Single-molecule localization microscopy and applications to visualize the accessible genome with ATAC-see

It has been thirty years since the first experimental observation of a single molecule, and thirteen years since the first few single-molecule super-resolution fluorescence microscopy methods were first published. Since these two key milestones, scientists have been developing and applying new optical methods to better understand the world around us, whether it is in crystals at cryogenic temperatures or in the crowded environment within living cells. In single-molecule localization microscopy, we first use fluorescent molecules to label the biological structure we are interested in studying. Next, instead of imaging all the fluorescent molecules at the same time in a single camera frame, we can use chemical means to make the molecules stochastically blink on and off. In this manner, instead of separating the overlapping fluorescent spots (or point spread functions) from each single emitter in space, we can now separate them temporally. Next, we can perform a process called superlocalization to measure their precise molecular coordinates in each camera frame, before recombining them spatially to create a super-resolution reconstruction. In my dissertation, I will describe how I have developed new capabilities for single-molecule localization microscopy and how I combined our super-resolution imaging methods with a new accessible chromatin labeling scheme to try to image the accessible genome in the nucleus. First, I will describe my attempts in using an engineered point spread function (PSF) called the corkscrew PSF to image labeled structures in 3D, but with a faster acquisition rate with a higher density of emitters per camera frame and fewer overlapping PSFs as compared to other engineered PSFs. From my work, I found that we had to use real-time Z drift correction to ensure that the amount of spherical aberration in our optical systems do not change while imaging a sample. A varying amount of spherical aberration changes the behavior of the corkscrew PSF over the imaging period which makes it challenging to accurately and precisely measure the locations of the emitters. Second, I will describe how I have fabricated a transmissive fixed dielectric phase mask that encodes for the Tetrapod PSF for use in our 4f optical systems. Microfabrication is a process that has to be done properly to create a functioning end product. I have tried to describe it with as many details as possible in Chapter 4. The fixed phase masks that I fabricated encode for a Tetrapod PSF that has an effective working axial range of 6 µm and work for emission wavelengths of 660 nm or 550 nm, and they have been used in multiple projects in the lab that resulted in several publications. Finally, I will describe how I have used our super-resolution imaging methods to visualize the accessible genome in the nucleus that has been labeled with a biochemical labeling method known as Assay of Transposase-Accessible Chromatin with visualization (ATAC-see). The two-meter long human genome is heavily compacted into a ten-micron-wide nucleus. But a part of the genome has to remain accessible to the biomolecular machinery that carry out gene regulation, transcription, and DNA repair. It would be useful if we could image the accessible genome within the nucleus and compare this map of accessible chromatin with other maps of labeled biomolecules. ATAC-see labels accessible chromatin in the nucleus with fluorophores. We can image the labeled fluorescence distribution with single-molecule localization microscopy to obtain super-resolution images of the underlying structure of accessible chromatin in the nucleus. This project had many twists and turns but I did make several interesting observations that may help to improve the ATAC-see labeling method so that future scientists may be able to accurately and reproducibly image the accessible genome in the nucleus. One observation was that ATAC-see appears to preferentially label the periphery of the nucleus. Another observation is that the Tn5 monomers do not readily dimerize after binding to the fluorescently-labeled dsDNA sequencing adapters. This greatly reduces the concentration of effective labels by about 30 times. Two ways to promote dimerization may be to use a peptide linker to connect two Tn5 monomers together or to connect the dsDNA sequencing adapters via extending the "reverse" strand.

Advisor: Moerner W. E. (William Esco)
Chang Howard Y. (Howard Yuan-Hao)
Greenleaf William James