy.

In conclusion, they propose that the physical information provided by holotomography microscopy is an effective procedure to investigate the intracellular materials of living cells quantitatively and statistically.

Figure5. Typical MIP image and X–Y cross section of TPM tomograms of a living diatom cell. (a) A schematic view of the cross-sectional analysis. (b)–(h) X–Y cross sections at depths of (b) 0 μm (cell surface), (c) 0.5 μm, (d) 1.0 μm, (e) 1.5 μm depth, (f) 2.0 μm, (g) 2.5 μm depth, and (h) 3.0 μm depth. Scale bar = 2 μm.
 

All the work described in this article is available for further reading through the link on our website – Publications