RARAF Imaging Capabilities on the Microbeam II Charged-Particle Microbeam End Station

Fluorescent Imaging

The primary mode of imaging on the Microbeam II end station is through fluorescent imaging. We have an Acticure mercury lamp light-guide coupled to our Nikon Eclipse e600 microscope with a Princeton Instruments PhotonMAX:512B EMCCD camera for image acquisition. The primary fluorescent stain for our users is Hoechst 33342, a DNA binding stain, which allows rapid location of the cell nuclei to be hit, or not hit, as the experiment calls for during irradiation. This equipment combination also allows us to image the full range of fluorescent proteins that have been developed. We encourage our users to contact us prior to scheduling experiments to discuss the imaging needs for the experiment. We currently offer a broad range of filtering options and look forward to assisting our users by expanding those options as needed.

Multi-Photon Imaging

Multiphoton microscope image acquired during an irradiation experiment using mitochondria sites (green) as targets. The cross-hairs (red) mark the center of the image, which coincides with the position of the ion beam.

We have a custom multi-photon imaging system built into our Microbeam II end station. The multi-photon system works by focusing a laser through the microscope optics into a very small volumetric space in the sample on the end station. In that volume, the photon density gets high enough that two photons combine to act as a single photon with twice the energy (half the wavelength). If at that location there is a fluorophore that can be excited by that wavelength, a fluorescent signal can be measured. By scanning this multi- photon volume in x, y and z, it is possible to construct a 3D fluorescent image of the sample on the end station. The multi-photon imaging is also available as an observational imaging technique when the microbeam end station is not in use for an irradiation experiment.

More information about out Multi-Photon Imaging is available here.

Oblique Illumination Imaging

	Non-stained cells imgaed using oblique illumination.

Oblique illumination imaging uses an off-axis light source from above the sample, passing through the sample, reflecting off a surface back through the sample into the microscope object, and forms a contrast image at the camera. This non-stain imaging, producing images similar to Normaski of Hoffman modulation, is primarily used as a long term observation technique. It can be used for low throughput, manually targeted irradiation where staining the sample is not practical for the experiment outcome.

Simultaneous Immersion Mirau Interferometry (SIMI)

SIMI uses Mirau interferometry to construct an image from interference patterns generated by the topological differences between the cells and the polypropylene films. The images show iso-lines of similar height above the background surface. For plated cells, the centers of the iso-lines are overwhelmingly the cell nucleus. For irradiation targeting, the center-of-mass of the central iso-lines is chosen as the irradiation location.