Building a Wavelength-Tunable Two Photon Microscope to Image GFP and mEos2 Fluorescent Proteins, Poster 44

The goal of this project is to build a wavelength-tunable two-photon microscope capable of imaging different fluorescent proteins. A multiphoton microscope uses a focused laser beam to produce non-invasive images of biological... [ view full abstract ]

The goal of this project is to build a wavelength-tunable two-photon microscope capable of imaging different fluorescent proteins. A multiphoton microscope uses a focused laser beam to produce non-invasive images of biological tissue. By scanning the focus of the laser within the sample, high quality three-dimensional fluorescence images can be created. Two-photon microscopy differs from confocal fluorescence microscopy because the wavelengths of the two exciting photons are much longer than the wavelength of the emitted light. Compared to confocal microscopy, this technique allows for deeper tissue penetration and reduced photobleaching. As different fluorescent dyes and proteins require laser excitation at varying wavelengths, the aim of this project is to image the green fluorescent proteins eGFP (peak wavelength 950 nm) and mEos2 (peak wavelength 780 nm). The microscope contains dispersive elements in which different wavelengths travel at different speeds, and our microscope was originally designed to operate at a wavelength of 800 nm. When the wavelength of the laser is tuned to 950 nm, the beam passes through the prisms at a different angle and no longer reaches the sample. By calculating the wavelength-dependence of the prism geometry using Matlab, we can quickly adapt our alignment when changing wavelength so that we can image various biological specimens. Research reported in this presentation was supported by an Institutional Development Award (IDeA) from the National Institute of General Medical Sciences of the National Institutes of Health under grant number P20GM103449.