Lattice LightSheet produces an ultra-thin light sheet to achieve single-molecule sensitivity for live cell research. Invented by Nobel Laureate Dr. Eric Betzig of the Howard Hughes Medical Institute Janelia Research Campus, this microscope has been applied to biological systems spanning four orders of magnitude in space and time.
An extremely sensitive primary objective coupled with a custom-designed illumination system allows optical sectioning using extremely low light doses for imaging with unprecedented duration. 3D experiments previously limited to just seconds or minutes due to phototoxicity can now be continued safely for hours or days. The combination of high spatiotemporal resolution, speed and sensitivity make the Lattice LightSheet the ultimate tool in a new era for living cell microscopy.
The creation of the lattice light sheet is far more complex than sheet creation in typical SPIM methods. Many SPIM methods sweep an apertured laser beam, limiting the thinness and flatness of the sheet. Lattice LightSheet uses a sophisticated 4-step procedure to create an exceptionally thin and flat sheet, resulting in extremely efficient illumination of only the plane of interest.
1. Cylindrical lenses stretch the input beam to form a sheet
2. SLM generates an optical lattice of Bessel beams
3. Annular mask removes artifacts and lengthens the sheet
4. Galvos sweep the sheet in z and dither the sheet in x
mCherry Lifeact expressed in NK cells
GFP Lifeact expressed in K562 cells
Lytic synapse formation with 50ms exposures
Because the light is entering the specimen along the plane of focus, the likelihood of a useful fluorescence event is far higher than other methods that illuminate through the cell.
IMAGING: High resolution 1.1NA water immersion objective with depth of field perfectly matched to the light sheet thickness, generating excellent optical sectioning.
ILLUMINATION: A custom-made 0.71NA LWD water immersion objective with field of view and approach angle perfectly matched to the 1.1NA imaging objective.
0.4µm lightsheet thickness
0.5µm objective depth of field
SPIM: 3µm sheet thickness at 50µm length
LLS: 0.4µm sheet thickness at 50µm length
A Bessel beam lattice creates an ultra-thin 0.4µm sheet to evenly illuminate the entire plane of interest.
Fully enclosed, temperature controlled, medical grade stainless steel specimen chamber. Cells are mounted on a standard, horizontally-oriented 5mm round coverslip.
Peak light intensity is greatest in point-to-point scanning methods. Total light dose increases in methods that illuminate the entirety of the cell.
Two-color volume rendering seen from two different orientations of the interaction of a T-cell expressing mEmerald-Lifeact (orange) with a target cell expressing a plasma membrane marker fused to tagRFP (blue) over 430 time points at 1.3 sec intervals. The target cell has been made invisible in the views in the right column (cf., Fig. 5A-C for a second example).
Lattice LightSheet uses ultra-thin sheets of light to image 3D cellular dynamics for hundreds of volumes at dozens of frames per second at diffraction-limited resolution and super-resolution. Invented by Nobel Laureate Dr. Eric Betzig of the Howard Hughes Medical Institute Janelia Research Campus, this microscope has been applied to biological systems spanning four orders of magnitude in space and time. An extremely sensitive primary objective coupled with a custom-designed illumination system allows optical sectioning using extremely low light doses for imaging with unprecedented duration. 3D experiments previously limited by phototoxicity in just seconds or minutes can now be continued safely for hours or days. The combination of high spatiotemporal resolution, speed and sensitivity make the Lattice LightSheet the ultimate tool in a new era for living cell microscopy.
Early successful applications of this instrument include the diffusion of single transcription factors in a spheroid of stem cells, 3D dynamic instability of microtubules during mitosis, formation of the immunological synapse in T cell/antigen presenting cell (APC) interactions, neutrophil motility in a 3D matrix, and embryogenesis in C elegans and Drosophila melanogaster.
Lattice LightSheet incorporates two highly specialized objectives at right angles in an aqueous interface above the specimen. The 25x/1.1NA imaging objective is a water immersion lens with correction collar and a long 2mm working distance.
A custom-designed 0.71NA long working distance illumination objective was constructed to fit immediately adjacent to the imaging objective and perfectly illuminate the imaging field for maximum signal and resolution. A third objective is located below the specimen chamber and serves as eyepieces for sample location.
Illumination is highly specialized for optimal power delivery in an exceptionally thin light sheet. A high-speed spatial light modulator (SLM) in combination with an annular mask allows spatially confined optical lattices to be projected onto the sample. A galvo mirror controls lattice movement, either dithering to form a uniform sheet or discretely stepping for super resolution structured illumination microscopy (SIM).
A high-speed high-resolution sCMOS camera is used to capture image data. SlideBook™ 6 software controls all aspects of the system allowing for intricate high-speed synchronization of laser firing, SLM pattern display, galvo movements and camera readout while providing the ability to de-skew raw data, deconvolve and view 3D renderings of the sample. The specimen chamber and system are environmentally controlled to enable long-term living cell experiments.
Read more about lattice light sheet microscopy in the October 2014 Science magazine here.
1.1NA water objective, 2 mm WD, 62.5x total magnification
0.71NA water objective, LWD
405nm 350mW, 445nm 100mW, 488nm 300mW, 515nm 150mW, 560nm 500mW, 594nm 500mW, 642nm 500mW
Hamamatsu ORCA-Flash4.0 v3 sCMOS
Single sCMOS, Single EMCCD, Dual sCMOS direct 1x projection, or Dual EMCCD relayed 2.5x projection
Medical grade stainless steel with temperature control and perfusion capabilities
Standard, horizontally-oriented 5mm round coverslip
Dual 12-Core Xeon 2.3GHz CPUs, 128GB RAM, 4GB GPU, 512GB Solid State Drive (for operating system), 2TB SSD RAID-10 Drive Array for acquisition, 10Gb Ethernet with optional multi-mode and single-mode fiberoptic connectivity