Multi-Photon Imaging

RARAF microbeam users are increasingly using and planning to use 3-D tissue samples and small organisms. To image their 3-D samples, our users require the multiphoton microscope that we developed and fully integrated into our main electrostatic-lens Microbeam II endstation at RARAF. A custom-design for the multiphoton microscope was necessary, given the geometrical constraints of the pre-existing microscope fitted at the terminus of the vertical ion beamline. Intended for detecting and observing short-term molecular kinetics of radiation responses in living tissue and in cell-culture samples, the multiphoton microscope at RARAF is the first of its kind to be assembled and implemented onto a microbeam cell-irradiation platform.

Shown above are multiphoton microscopy images of HUVEC tissue samples and a c.elegansnematode through fluorescence, autofluorescence (AF) and second harmonic generation (SHG). The cell nuclei, are stained with YOYO-1. Imaging modes: Cell nuclei imaged by YOYO-1 (green) and cell cytoplasm imaged with AF (blue); Cell nuclei imaged by YOYO-1 (green), cell cytoplasm imaged with AF (blue), and collagen imaged by SHG (red). The c. elegans is an image of the pharynx section of a wild-type specimen. SHG (red) and AF (blue) were used to produce this image.

While the multi-photon microscope is integrated into the Microbeam II end station, it is available as a stand-alone system when there are no irradiation experiments requiring the other integrated facilities.

Microbeam Imaging and Targeting of 3D Structures

The 3D nature of cells and the pencil-beam nature of microbeam charged-particle tracks need to be considered in all microbeam irradiations, and indeed this has been a constant theme from the outset of modern microbeam systems. Different users approach this issue in a variety of different ways. For example, some users use highly flattened cells achieved with optimized plating techniques, or targets can be isolated in two dimensions by cell-cycle specificity – for instance, CP2 targets a specific chromosome that is condensed at metaphase with limited overlap with other chromosomal domains.

Another approach used at RARAF and illustrated here is to use 3D imaging to “see” what is above and below the target locations, and to use this information to choose microbeam irradiation locations. For example, the image shows a 3D multi-stack image of the mitochondria of a target cell, obtained with our multi-photon microbeam microscope; the upper image is a z max-projection of the 3D image and the lower image is an x-z cross-sectional plane of the cell through the sub-cellular targets. Given such information, users can, in real time, designate microbeam locations.

For example, targeting a mitochondrial location above the nucleus (Track a); traverse the nucleus but hit no mitochondrial locations (Track b); traverse the cytoplasm but miss all mitochondrial locations (Track c), or traverse the cytoplasm and target a mitochondrial location (Track d).