Selene Flemming

I am a transgender first year GSBS student who uses she/her pronouns.

I love all things microscopy and have started development of Cryo-5D (xyzθλ), a cryogenic super resolution fluorescence microscope for correlated light and electron microscopy (CLEM). It will perform single molecule localization microscopy (SMLM) at 10E-7 torr and 80K and will be able to detect the azimuthal orientation (θ) of single fluorescent proteins via polarization modulation/demodulation and linear dichroism. Using a high numerical aperture reflective objective and off axis parabolic relay mirrors with astigmatic blazed grating point spread function (PSF) engineering, it is our hope to achieve under 20nm lateral (xy) and 50nm axial (z) localization precisions with 10nm spectral (λ) resolution and zero chromatic aberration. 

To combat devitrification due to the high intensity laser illumination typical of SMLM, a digital micromirror device (DMD) will be used to selectively photobleach sparse fluorophores back to their dark state while limiting heat absorption in the sample. In addition to encoding the PSF with axial and spectral information, a liquid crystal phase spatial light modulator will also adaptively compensate for aberration imparted by the reflective objective’s secondary mirror obscuration and non planarity of cryo-EM samples. A dedicated tracking camera will be employed to compensate for thermal drift in three dimensions. The Cryo-5D microscope has been designed to image Autogrids on the Aquilos fib shuttle, allowing for quick and contamination free transfer between instruments in the cryo-CLEM workflow.

I am conducting this research jointly in the Grigorieff lab and Grunwald lab.