STED Nanoscope – MS Thesis

The use of optical fiber to deliver STimulated Emission Depletion (STED) patterned light to achieve super-resolved images has been an objective of nanoscopy. The ability to do so would represent a new modality for the delivery and use of STED nanoscopy in medical applications. My MS Thesis research demonstrates the successful ability of Endlessly Single Mode (ESM) fiber to deliver excitation and STED ‘doughnut’ patterning to 100nm fluorescent nano-bead samples using ESM fiber optics.

Thesis available here.

Simplified Jablonski diagram of electron energy shell orbitals showing the interaction of an excitation photon (blue) with the ground state energy levels (S0), the excitation of the electron (yellow arrow) to the excited state (S1), thermal relaxation (purple line), and the de-excitation of the electron (red arrow) back to the ground state with the emission of a lower energy photon (green).

Gold-Bead Images. A) X-Y planar images showing the STED beam (top) and the excitation beam (bottom). B) X-Z plane images showing the STED beam (top) and the excitation beam (bottom). C) Y-Z plane images showing the STED beam (top) and the excitation beam (bottom). D) Overlapped contrast and brightness enhanced images of the excitation (blue) and STED (red) beams. The maxima/minima of the frequencies overlap within 11.178 nm in both axes. Length scales are as indicated. Heat color scale is in volts collected from a photo-multiplier tube. Both excitation and STED images overlap well.

100μm Florescent Bead Images: A) X-Y planar images showing 100nm fluorescent bead. F) X-Z plane images showing 100nm fluorescent bead. E) Y-Z plane images showing 100nm fluorescent bead imaged using the ESM fiber with the STED light off. Length scales are as indicated. Heat Scale is in photon counts collected from an avalanche photo diode (APD).

Full schematic of the generation and detection apparatus. Pre-fiber laser alignment and collection. Post fiber optics and sample stage.

STED nanoscopy has expanded into many area of biological imaging since its inception. The ability to detect fluorophores in near real time with nanometer accuracy has numerous applications in the realm of biology where the sizes of proteins and other biologically relevant molecules are diffraction limited. Many STED systems are very large in size and take up entire vibration isolation tables with very finicky optical elements. These requirements have thus far limited the use and wide adoption of STED nanoscopy in research. Using a fiber to deliver the light sources and then collect the resultant fluorescent light will help to alleviate the space and size restraints of current STED systems. My Masters research work demonstrates the practical implementation and components of a fiber based STED system that can achieve super-resolution.