The 100-fold mismatch in size between the resolution of an optical microscope and the length scales of biomolecular complexes hinders direct observation of subcellular phenomena. Imaging beyond the diffraction limit is thus of great practical interest in cell biology, and the last decade has seen an explosion in ‘super-resolution’ optical imaging techniques1
. Despite these advances, long-term live imaging of thick samples still presents challenges for such methods.
Photoactivated localization microscopy (PALM)2
and related single-molecule imaging techniques3,4
enable imaging with lateral resolutions down to 20 nm (sub-100 nm axially) on 3D samples such as whole fixed cells5,6
and much thicker cellular spheroids7
. While a few live cell experiments have been performed at slow imaging rates over extended durations8
and at rates up to 2 Hz for very short periods9
, the current requirements for ~kW/cm2
excitation intensities and thousands of frames have confined most efforts to fixed cells.
Stimulated emission depletion microscopy10
(STED) enables cellular imaging with ~20-100 nm resolution11
. STED has also been applied to thicker samples12,13
, although so far only providing 2D super-resolution. STED resolution scales with illumination intensity, constraining the choice of dyes and entailing a compromise between spatial resolution and phototoxicity in live experiments.
In structured illumination microscopy (SIM)14,15
, spatially patterned light is used to excite sample fluorescence. Frequency-mixing between the excitation pattern and fluorophore density moves normally unobservable high resolution information in the sample into the observable passband of the microscope. By varying the pattern orientation and phase, recording the resulting fluorescence, and appropriately post-processing the resulting multi-image datasets, it is possible to obtain images with ~100 nm lateral and ~300 nm axial resolution16
for linear SIM. While this resolution is more modest than that of PALM or STED, SIM offers other advantages. The number of raw images required for a single SIM image is far fewer than in PALM, and the illumination intensity is far less than in STED. These benefits allow increased imaging speed and duration, and enable live SIM at 11 Hz in 2D17
and 0.2 Hz in 3D on whole cells18
for hundreds of time points. Linear SIM is also compatible with the full array of conventional fluorescent dyes, unlike PALM or STED.
In addition to resolution-doubling, 3D SIM offers another major advantage over conventional widefield imaging: computational optical sectioning (the removal of out-of-focus blur). While highly effective on thin samples, SIM retains the shot noise associated with the computationally removed background, and is unsuitable for thick or very densely-labeled samples. Confocal microscopy physically rejects out-of-focus light with a pinhole, and provides higher contrast images in thick samples. Confocal microscopes, however, provide at best a √2 improvement in lateral resolution with a small pinhole, and this enhanced resolution is difficult to attain as the corresponding loss in signal is prohibitive.
We present a hybrid technique, multifocal SIM (MSIM), that combines the resolution-doubling characteristics of SIM with the physical optical sectioning of confocal microscopy. MSIM uses sparse 2D excitation patterns generated with a digital micromirror device (DMD) integrated into a conventional wide-field microscope and digital post-processing to obtain optically-sectioned images with ~145 nm lateral and ~400 nm axial resolution at 1 Hz frame rates. Relative to existing SIM, our implementation is easier to integrate onto existing microscopes and is considerably cheaper than commercial SIM. We obtain dual-color, volumetric images of whole fixed cells, and extend SIM to live samples 8-fold thicker than previous experiments on whole cells. We apply 4D MSIM to study the evolution of the posterior lateral line primordium in live zebrafish embryos and also obtain multicolor, 4D super-resolution datasets on cells embedded in collagen gels.