Nanoscale View on Mammalian Cells

This super-resolved 3D-reconstruction of the entire nuclear lamina of a mammalian cell was acquired using TILT3D. (Source: A.K. Gustavsson, Moerner Lab.)

In 2014, W. E. Moerner, the Harry S. Mosher Professor of Chemistry at Stanford Univer­sity, won the Nobel Prize in chemistry for co-deve­loping super-reso­lution micro­scopy. Now, he and his lab have created a new micro­scope that produces 3D nanoscale images of mamma­lian cells in their entirety. “A cell has a whole city of proteins, enzymes and structures working all the time,” Moerner said. “We have some idea of what’s in a cell – many of us are familiar with drawings of mito­chondria or of the endo­plasmic reticulum – but it’s an average idea. When we look at indi­vidual cells, we recognize that they all aren’t exactly like the pictures we have in text­books.”

The new micro­scope, TILT3D, combines two new imaging tech­niques with super-reso­lution micro­scopy to capture very clear 3D images of structures and indi­vidual molecules within a cell. One of the two new techniques, tilted light sheet illu­mination, addresses problems with focus and func­tionality that occur with existing illu­mination techniques. In most light micro­scopes, the cell sample is lit from below. “This is a problem if you want to inves­tigate the details of a cell because it leads to visually hazy images where only some parts are in focus – like a photo taken over a long distance,” said Anna-Karin Gus­tavsson, a post­doctoral fellow in the Moerner lab.

Standard light sheet illu­mination gets around this problem by shining only a slice of light in from the side to obtain a pancake-like illumi­nation of the sample. Even with this advan­tage, if you try to get a light sheet to shine on the very bottom of a cell, it bounces off the corner of the chamber con­taining the sample, which distorts the image. By tilting the light sheet, the Moerner lab avoids hitting the corner.

In addi­tion to clearing the visual clutter by tilting the light sheet, the new micro­scope includes an optical method for imaging in 3D. To achieve this, the researchers tag molecules in the cell sample with chemicals that fluoresce when lit and use chemical addi­tives to make them blink brightly. Then, the group adjusts the micro­scope to convert each fluorescent blink into two spots of light at different angles. With these two spots, the researchers can get the position of each molecule in three dimen­sions, which informs the final 3D image.

Stacking their pancaked 3D images on top of one another, the researchers can create a top-to-bottom recon­struction of a cell. Tilted light sheet imaging also makes it possible to track the 3D movement of molecules over time with a preci­sion of tens of nano­meters, which could capture molecules bonding, moving by motors or traveling randomly through structures of the cell. Combining TILT3D’s clear image and 3D capabi­lities with existing super-reso­lution techniques, the micro­scope can create precise images at super-reso­lution. This opens up new oppor­tunities to produce detailed 3D images of mammalian cell structures, even of ones that were pre­viously too dense to image clearly.

In addition, Moerner and his lab members success­fully tested their micro­scope on known cellular structures. They are already walking other labs through the process of dupli­cating this micro­scope. The design can be a modular addi­tion to existing light micro­scopes. In the future, they hope that their 3D tilted light sheet illumi­nation imaging will be used for any number of projects. “TILT3D is simpler than other micro­scopes that have been designed for imaging of these chal­lenging samples, and it can be used for imaging both of static structures and of moving molecules” said Gustavs­son, who is partly suppor­ted by a post­doctoral fellowship from the Karo­linska Insti­tutet in Sweden. “We designed it to be versatile, not bound to a specific question.”

The researchers will continue to work on TILT3D, parti­cularly on combining static and dynamic infor­mation from several different proteins. Along­side their many other inno­vations and studies in cellular imaging, they hope this tech­nology can enable them and others to learn more about the structures and processes of cells, one molecule at a time. (Source: Stanford U.)

Reference: A.-K. Gustavsson et al.: 3D single-molecule super-resolution microscopy with a tilted light sheet, Nat. Commun. 9, 123 (2018); DOI: 10.1038/s41467-017-02563-4

Link: Moerner Lab, Stanford University, Stanford, USA

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