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Unique System Features

  • Combines multiple imaging modalities in a single instrument (see below)

  • Multiplex different imaging modes in a single experiment and can swap between modalities in seconds

  • Achieves previously established benchmarks of all imaging modes (e.g. spatial resolution, light sheet thickness) in a single instrument

  • Extended light sheet imaging tile size (220 um)

  • Contains light sheet, inverted, and upright imaging objectives

  • Uses a 25 mm coverslip

  • Coverslip "quick scan" for optimal sample finding

  • Temperature-controlled sample chamber with CO2 gas control

  • Seven illumination wavelengths

  • Simultaneous two-color or sequential multi-color illumination for all imaging modes

  • Adaptive-optics correction (wavefront sensing) for both excitation and emission light paths for all imaging modes

  • Two sCMOS cameras for multiple color (simultaneous) imaging, FRET, and spectra separation

Multi-Modal Imaging

  • Lattice light sheet

  • Expansion microscopy (ExM) imaging with lattice light sheet

  • 3D structured illumination super resolution

  • Multifocal image scan/pixel reassignment super resolution

  • Two photon (2P) point scan

  • 2P Bessel fast functional imaging (upright objective)

  • 2P Bessel light sheet

  • 2P Bessel point scan

  • Phase imaging

  • Patterned photostimulation

  • 3D single-molecule localization

System Specifications

Available Lasers (in nm):

  • 405 (Oxxius diode laser, rated 100 mW)

  • 445 (Oxxius diode laser, rated 100 mW)

  • 488 (MPB fiber laser, rated 500 mW)

  • 514 (MPB fiber laser, rated 1 W)

  • 560 (MPB fiber laser, rated 1 W)

  • 607 (MPB fiber laser, rated 1 W)

  • 642 (MPB fiber laser, rated 2 W)

  • Custom Chameleon 2P laser (900 nm - 1050 nm)


Objectives:

  • Light Sheet Excitation: Special Optics 0.65 NA, 3.74 mm working water dipping lens

  • Light Sheet Detection/Inverted/Upright: Zeiss 1.0 NA water-dipping objectives with 2.2 mm working distance


Cameras

  • 2x Hamamatsu Orca Flash 4.0 v3 sCMOS

Limitations

Although a novel and powerful tool for biomedical imaging, the scope does have limitations.

  • It can take several seconds to change imaging modes; this has to be accounted for when planning complex, high temporally resolved experiments.

  • Simultaneous multi-color imaging mode is limited to two colors.

  • Although capable of performing more conventional "cell on a coverslip" volumetric imaging, these experiments are better suited to the LLSM (more info here).

  • 2P imaging is done with a custom Chameleon laser and has a more limited wavelength range than 2P systems found on commercial instruments.

Instrument Summary

At the leading edge of microscopy is the quest to capture biology as it occurs in its natural environment. It is clear that for decades studies have yielded excellent information from interrogating cells on a coverslip, but truly understanding biological systems requires observation in more natural conditions. The advent of modern fluorescent light sheet microscopy has made it possible to capture live biology with much less perturbation due to laser light intensity. This also improved diffraction-limited image quality by only illuminating within the focal depth of the detection objective, thereby improving signal-to-background [1,2]. However these instruments still have limitations. Light experiences significant distortions when going through thick (>100 um) tissues; this causes unacceptable image degradation. 

Recently, the Betzig lab at HHMI Janelia coupled the principles of adaptive optics with lattice light sheet microscopy [3]. By using a 2P laser spot (a "guide star") that is focused in the imaging volume, wavefront sensing allows for quantification of the aberration the light experiences. This aberration can then be corrected by a deformable mirror (emission correction) or adjusting the shape of the incoming light with a spatial light modulator (excitation correction). This creates an instrument that is capable of gentle illumination, high contrast imaging, and improved imaging penetration depth. With the new Multimodal Optical Scope with Adaptive Imaging Correction (MOSAIC), the Betzig lab has achieved integration of adaptive optics with many imaging modes on the same instrument. Users can take advantage of multi-modal imaging to, for example, interlace super-resolved 3D-SIM images with long-term volumetric, gentle imaging with LLSM, using adaptive optics correction in both modes. Additionally, with simultaneous two-color imaging in all modes and large tile sizes, volumes can be captured several times faster than conventional, sequentially scanned imaging modalities. Users interested in leveraging the multi-modal nature of this instrument must discuss their project with the AIC team prior to submitting a proposal.

Suggested Reading

  1. Planchon, T. A., et al. Rapid three-dimensional isotropic imaging of living cells using Bessel beam plane illumination. Nat. Methods 8, 417–23 (2011).

  2. Chen, B.C., et al. Lattice light-sheet microscopy: imaging molecules to embryos at high spatiotemporal resolution. Science 346, (2014).

  3. Liu, T.-L., et al. Observing the cell in its native state: Imaging subcellular dynamics in multicellular organisms. Science 20, (2018).