Microscope Provides Precise 3D Imaging of Thick Mammalian Cells

Facebook X LinkedIn Email
A microscope developed by researchers at Stanford University produces 3D nanoscale images of mammalian cells in their entirety. 

TILT3D — tilted light-sheet microscopy with 3D point spread functions (PSFs) — combines a tilted light-sheet illumination technique with long axial range PSFs for low-background, 3D super-localization of single molecules and for 3D superresolution imaging in thick cells. Because the axial positions of the single emitters are encoded in the shape of each single-molecule image rather than in the position or thickness of the light sheet, the light sheet need not be extremely thin.

TILT3D microscope produces 3D images in nanoscale, Stanford  University.
This super-resolved 3D reconstruction of the entire nuclear lamina of a mammalian cell was acquired using TILT3D. Scale is in micrometers. Courtesy of Anna-Karin Gustavsson, Moerner Laboratory.

Tilted light-sheet illumination is used to address issues with image distortion that can occur with techniques that illuminate the cell sample from below. By tilting the illumination plane, this imaging technique allows for sectioning and imaging of cells all the way down to the coverslip. 

The microscope uses an optical method for imaging in 3D. The research team from Stanford tagged molecules in cell samples with chemicals that fluoresce when lit, and they used chemical additives to make the molecules blink. The team adjusted the microscope to convert each blink into two spots of light at different angles. The light spots are used to acquire the position of each molecule in 3D.

By stacking pancaked 3D images on top of one another, the researchers can create a top-to-bottom reconstruction of a cell. The implementation of the simple tilted light sheet in combination with PSF engineering significantly improves the localization precision of single molecules, making it possible to track the movement of molecules over time in 3D with a precision of tens of nanometers.

To validate TILT3D for 3D superresolution imaging in mammalian cells, researchers imaged mitochondria and the full nuclear lamina using the double-helix PSF for single-molecule detection and the tetrapod PSF for fiducial bead tracking and live axial drift correction.

The researchers are now walking other labs through the process of duplicating the TILT3D. The design can be a modular addition to existing light microscopes; TILT3D is built on a standard inverted microscope and has minimal custom parts.

Professor W. E. Moerner, left, and postdoctoral scholar Anna-Karin Gustavsson position a sample on the new TILT3D microscope. Stanford University.
Professor W.E. Moerner, left, and postdoctoral scholar Anna-Karin Gustavsson position a sample on the new TILT3D microscope. Courtesy of L.A. Cicero/Stanford News Service.

“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 researcher Anna-Karin Gustavsson. “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. The microscope could open up new opportunities to produce detailed 3D images of mammalian cell structures, even of cells that were previously too dense to image clearly.

The TILT3D was developed in the lab of W.E. Moerner, who in 2014 won the Nobel Prize in chemistry for codeveloping a way to image shapes inside cells at very high resolution, called superresolution microscopy.

The research for this article was published in Nature Communications (doi: 10.1038/s41467-017-02563-4).

Published: March 2018
Superresolution refers to the enhancement or improvement of the spatial resolution beyond the conventional limits imposed by the diffraction of light. In the context of imaging, it is a set of techniques and algorithms that aim to achieve higher resolution images than what is traditionally possible using standard imaging systems. In conventional optical microscopy, the resolution is limited by the diffraction of light, a phenomenon described by Ernst Abbe's diffraction limit. This limit sets a...
Nanopositioning refers to the precise and controlled movement or manipulation of objects or components at the nanometer scale. This technology enables the positioning of objects with extremely high accuracy and resolution, typically in the range of nanometers or even sub-nanometer levels. Nanopositioning systems are employed in various scientific, industrial, and research applications where ultra-precise positioning is required. Key features and aspects of nanopositioning include: Small...
An SI prefix meaning one billionth (10-9). Nano can also be used to indicate the study of atoms, molecules and other structures and particles on the nanometer scale. Nano-optics (also referred to as nanophotonics), for example, is the study of how light and light-matter interactions behave on the nanometer scale. See nanophotonics.
Research & TechnologyeducationAmericasImagingMicroscopysuperresolutionNanopositioningmedicalBiophotonics3D imagingnanolight-sheet microscopyBioScan

We use cookies to improve user experience and analyze our website traffic as stated in our Privacy Policy. By using this website, you agree to the use of cookies unless you have disabled them.