High-resolution, low light-dose lightsheet microscope

First developed by Nobel Laureate Dr. Eric Betzig, the 3i Lattice LightSheet microscope is capable of imaging biological systems spanning four orders of magnitude in space and time. The system generates an optical lattice to create an ultra-thin light sheet to image biological samples over long periods of time and with very fine resolution. This allows for 4D living cell imaging, where experiments limited to seconds or minutes on other imaging platforms can be extended to hours or even days. The combination of high spatiotemporal resolution, imaging speed and sensitivity make Lattice LightSheet the ultimate imaging tool for a new era of living cell microscopy.

Ideal Lightsheet for Live Cell Imaging

Lightsheet imaging offers fast fluorescence imaging with decreased light dose at the sample plane compared to confocal imaging. Conventional selective plane illumination microscopy (SPIM) methods use a Gaussian beam where light sheet thickness is proportional to light sheet length, leading to a beam that is typically too short or thick for ideal sub-cellular imaging. The innovative Lattice LightSheet combines the flexibility of a Gaussian light sheet with the thin optical sectioning of a Bessel beam to achieve the best optical sectioning of any light sheet microscope.

MAKING A LATTICE LIGHTSHEET

1. Cylindrical lenses stretch and collimate the beam to form a sheet projected onto a spatial light modulator (SLM).

 

2. SLM generates an optical lattice of Bessel beams.

 

3. Annular mask acts as a zero order filter, removing artifacts and lengthening the sheet.

 

4. Galvos dither the sheet in X and sweep in Z.

Gaussian

Bessel

Lattice

Low Phototoxicity for Prolonged Living Cell Imaging

Mouse embryonic fibroblasts expressing SiR-Actin (642 nm) imaged with 25 ms exposures. After 8 minutes, the cells died on the spinning disk confocal but the Lattice LightSheet cells remained viable.

Lattice LightSheet
Spinning Disk Confocal
HeLa cells transfected with GFP-Lifeact (488nm) to visualize actin dynamics and stained with DiL (561nm red)(Thermo) to visualize membrane dynamics. 
Courtesy of James Springfield, Institute for Molecular Bioscience (IMB) at the University of Queensland

Optimal Lightsheet Illumination

ILLUMINATION

Custom-made 0.71NA long working distance water immersion objective for lightsheet illumination, mechanically and optically matched to the imaging objective.

IMAGING

High-resolution 1.1NA water immersion objective with depth of field matched to lightsheet thickness for excellent optical sectioning.

LIGHTSHEET EXCITATION EFFICIENCY REDUCES PHOTOTOXICITY
Because the light is entering the specimen along the plane of focus, the likelihood of a useful fluorescence event is far higher than in other methods that illuminate through the cell.

 0.4µm lightsheet thickness      0.5µm objective depth of field

Distinct Capture Modes

SHEET SCAN

The light sheet and objective are moved in tandem to form a 3D image.

SAMPLE SCAN

For larger imaging areas, the sample is moved while the light sheet and objective are stationary.

STRUCTURED ILLUMINATION MICROSCOPY (SIM)

The light sheet is stepped along 5 discrete phase steps on the X axis in either sheet scan or sample scan mode. Five raw images are collected that are reconstructed to produce an image that is beyond the diffraction limit of the detection objective.

Mouse embryonic fibroblasts expressing SiR-Actin (642nm, red) labeled with Tubulin Tracker (488nm, green) and NucBlue (405nm, blue). 3 minutes into a 10 minute live capture with 25ms exposures for all channels.

Features

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Motorized Sample Chamber

Objectives remain fixed and aligned

Rapid and repeatable sample changing

Expanded specimen access when loading

Load and home positions for 2-move sample change

Removable inner chamber for easy cleaning

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SOLID STATE HEATING SYSTEM

Accurate and stable temperature setting for the objectives and sample holder

No water, tubing, heaters or pumps

Active feedback for improved thermal stability

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OKOLAB INCUBATOR UPGRADE

Complete Okolab Incubator with Bold Line temperature controller, heater and digital CO2 gas mixer for comprehensive environmental control

Includes active humidity controller (50-95%) and heated sample chamber and objective assembly with easy to use Oko-Touch controller.

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Motorized Annular Mask

Fast, automated changing of annuli

Easy testing of various mask positions and patterns on a single biological sample

No alignment required after change

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LED BRIGHTFIELD & EPI-FLUORESCENCE ILLUMINATION

Proper epi-fluorescence illumination via LEDs, mirrors and filters behind
the vertical board

Significant reduction in light dose compared to laser epi-excitation

Multi-band dichroic replaces 90/10 beamsplitter

Multi-channel LED illuminator to match the imaging lasers

White brightfield LED illumination through excitation objective

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IMPROVED OPTICAL MOUNTS
AND BEAM SHIELDING

LASER LAUNCH SOLID BASEPLATE

• Solid aluminum baseplate with no anodization and a clear alodine treatment to prevent oxidation
• Large heatsink increasing thermal stability
• Counter-bored bolt passthroughs for better thermal conductivity
• Less than 1°C gradient across the baseplate resulting in less than 1 mrad of beam shift between startup and equilibrium

• Front and back plates mechanically secured with metal rivets
• Breadboard extended down to contact air table for increased stability
• Holes for cable management and access to rear-mounted epi-fluorescence light path

• 12 mL chamber for increased resistance to evaporation
• 3 mL chamber for reduced cost of expensive media and reagents
• Two ports for active perfusion of media
• Slots into lower assembly for easy exchange and cleaning

• Cylindrical lens assembly redesigned to replace large, glued elliptical mirror at 37° angle with a pair of mirrors in adjustable 90° mounts
• Precise alignment with improved optical stability
• Covers prevent incidental contact with galvo assembly, SLM and emission path
• Beam shielding increases laser safety and keeps dust from disturbing the optical path

NEW VERTICAL MOUNTING BOARD

MULTIPLE SAMPLE CHAMBERS

Slider

Slidebook Software

SINGLE CONTROL PANEL

Streamlined control module for image loading, sample finding, and image acquisition

 

Tools for daily alignment adjustment

 

SLM presets for rapid switching between exposures, wavelengths, and ROIs

 

Integrated SIM capture and processing

LIGHTSHEET TRACKING

SlideBook monitors lightsheet position during capture

 

Dynamic adjustment of objective piezo maintains a focused image on the camera

INTEGRATED SLM PATTERN GENERATION

SLM pattern generator for complete adjustment of pattern parameters
Pattern defaults for quick starting points
Pattern generation tied to specific position on motorized annular mask for rapid annulus changes

Microvolution GPU based deconvolution package integration for fast, post-acquisition processing

Cell Viability

Peak light intensity is greatest in point-to-point scanning methods.Total light dose increases in methods that illuminate the entirety of the cell
*Acquisition speeds are based upon the lowest exposure time needed to image a 10µm cell with a step size of 0.2µm with a similar signal-to-noise ratio.

LIGHT DOSE / PHOTOTOXICITY

Specifications

LIGHT SHEET THICKNESS

0.4µm at 50µm length

 

DETECTION OPTICS

1.1NA water objective, 2.0mm WD, 62.5x total magnification

 

ILLUMINATION OPTICS

0.71NA water objective, 3.7mm WD

 

LASER OPTIONS

405 350mW, 445 100mW, 488 300mW, 515 150mW, 560 500mW, 592 500mW, 642 500mW

 

STANDARD CAMERA

Hamamatsu ORCA-Flash 4.0 v3 sCMOS

 

CAMERA OPTIONS

Single sCMOS, Dual sCMOS direct 1x projection, Dual EMCCD relayed 2.5x projection

SAMPLE CHAMBER

Medical grade stainless steel with TEC temperature control and perfusion capabilities

 

SPECIMEN MOUNTING

Standard, horizontally-oriented 5mm round coverslip

 

ACQUISITION COMPUTER

Dual 10-Core Xeon 2.4GHz processors, 128GB RAM, NVIDIA Quadro P4000 8GB workstation graphics card, 1TB OS SSD and 6TB SSD array, 10GbE (copper) Adapter

 

ANALYSIS COMPUTER

Dual 10-Core Xeon 2.4GHz processors, 256GB RAM, NVIDIA Quadro P6000 24GB workstation graphics card, 1TB OS SSD and 6TB SSD array, 10GbE (copper) Adapter

 

STORAGE SOLUTIONS

DDN® unified storage systems for direct full-speed acquisition and analysis starting at 300TB. DDN systems utilize a BioScaler GPFS file system and are easily expandable to multiple petabytes

Find out more about
Lattice LightSheet






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Email: sales@intelligent-imaging.com

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Email: support@intelligent-imaging.com

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