Time-lapse two-color 3D imaging of live cells with doubled resolution using structured illumination

Abstract

Previous implementations of structured-illumination microscopy (SIM) were slow or designed for one-color excitation, sacrificing two unique and extremely beneficial aspects of light microscopy: live-cell imaging in multiple colors. This is especially unfortunate because, among the resolution-extending techniques, SIM is an attractive choice for live-cell imaging; it requires no special fluorophores or high light intensities to achieve twice diffraction-limited resolution in three dimensions. Furthermore, its wide-field nature makes it lightefficient and decouples the acquisition speed from the size of the lateral field of view, meaning that high frame rates over large volumes are possible. Here, we report a previously undescribed SIM setup that is fast enough to record 3D two-color datasets of living whole cells. Using rapidly programmable liquid crystal devices and a flexible 2D grid pattern algorithm to switch between excitation wavelengths quickly, we show volume rates as high as 4 s in one color and 8.5 s in two colors over tens of time points. To demonstrate the capabilities of our microscope, we image a variety of biological structures, including mitochondria, clathrin-coated vesicles, and the actin cytoskeleton, in either HeLa cells or cultured neurons.extended resolution | frequency mixing | multicolor | patterned excitation F luorescence microscopy allows noninvasive 3D imaging of the interior of living specimens with molecular specificity, and is therefore an invaluable resource to the biological sciences. Unfortunately, the resolving power of fluorescence microscopy is fundamentally limited by the diffraction of light.Lately, many techniques have been introduced to extend the resolution beyond the classic diffraction limit (1-8). Although the improvement of spatial resolution is impressive, the practical impact of these new techniques will largely depend on whether they can keep the two key advantages of fluorescence microscopy, namely, the capability of imaging living cells in three dimensions and multicolor labeling. Live-cell imaging over many time points has been demonstrated for some resolution-extending techniques (9-15) but, to our knowledge, not for multicolor 3D imaging of whole cells.Stimulated emission depletion (STED) microscopy increases the spatial resolution by suppressing the fluorescence emission on the rim of a focused laser spot, theoretically enabling unlimited resolution (16). STED microscopy has been demonstrated for live imaging at high frame rates (12, 15), but its point-scanning nature makes it unsuitable for fast volumetric imaging over large fields of view. Furthermore, to achieve high spatial resolution, very high power densities (38-540 MW/cm 2 ) (12) are necessary for the STED laser beam, which may cause increased photobleaching and phototoxicity, thus limiting the application of live-cell STED microscopy.In localization-based microscopy, only sparse subsets of photoswitchable fluorophores in a sample are imaged at a time, which allows the isolation and precise localizati...

A Guide to Structured Illumination TIRF Microscopy at High Speed with Multiple Colors

“…The first in vivo images obtained with TIRF-SIM were reported in 2009 16 achieving frame rates of 11 Hz to visualize tubulin and kinesin dynamics, and two color TIRF-SIM systems have been presented 17,18 . Most recently, a guide for the construction and use of a single color two-beam SIM system was presented featuring frame-rates of up to 18 Hz 19,20 .…”

Saturated excitation of fluorescent proteins for subdiffraction-limited imaging of living cells in three dimensions

“…The key element common to these super-resolution techniques for breaking the diffraction limit is the use of the photoswitching property of fluorophores, which is realized by using various optical phenomena such as stimulated emission, cis-trans isomerization, triplet pumping [14] and saturated excitation (SAX) [15][16][17]. Structured illumination microscopy (SIM), while not breaking the confocal microscopy diffraction limit, is also well known as a high-resolution imaging technique which doubles the spatial resolution by overcoming the diffraction limit in classical wide-field fluorescence microscopy [18,19].…”

Super‐Resolution Microscopy: Going Live and Going Fast

“…The imaging speed in SIM is limited by the speed with which the illumination pattern can be modulated and the camera speed [8] . A temporal resolution of 100 ms in 2D [9] , 4 s in 3D [10] and 8.5 s in multi-colour 3D [10] imaging (Figure 1) has been achieved in living cells at the 2-fold enhanced spatial resolution. The achieved spatiotemporal resolution allowed observation of clathrin coated vesicle dynamics such as splitting and fusion events as well as clathrin mediated endocytosis (Figure 1).…”