Biological Physics Lab

Microfluidics regards miniaturized fluid handling devices in which the flow channels typically have one of their dimensions in the range of 10 − 500 µm. We develop tailored microfluidic structures and pumping systems for dynamic control of microenvironments. The microfluidic devices are used in intra- and intercellular single-cell response studies, as well as for biomimetics. Cells are trapped and hosted inside the devices and enable us to follow cell behaviour, on both short- and long-term basis, by microscopy and bio-chemical off-chip analyses.

The two distinct directions of tailored microfluidics are described below:

Organ-on-chip

Using microfluidics, we mimic the smallest functional unit of the human liver, the liver lobule, and recapitulate the physiologically relevant environment crucial for specialised cell behaviour. It has been shown, the functionality of cultured hepatocytes is improved when the different types of liver cells are co-cultured, a feature facilitated by tailored microfluidics. Furthermore, the technology enables fine-tuned exposure of the various fluid shear stresses optimal for the different cell types. In combination with off-chip analyses of the flow-through media and 3D imaging of the formed liver tissue, we aim to delineate the cellular development of functional liver tissue and its down-stream use as “disease-on-chip”. We will also investigate the tissue response to novel drug candidates in healthy and diseased tissue, in particular, focusing on the liver disease non-alcoholic fatty liver disease (NAFLD). Furthermore, combined with stem-cell technology, we envision our organ-on-chip technology will be used for personalised medicine.

Single-cell analysis

Single-cell analysis enables capturing the heterogenic responses in a sample population. We have established a single-cell analysis methodology relying on microfluidics and microscopy, sometimes also taking advantage of optical tweezers. The biological sample is constituted either by yeast cells, algae or mammalian cells. The image analysis of the microscopy data regards traditional tracking and monitoring of intra- and extracellular signalling events. Usually, some kind of fluorescently labelled or autofluorescent molecule is traced, but with the development of AI-supported image analysis software, also label-free detection is used.

Much focus has been towards the metabolic pathways, but also pathological behaviour of specific cancer cell types and studies of active matter is on the agenda.

The technology of optical tweezers is based on the original work of the Nobel laureate Prof. Arthur Ashkin in which the momentum of light is used to exert forces, or radiation pressure, on a matter. Today, this technology is used in many biological and life-science oriented applications. We have built and use laser-based optical setups for non-invasive and sterile manipulation and probing of materials and single cells.

By using infrared laser light, absorption and optical damage are low, which allow cells to reproduce within the optical trap. A key component in the optical setup is a powerful lens, able to strongly focus the light and enabling stable three-dimensional trapping. This is possible using a high numerical aperture microscope objective. After calibration of the force exerted by the trap at known experimental conditions, the optical tweezers can also be used as a tool to measure or exert precise forces in the order of pN.

With holographic optical tweezers (HOT), several optical traps can be created on-demand and repositioned independently in three dimensions. Holographic beam steering is achieved using a spatial light modulator (SLM) connected to a personal computer. Traps are created, repositioned or removed by generating new holograms that are sent to the SLM over the PCI or PCIe bus (or in some cases, over DVI). As optical trapping preferably can be performed with the same microscope objective as is used for imaging of the sample, a combinatory setup of optical tweezers and fluorescence microscopy is optimal for our single-cell studies.

Advanced imaging for temporal and spatial visualisation constitutes a vital part of our research. Different projects demand different imaging techniques, which are briefly described below:

Fluorescence microscopy

By the use of fusion-proteins, or, by monitoring naturally occurring autofluorescent molecules, migratory or fluctuating behaviours can be observed and analysed. Microfluidics enables control of the extracellular environment, and therefore, external perturbations on cellular response can be studied in real-time in dual channels. This methodology works perfectly on subcellular levels but is diffraction limited.

dSTORM

In super-resolution microscopy, is light microscopy techniques where mages can be acquired with a higher resolution than the one imposed by the diffraction limit. In dSTORM, or, direct stochastically optical reconstruction microscopy, one takes advantage of the photoswitching of a single fluorophore.

Thanks to super-resolution microscopy techniques, one can acquire images with higher resolution than the one imposed by the diffraction limit. One of the most popular super-resolution techniques is dSTORM (direct stochastic optical reconstruction microscopy). This technique is based on the photoswitching properties of fluorophores which usually is achieved by illuminating the sample with one specific wavelength of light and an appropriate buffer condition. Then, after recording a stack image of thousands of frames, all fluorophores are localised and subsequently combined to form one super-resolved image.

Light sheet microscopy (3D imaging-link)

The laser light in light-sheet microscopy is focused only in one direction and results in a thin slice of light illuminating sections of the sample. This feature offers low photodamage and stress to the sample, 3D imaging with intermedium-to-high resolution and high imaging speed. In a collaboration project, we have a set-up in which the sample volume is scanned by changing the relative position of the light-sheet and the sample. In that way, only the fluorophores that are excited by the laser light will emit fluorescence and will be collected by the camera.

HepVisage

Liver-on-chip for drug discovery and personalized medicine, 2018-

(SSF Works; Instrument, Technique, and Method Development Projects 2017)

An interdisciplinary research project in which a realistic model of the human liver tissue will be monitored in real-time with light-sheet microscopy and validated by studying liver metabolism and disease, in particular NAFLD, fibrosis and liver cancer.

Collaborators:

Senior lecturer Giovanni Volpe, Department of Physics, GU

Prof. Stefano Romeo, Wallenberg Laboratory, SA, GU

Assistant Prof. Daniel Midtvedt, Department of Physics, GU

Individual Glycolytic Oscillations to Propagating Waves

2011-

Delineating the metabolic pathway of glycolysis present in eukaryotic cells. So far, our focus has been to investigate the individual cellular glycolytic oscillations, their entrainment of each other to reach a synchronized bulk behaviour and the network regulation. The single-cell data are vital to understanding the transitions between “out-of-phase” to “in-phase” oscillations. Experimental data complemented with numerical analysis and systems biology.

Collaborators:

Prof. Jacky L. Snoep, Stellenbosch University, South Africa

Prof. Bernhard Mehlig, Department of Physics, GU

Senior Lecturer Giovanni Volpe, Department of Physics, GU

DeLiver

2018-

ITN funded by the European Union’s Horizon 2020 research and innovation program under grant agreement No. 766181

The liver contains a large number of very fine capillaries (the sinusoids), which are lined by endothelial cells. In the liver, these endothelial cells are called liver sinusoidal endothelial cells (LSEC) and they contain thousands of nanosized pores (fenestrations) that enable the clearance of molecules and small particles from the blood. The size of these fenestrations is well below the optical diffraction limit, and consequently, very little is known about the essential physiological function of these unique structures and their role in the transfer and/or clearance of metabolites and pharmaceuticals to vital organs. LSECs are also responsible for the removal and degradation of pharmaceuticals, virus and waste macromolecules, making them a highly relevant type of cell for the study of pharmacological drug uptake. Because of the issues mentioned above, however, most pharmaceutical companies currently cannot assess the effect of drugs on these cells. Optical nanoscopy techniques and microfluidic chip fabrication aid the studies and characterization of the fenestrations in different microenvironmental conditions.

Collaborators: Consortia members distributed at 6 universities and 3 SMEs across Europe with additional partners from across the world.

Activematter

2019-

ITN funded by the European Union’s Horizon 2020 research and innovation program under grant agreement No. 812780.

The project focuses on experimental, theoretical and computational aspects of active matter. The specific aim of the ACTIVEMATTER network is to “prepare a new generation of physicists and engineers with the scientific insight and managerial skills to harness active matter at mesoscopic and nanoscopic length- scales and to exploit it in high-impact applications (e.g. the design and fabrication of biomimetic materials, the targeted localization, pick-up and transport of nanoscopic cargoes in drug delivery, bioremediation and chemical sensing)”.

Collaborator: One external PhD student at the SME Elvesys and consortia members from 9 different countries.