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Biocenter Oulu: technology services for biomedical research

Biocenter Oulu offers services for the study of proteins, cells and genes, and the generation creation of transgenic animals. One of its strengths is the light and electron microscope imaging of tissues and cells.

 

According to Professor Lauri Eklund, Coordinator in Light Microscopy at Biocenter Oulu, genetically modified model animals and mice in particular have helped researchers to understand more about phenomena related to the normal development and tissue operation of mammals than of any other organism. In addition to better understanding of developmental processes, these can be used as model organisms to study human diseases.

“Many imaging projects in Oulu concern the study of genetically modified mice. Mouse embryos and organs are imaged in Oulu either in whole, or by means of tissue specimens and light microscopy down to the accuracy level of individual cells, or even in higher resolution using electron microscope. We have also introduced methods to obtain images of cells and macromolecular structures in aestheticized mice, enabling us to see living tissues at high resolution. For this purpose, an intravital imaging laboratory has been set up, which enables cell examination in animals under anaesthesia. Small surgical procedures can also be performed in the laboratory.

“In addition, we use image data to create 3D models by means of optical sectioning. In addition to imaging of relatively large volumes, motorised microscopes can also be used to create tissue images of a large surface areausing tile scanning.”

The core facility service of Biocenter Oulu’s light microscopy specialises in what is known as mesoscopic imaging. Mesoscopic imaging helps to understand interaction between cells in a complex tissue environment, or even in entire organisms. Samples of mesoscopic scale are larger in volume and area than normal microscopy —ranging from a few millimetres to a couple of centimetres. These include mouse embryos, organoids resembling three-dimensional organs, and entire small model organisms, such as flies and fish emryos.

“In technical terms, mesoscopic imaging often requires a tissue culture environment suitable for microscopic imaging, specially designed 3D imaging equipment, tissue clarification methods, and advanced image analysis and processing capacity,” says Eklund.

Locating cellular and tissue structures and events

Mouse embyonic kidney (14.5 days old) imaged with a light sheet fluorescence microscope. Picture: Veli-Pekka Ronkainen.

Biocenter Oulu has a range of microscopes that enable various imaging methods and can pinpoint the location of various events in cells and tissues. A time dimension (4D imaging) can be added to three-dimensional images in living samples. Image sequences can be created, for example to show how cells differentiate and grow into, say, embryos or organoids.

“Thanks to the work done by Professor Seppo Vainio’s research group, we can grow an organoid within a few days, using various types of renal progenitor cells. This is something that has attracted international interest. Many researchers have come to Oulu to learn about the technique.”

Confocal and light sheet fluorescence microscopes are suitable for the imaging of three-dimensional and living samples. Especailly light sheet fluorescence microscope can scan the samples quickly without phototocix effect. Electron microscopY, on the other hand, can be used to find changes in the structures of cells and extracellular spaces, which are beyond the resolution of optical microscopy. However, this technology requires that the samples are fixed into place.

Although light waves cannot create magnifications in the same way as electron microscopes, innovative use of excitation laser light, fluorescently labelled molecules and image data processing, can achieve a level of resolution in optical microscopy which allows the examination of individual cells, cell organelles and macromolecular structures.

In most cases such objects must be made fluorescent in order to become visible in 3D microscopy. In the living cells or organism a fluorescent protein can beattached genetically, to the molecule to be studied. The fluorescent compounds (fluorophores) absorb energy from the excitation light and release part of this energy in the form of longer light wavelengths. This quantum mechanics phenomenon, which is visible to the human eye in certain range, is known as fluorescence.

It is also possible to search for specific proteins in cells and tissues by using antibodies to which a fluorescent marker has been attached. The antibody will identify its epitope in a certain protein and attach to it. Once it has attached, the marker can be detected with a microscope. The marker is chosen based on the kind of microscope that will be used to study the sample.

“We have microscopes equipped with spectral detectors and continuous laser light that enable us to study more than one fluorescent label simultaneously. This enables the study of complex interactions and multiple proteins.”

In fluorescence microscopy, fluorescent molecules are used as markers, while electron microscopy uses electron dense material, such as gold, for example.

”Oulu also has what is known as a label free imaging method that does not require additional markers or contrast agents, but can image endogenous molecules These include connective tissue collagen made visible with multiphoton technology, or other body’s own molecules, such as haemoglobin, which can berevealed through photoacoustic imaging. In the case of the latter technology, by combining excitation lasers, structural and functional information can be obtained from tissues, such as the structure of blood vessels and the blood’s oxygenation level.

“These technologies are very useful when imaging living tissues into which it is difficult to introduce markers.”

In terms of electron microscopy, Oulu specialises in the ultrastructural pathology in model organisms and immunoelectron microscopy of tissues and cultured cells,. These techniques provide information on the minutest details and the exact position of the proteins being studied in cell and tissue structures.

In immunoelectron microscopy, a metal labelled antibody or other reagent is related to the protein being studied, which means that the protein’s location can be determined very accurately, in a nanometer scale. This can provide new information on, for example, cell structures and protein interactions or orientation.

“The use of electron microscope methods to examine ultrastructures has been particularly fruitful in terms of the study of macromolecular structures of the extracellular matrix, which cannot be seen using optical microscopy. A fairly new area of study involves extracellular vesicles, “exomes”, which can be imaged by means of electron microscopy.

Data analysis challenges

Image of mouse brain blood vessels and related cells obtained with multiphoton microscopy. Picture: Veli-Pekka Ronkainen and Mika Kaakinen.

The problems with traditional imaging have been low resolution, low imaging depth, and lack of effective and quantitative analytics for the image data. In addition, special experience is required in order to extract biological data from the images.

Computer learning and machine vision methodsfor image interpretation has been developed in Oulu. In the approaches Biocenter Oulu has been collaborating with Professor Janne Heikkilä from the University of Oulu’s Center for Machine Vision and Signal Analysis.

“Data storage, transfer and analyses are challenging with respect to the 3D and 4D imaging of large samples. When data is transferred from microscope to user, one should be able to analyse it. Depending on the data, analyses may require a high amount of computing power. If the original data is stored somewhere distant, image processing may be slowed down due to insufficient data transfer speeds.”

Lauri Eklund regards the infrastructure provided by ELIXIR Finland’s CSC as the best solution, in national terms, for raw data storage and the reuse of open data.

Although metadata is linked to image data, there still can be many problems with data management.

“In order for image data to be reusable, it should conform to certain standards and be curated and annotated. Ideally, research infrastructures should provide image data “librarians” and “image information specialists”.

Ari Turunen

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For more information:

Biocenter Oulu

Biocenter Oulu is part of Biocenter Finland, which coordinates infrastructural activities for major national research. It is also a member of various European research infrastructures. These are Infrafrontier (transgenic mice), Euro-BioImaging (biological imaging) and Instruct (protein structure research).

https://www.oulu.fi/biocenter/

CSC – IT Center for Science Ltd

CSC – IT Center for Science Ltd is a state-owned, non-profit public limited company run by the Ministry of Education and Culture. CSC maintains and develops the state-owned centralised IT infrastructure.
http://www.csc.fi
https://research.csc.fi/cloud-computing

ELIXIR

ELIXIR is building an infrastructure to support research and the bio sector. It combines the leading organisations of 21 European countries and the European Molecular Biology Laboratory (EMBL) into a single infrastructure for biological information. Its Finnish centre is CSC – IT Center for Science Ltd.
http://www.elixir-finland.org
http://www.elixir-europe.org

Confocal microscopy: 3D

Picture: Veli-Pekka Ronkainen.

In standard bright-field and fluorescence microscopy, light goes through the entire sample, and in doing so the light wave becomes dimmer and is diffracted in the tissues, causing the imaged object to become blurred, and resulting in poor imaging depth and resolutionin thick samples.

To overcome these shortcomings, a confocal microscope uses a narrow laser beam to scan a small part of an sample one depth layer at a time. Pinhole filters out emitted light that is not on the focal plane, thereby achieving greater resolution in samples that are too thick for traditional wide-field fluorescence microscopy.

A confocal microscope creates the final image from small aligned areas. Three-dimensional images are created by reconstructing two-dimensional optical sections from various depths of the sample. Three-dimensional modelling combines several optical layers to create visual structures that cannot be viewed with traditional optical microscopy.

“The multi-disciplinary approach of the University of Oulu has been used in the adoption of new technologies. In terms of light sheet fluorescence microscopy and photoacoustic microscopy, for example, we cooperated with Docents Matti Kinnunen and Teemu Myllyllä of the University of Oulu and the Optoelectronics and Measurement Techniques Laboratory before such technologies were commercially available. This gives researchers a competitive edge,” says Dr Eklund.

 

Light sheet fluorescence microscopy and time series for the imaging of living samples

Picture: Veli-Pekka Ronkainen.

Light sheet fluorescence microscopy can be used to obtain microscopic images of light-sensitive samples or rapid biological processes within large living specimen. The sample is illuminated with excitation light one layer at a time, and the signal created by the sample is collected with another objective. The microscope has continuous optical sequencing: when the sample is moved on the light sheet, individual optical levels can be saved as 3D images. Large 3D samples can be scanned more quickly but with somewhat lower resolution than with a confocal microscope.

“Biocenter Oulu was the first laboratory in Finland to adopt this technology. The light sheet fluorescence microscope can take images of mesoscopic samples with clarified tissues, as well as living three-dimensional samples, whereby a time dimension is given for the images. This means that we can follow, for example, the genetically labelled cells during the growth of embryo or organoid within a specific time frame,” says Eklund.

Thanks to a new light sheet fluorescence microscope developed in Heidelberg, in 2015 researchers in EMBL were the first to observe how a fertilised egg developed into mouse embryo in a few days.

In 2018, the Howard Hughes Medical Institute in the United States introduced a microscope that utilises multiple angles of vision, enabling the imaging of an embryo’s growth at the level of individual cells. The researchers followed the embryos to see which genes were activated and which cells connected to each other.

Two light sheets illuminated the embryo, while two cameras recorded the early development of organs. The embryo’s location and size were tracked by algorithms. Algorithms observed how the light sheet was moving in the sample, deciding how to obtain the best images while ensuring that the embryo remained in focus. Because an embryo changes constantly, the microscope must continuously adapt andvery fast select from hundreds of images and time windows.

”The advanced mesoscopic methods of the future may be able to use non-diffractive excitation light (Bessel Beam and Airy Beam). Unlike ordinary light, this type of excitation light retains a uniform intensity in thick tissue samples. Moreover, the excitation light’s asymmetric form and ability to re-shape improve the imaging resolution in non-homogenous tissue samples with a high level of light diffraction.

By reshaping we mean that, unlike ordinary light, the excitation light can return to normal despite partially hidered by an obstacle.

According to Lauri Eklund, the rapid development of techniques for 3D imaging of living samples has led to an enormous increase in the amount of stored image data. Demand for quantitative analysis software for image data is also high.

“We can get the best out of these new imaging technologies if we also master image processing and analysis. In the case of mesoscopic imaging in particular, the large size of the samples requires effective image analytics and processing tools that can, for example, remove image noise and enable accurate 3D modelling. Smart computer software can also analyse cell behaviour and identify cell properties. In some applications you can, for instance, distinguish between cell types, determine cell division activity and analyse cells’ movement and viability.”