Jesse D. McMullen

Feasibility of using multiphoton excited tissue autofluorescence for in vivo human histopathology.

Rapid and direct imaging of microscopic tissue morphology and pathology can be achieved by multiphoton imaging of intrinsic tissue fluorophores and second harmonic signals. Engineering parameters for developing this technology for clinical applications include excitation levels and collection efficiencies required to obtain diagnostic quality images from different tissue types and whether these levels are mutagenic. Here we provide data on typical average powers required for high signal-to-noise in vivo tissue imaging and assess the risk potential of these irradiance levels using a mammalian cell gene mutation assay. Exposure times of ~16 milliseconds per cell to 760 nm, ~200 fs raster-scanned laser irradiation delivered through a 0.75 NA objective produced negligible mutagenicity at powers up to about 50 mW.

Non-ionic photo-acid generators for applications in two-photon lithography

Non-ionic photoacid generators (PAGs) have been designed and synthesized for use in two-photon lithography (TPL). The chromophores in these new PAGs are covalently linked to the photocleavable group by a flexible joint. Their thermal stability, solubility and efficiency to produce acid under both one- and two-photon excitation were characterized. The potential of these PAGs for TPL was tested in two negative-tone resist systems relying on different mechanisms: free-radical/cationic polymerization or a cationically initiated cross-linking reaction. These PAGs needed lower threshold power for polymerization compared to a commercially available photoinitiator, isopropylthioxanthone, and a photoacid generator, N-hydroxynaphthalimide triflate. Microstructures with a resolution of 0.6 µm were fabricated and the threshold power for polymerization was found to be below 2 mW.

Comparison of objective lenses for multiphoton microscopy in turbid samples.

Optimization of illumination and detection optics is pivotal for multiphoton imaging in highly scattering tissue and the objective lens is the central component in both of these pathways. To better understand how basic lens parameters (NA, magnification, field number) affect fluorescence collection and image quality, a two-detector setup was used with a specialized sample cell to separate measurement of total excitation from epifluorescence collection. Our data corroborate earlier findings that low-mag lenses can be superior at collecting scattered photons, and we compare a set of commonly used multiphoton objective lenses in terms of their ability to collect scattered fluorescence, providing guidance for the design of multiphoton imaging systems. For example, our measurements of epi-fluorescence beam divergence in the presence of scattering reveal minimal beam broadening, indicating that often-advocated over-sized collection optics are not as advantageous as previously thought. These experiments also provide a framework for choosing objective lenses for multiphoton imaging by relating the results of our measurements to various design parameters of the objectives lenses used.

Enhancing collection efficiency in large field of view multiphoton microscopy

Many multiphoton imaging applications would benefit from a larger field of view; however, large field of views (>mm) require low magnification objectives which have low light collection efficiencies. We demonstrate a light collection system mounted on a low magnification objective that increases fluorescence collection by as much as 20‐fold in scattering tissues. This peripheral detector results in an effective numerical aperture of collection >0.8 with a 3–4 mm field of view.

A multiphoton objective design with incorporated beam splitter for enhanced fluorescence collection

We present a de novo design of an objective for use in multi-photon (MPM) and second harmonic generation (SHG) microscopy. This objective was designed to have a large field of view (FOV), while maintaining a moderate numerical aperture (NA) and relative straight forward construction. A dichroic beam splitter was incorporated within the objective itself allowing for an increase in the front aperture of the objective and corresponding enhancement of the solid angle of collected emission by an order of magnitude over existing designs.