3D Microbeam Studies in vivo

RARAF – 3D Microbeam Studies in vivo

In-vivo Irradiation Facilities

This movie shows the pharynx section of a wild-type C. elegans specimen. Second harmonic generation (red) and autofluorescence (blue) were used to produce this movie. Following the anesthesia period and the multiphoton imaging process, the C. elegans specimen regained normal motion and health. This is a testament to the non-damaging quality of multiphoton microscopy.

One prominent theme of research that is under taken using the Columbia microbeam is damage signal transduction, both within cells and between cells. While early inter-cellular signal transduction studies were done with cells plated in 2D monolayers, there is an increased interest in irradiation of in-vivo 3D tissue systems. Our multiphoton microscope allows us to use the RARAF microbeam for in vivo microbeam studies. Researchers at RARAF are now irradiating small, living organisms, such as the C. elegans nematode. Of the various small-animal systems suggested by our users, we are developing microbeam support for the following three well-characterized and widely-used in-vivo systems.

C. elegans

The advantages of C. elegans as a research tool are well established, being a multicellular eukaryotic organism that is simple enough to be studied in great detail. From a practical perspective it is small enough to be compatible with microbeam irradiation and a wide variety of mutants and transgenics are readily available, as is a large community of C. elegans researchers. C. elegans is particularly well adapted to microbeam irradiation due to its thin body, allowing particle penetration to targets throughout the entire animal with minimal beam scattering. The abundance of genetically modified worms with fluorescent markers provides an easy way to target individual organs and quantify the required endpoint. Currently we can irradiate either chemically anesthetized C. elegans worms, in our standard microbeam dish, or mechanically constrained worms in a microfluidic channel.

The first RARAF microbeam irradiation studies with C. elegans were recently published. This study used C. elegans strain SJ4005 hsp-4::gfp(zcls4)V, which has a GFP reporter for the hsp-4 heat-shock gene. Heat shock proteins (HSPs) are a ubiquitous family of gene products present in cells under unstressed conditions that are expressed in much higher concentration in the presence of stress. HSP responses can be induced by diverse stressing agents including heat, UV irradiation, γ-rays irradiation and chemicals.

In this study, young adult C. elegans hermaphrodites were used. Before exposure, worms were individually imaged using an epifluorescent microscope. For microbeam irradiations, worms were anesthetized and placed in a customized microbeam dish with a micro cover-slip for individual exposure. Animals were irradiated at the tail, in the center of the GFP expression region, with 0, 25, 50, and 75 3-MeV protons (LET 12.5 keV/µm, max. penetration 140 µm). The microbeam diameter was 1 µm. (This is indicated by a red dot on the figure below). Control worms were mock-irradiated, by targeting the microbeam just outside the worm (~ 200 μm) and using the same set-up time for anesthetic exposure and concentrations. After exposure, the micro cover-slips were removed using a micro aspirating pump, and worms were washed with buffer and re-cultured in standard agar/covered Petri dishes. Based on well established protein kinetic studies and our preliminary expression time studies, we selected a twenty four hour time point for GFP expression evaluation.

Microbeam exposed worms showed a different GFP stress response in terms of intensity and localization, compared with the control group. Increase in expression was noted only after delivering 50 and 75 protons. Mock irradiated worms did not exhibit any GFP stress response after 24 hours (Fig. a). No apparent stress response was detected when 25 protons were delivered (Fig. b). However, when the worms were exposed to 50 or 75 protons, a strong stress response in the posterior intestine was observed, between the spermatheca and the microbeam targeted area (Fig. c-d). A one micrometer diameter proton beam was able to induce tail region in situ GFP over-expression as well as distal stress response as far as >150 µm away from the irradiated spot. No stress response was seen in other regions of the body of exposed worms, and only basal levels of GFP expression were detected at the pharynx and spermathecae regions.

Sugimoto previously used wild type and a mutant strain C. elegans as an in vivo model for microbeam irradiation. However, in those experiments C. elegans were irradiated with a collimated ~ 20-50 µm diameter microbeam of 220 MeV carbon particles (LET of 120 keV/µm). Worms were exposed to 1500 carbon ions delivered to an extended worm body area. While these irradiation techniques are useful to study organ or region exposures, they do not allow the exposure of a restricted number of cells or small tissue sections in a living organism. Our technique is capable of delivering, with high accuracy, small numbers of particles at the sub-cellular and cellular level. Many phenomena under study using microbeams, such as the bystander phenomenon, must involve cell-to-cell communication, extracellular environment, and functional integrity. The capability to target individual tissue components is quite compelling for such studies.

We show that low doses of protons delivered at the tail region, where different organs and cell populations are located, are capable of inducing both local and distal GFP over-expression. This response is remarkable since the microbeam used is estimated to traverse a series of spatially co-localized cell structures. How this spatially restricted stressor is capable of inducing a distal stress response is not understood. However, it is worth noting that among the organs potentially irradiated are the intestine and the stomato-intestinal muscle. Cell-cell communications as well as tissue damage signaling are potential mechanisms involved in these responses. Belyakov et al suggested that autocrine/paracrine mechanisms or juxtacrine signaling are potential bystander pathways to explain long range bystander phenomena in complex tissues. Future studies will be focused on the identification of cell targets and signal mechanisms using different biological endpoints.

Use of this targeted irradiation method provides a new tool to investigate complex long-range biological responses such as the bystander effect in living organisms. Future developments include a microfluidics-based worm clamp for microbeam irradiation without anesthesia as well as microbeam modulation techniques to adjust the position of the Bragg peak to different locations within the target organism.

More detail can be found in A. Bertucci, R.D. Pocock, G. Randers-Pehrson, and D.J. Brenner. Microbeam irradiation of the C. elegans nematode. J. Radiat. Res. (Tokyo) 50 Suppl A: A49-54 (2009).

Medaka Fish Embryos

The medaka (Oryzias latipes) is a small egg-laying freshwater fish, which is increasingly being used as model system for developmental genetics and evolutionary biology. The medaka embryo is particularly well suited for microbeam studies, due to its small size and optical clarity for imaging internal tissues and organs. The medaka genome has recently been sequenced, and many transgenics and mutants are now available. We have focused on the medaka rather than zebrafish, because it is somewhat more hardy, but essentially all the irradiation protocols are directly applicable for zebrafish.

We have incorporated an online system for Medaka embryo orientation into the microbeam end station. This allows targeting organs, by using their position related to prominent physiological features (e.g. the eyes), which are easily visible using our microbeam imaging system.

Mouse Ears

We are currently in the process of developing a system for irradiating hairless mouse ears, as an in-vivo skin model. The mouse will be anesthetized and the ear flattened in a custom designed fixture, using light suction. The ear can then be presented to the microbeam for irradiation.

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