American 3i Lattice LightSheet Microscope

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 experiment previously limited by phototoxicity can now be continuously monitored for hours or days. The combination of high spatiotemporal resolution, speed and sensitivity makes the lattice LightSheet the ultimate tool in a new era for living cell microscopy.

Top and side view volume renderings of HeLa cells expressing mEmerald-Lifeact, demonstrating the higher speed and reduced phototoxicity of the lattice light (right) compared to Bessel beam plane illumination (left) when each is applied to super-resolution structured illumination microscopy (SR-SIM).

2x increase in axial resolution

10x to 20x reduction in phototoxicity and photobleaching

Conventional sample preparation

Conventional light sheets created with Gaussian beams are too thick over cellular dimensions to allow subcellular imaging. Nondiffracting beams swept across the imaging field creates a virtual light sheet of

submicrometer thickness, well suited to noninvasive highspeed 4D imaging.

By spreading multiple ultrathin, nondiffracting light sheets of low intensity across a broad area, the intensity at any single plane is reduced, and the effect of nonlinear photodamage mechanisms is

minimized.

Specimens are cultured or mounted on5mmdiameter cover slips. No special dye or

sample treatment is required. The sample holder has inlet and output ports for perfusion of heated or cooled media.

20x increase in speed relative to spinning disc confocal microscopy

The high axial and temporal resolution is achieved simultaneously - which is not possible under either the

spinning disk confocal or Gaussian light-sheet paradigms.

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 long2mmworking distance. A custom-designed 0.71 NA 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. Access ports allow perfusion of stimulation agents, with delivery schedule digitally controlled by SlideBook™6 and synchronized with imaging.

Our collective aim is to provide advanced multidimensional microscopy platforms that are intuitive to use, modular in design, and meet the evolving needs of investigators in the biological research community.

3i’s headquarters inDenver,Colorado(USA) is a repurposed taxi repair garage modernized using 100% reclaimed materials. The building features – among other recycled and reused items – hockey glass, reclaimed pallet racks, rail boxcar floorboards, and a ‘cellular’ wall system made from 20,000 recycled PET plastic water bottles.

3i’s European headquarters are located inGöttingen,Germany, home of the renown Georg-August-Universität Göttingen. Additionally, 3i has a wide global presence with application and research scientists throughout theUnited States(San Diego,Los Angeles,Baltimore) and Europe (London,Paris). 3i has partnered with numerous dealers located around the world includingChina,Japan,India,Taiwan, andIsrael.

Intelligent Imaging Innovations (3i) designs and manufactures cutting edge live cell and intravital microscopy imaging platforms driven by our SlideBook™software. 3i was established in 1995 by a group of scientists whose wide range of research activities includes cell biology, immunology, neuroscience and computer science.