Deep Tissue Imaging

From Visible to IR Wavelengths

November 02, 2010

Embryonic development relies on genetic coding and non-coding informations. In particular, physical forces generated by blood flow are critical for proper development of the cardiovascular system. To gain new insight into the fundamental control of cell response to physical changes and to study the dynamics and roles of biological flow during the development of the zebrafish, Dr. Julien Vermot established his lab in 2009 at the Institute of Genetics and Molecular and Cellular Biology (IGBMC) in Strasbourg, France. He belongs to the first lab to use the Leica DM6000 CFS equipped with an OPO/Ti:Sa infrared source for deep tissue imaging and infared excitation wavelengths up to 1300 nm.

The IGBMC, one of the leading European centers of biomedical research, is devoted to the study of higher eukaryotic genomes, the control of genetic expression, and the analysis of gene and protein functions. Dr. Vermot, group leader and scientific coordinator of the IGBMC imaging facility, coordinates the development of the light imaging techniques scientific program in collaboration with the board of users and the imaging facility.

His research focuses on the roles of fluid flow during embryogenesis. He is interested in characterizing fluid motion at a detailed level, such as watching blood cells flow, using resonant true confocal scanning. Dr. Vermot explains why the zebrafish is the optimal organism for studying in vivo fluid mechanics, as well as why he chose IR imaging to accomplish this and gives an outlook for the future:

Current research topics in the area of flow-controlled differentiation

Our lab is interested in addressing embryonic development; in particular, we try to understand what are the roles of biological flows during organogenesis and their connections to the developmental program encoded in DNA. More precisely, we want to understand the effects of biological flow at the cellular and tissue scale and find out how cells interpret the physical information provided by their environments, which is dominated by mechanical stress. We principally use zebrafish as a model organism and are keen to use and develop quantitative approaches based on live cell imaging. "How are flows generated in embryonic cavities?" is another question we try to answer. We usually deal with micrometer size structures and need high-speed imaging that is safe for the animal.

Basically, we explore the limits of the models, propos-ing that genes are the only driver of morphogenesis. More and more, we see that emerging complexity is dependent on the physical environments of the cells, flow being one of them. Practically, we look at the role of blood flow during cardiovascular development because it is related to human diseases, but there are many other organs whose development strictly depends on biological flows.

For example, we look at the role of cilia driven flow, which happens at a smaller scale compared to blood flow. Blood flow is controlled by heart contractions, which is about two orders of magnitude bigger than cilia. As a result, cilia generate a slower, smaller flow profile.

We found that the inner ear of zebrafish relies on motile cilia activity, which is important for the development of the sensory organ. Another example is the "left-right-organizer", present very early in development to break the embryonic left-right symmetry. Importantly, we can differentiate the different types of flow depending on the type of cilia beat. To see at this scale in 3D we need a very fast true confocal scanning instrument.

Most of us at IGBMC work on basic research. However, many of our projects are linked to human diseases. Most of the basic mechanisms in biology, when they go wrong, lead to such problems. We look at the origin of those diseases and do work that will lay the foundation for further and specific research to develop therapies. To do so, imaging is key and will be even more important in the future.

Fig. 1: Zebrafish embryo expressing the DsRed fluorescent protein under the control of a blood cell specific promoter. Fast imaging with resonant scanner at 167 frames per second at 512 x 64 pixels. Courtesy of Julien Vermot, IGBMC Imaging Center, Strasbourg, France.
A time-lapse sequence of images showing red blood cells flowing in Zebrafish embryo. Fast imaging with Leica TCS SP5II OPO at 167 frames per second. Red blood cells expressed DsRed, imaged by near IR excitation.
Fig. 1: Zebrafish embryo expressing the DsRed fluorescent protein under the control of a blood cell specific promoter. Fast imaging with resonant scanner at 167 frames per second at 512 x 64 pixels. Courtesy of Julien Vermot, IGBMC Imaging Center, Strasbourg, France.
A time-lapse sequence of images showing red blood cells flowing in Zebrafish embryo. Fast imaging with Leica TCS SP5II OPO at 167 frames per second. Red blood cells expressed DsRed, imaged by near IR excitation.

Multiphoton microscopy with OPO in developmental biology

Zebrafish is a very imaging friendly animal. The larvae are transparent and easy to culture under a microscope. However, structures that generate flows are often localized in deep, light scattering tissues. In this case two-photon imaging is the modality of choice because it allows deep imaging with limited phototoxicity. A confocal microscope with fixed stage and illuminated by an OPO helps to perform multicolor two-photon imaging using conventional fluorescent proteins, such as GFP and RFP. Second Harmonic Generation is possible.

It also allows us to manipulate the tissue through femtosecond cell ablation where you can target single cells in the tissue and perform imaging. This technique is challenging, it may not work all the time but can give interesting results. Two-photon microscopy is usually used to look deep into the sample. Furthermore, as opposed to single-photon microscope techniques, two-photon imaging illuminates only the part of the sample you image, thereby it limits photobleaching and photodamage.

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