Revolutionary light sheet microscope with spatiotemporal super-resolution and low photodamage

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 with unparalleled optical sectioning at extremely low photo- dosage and phototoxicity. 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.

Imagine 3D TIRF

The creation of a lattice light sheet is more complex than the creation of light sheets by typical selective plane illumination microscopy (SPIM) methods. Conventional SPIM methods use an apertured Gaussian beam to form a sheet that is too thick to allow excitation of isolated sub-cellular events. Lattice LightSheet employs a sophisticated multi-step process to create a light sheet consisting of a parallel linear array of coherently interfering Bessel beams. The resulting light sheet is exceptionally thin and flat with unequaled optical sectioning and essentially no out-of-focus light.

Minimum Sheet Thickness

Regularly spaced Bessel beams interfere thus producing a non-diffracting, bounded lattice light sheet that carries the vast majority of the energy in the central core and little energy in the side lobes.

HOW IT’S MADE:

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.

A. Standard 4µm Gaussian beam.
C. Non-interfering Bessel pattern with sub-optimal 4µm sheet thickness.
D. Optimized, optically-confined Lattice LightSheet Bessel pattern with 0.4µm sheet thickness.
B. Thin 0.4µm non-diffracting Bessel beam.

An Unprecedented  Combination of  Imaging Duration and Resolution

Mouse Embryonic Fibroblasts expressing SiR-Actin (642nm)

Acquired on spinning disk confocal with 25ms exposures.
Acquired on Lattice LightSheet with 25ms exposures.

Extremely Efficient Illumination

IMAGING

High resolution 1.1NA water immersion objective with depthof 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.

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.

A Bessel beam lattice creates an ultra-thin 0.4µm
sheet to evenly illuminate the entire plane of interest.

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.

 0.4µm lightsheet thickness

 

  0.5µm objective depth of field

Distinct Capture Modes

Dithered Sheet Scan

The light sheet is rapidly dithered along the X-axis and one image is captured per Z plane.

Structured Illumination Microscopy (SIM)

During SIM acquisition, the light sheet is moved in 5 discrete phase steps along the X-axis. Five raw images are collected that are reconstructed to produce an image that is beyond the diffraction limit of the detection objective.

The advantage to SIM acquisition is an increase in axial resolution from approximately 370nm to 280nm, and X resolution from approximately 230nm to 150nm.

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

Technique Peak Light Intensity Total Light Dose Axial Resolution Temporal Resolution
Widefield (WF) Better Poor Good Better
Multiphoton (2P) Poor Better Poor Good
Laser Scanning Microscopy (LSM) Poor Good Good Poor
Scanning Disk Confocal (SDC) Good Good Better Better
Selective Plane Illumination Microscopy (SPIM) Better Better Better Better
Lattice LightSheet (LLS) Best Best Best Best

Updates

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INDEPENDENT EPI-FLUORESCENCE LIGHT PATH

A redesigned epi-fluorescence light path and LED light source allows users to rapidly find samples with a 20x objective.

LED light source is independent from the laser launch and controlled in SlideBook, eliminating the need for a flip mirror.

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THERMAL REGULATION

Fully enclosed, medical-grade stainless steel specimen chamber with thermoelectric cooling (TEC) temperature control.

Rapid heating and cooling of the upper and lower sample chambers allows for a quick transition between the imaging of specimens with different optimal temperature.

Cells are mounted on a standard, horizontally-oriented 5mm round coverslip.

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MOTORIZED SAMPLE CHAMBER

Motorization of the lower sample chamber allows for easy access to the sample holder, precisely returning the specimen to the prior Z position.

Maintains the integrity of the optical path by leaving the objectives stationary.

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MOTORIZED ANNULAR MASK

A motorized annular mask contains 17 of the most commonly used annuli and one blank position enabling rapid and straightforward switching to a different annulus.

Lattice pattern generation in SlideBook combined with the motorized annular mask allows for real-time biological evaluation of different annuli and patterns.

Slider
TYPICAL ANNULI SELECTIONS
Outer NA Inner NA Ideal For
0.55 0.513 Ultra-thin sheet for single cell monolayers, lamellipodia, axons, and organelles
0.45 0.402 Thin sheet for single cell monolayers, granular cells, lymphocytes, organelles
.35 0.302 Intermediate sheet for multi-cell layer tissue, samples 20µm to 35µm thick
.30 0.270 Thick sheet for dense tissue, connective tissue, samples 30µm to 50µm thick

SlideBook Control

Alignment Tab

User controls all hardware necessary to perform daily alignment and acquire standard PSFs. Allows for the capture of dithered or SIM PSFs.

Biology Tab

User controls all hardware necessary to perform acquisition of biological samples. Allows for the capture of dithered or SIM data.

Feature Benefits
Pre-Sets PSFs: Allows for rapid of PSFS for daily alignment and calibration. Biology: Allows the user to define commonly used imaging parameters to allow for rapid acquisition.
Pattern Generation User can generate new patterns for usage in conjunction with the motorized annular mask for rapid and real time evaluation of optimal patterns/mask combination for specific biological samples
Sheet Tracking SlideBook monitors the position of the lightsheet and adjusts the imaging objective position to maintain focus throughout time-lapse capture.
Post Acquisition Processing Data generated can be analyzed using a streamlined workflow within SlideBook. Data can be acquired, deskewed and deconvolved directly in SlideBook. In addition, processed data can be seamlessly piped into a user chosen rendering package.

Specifications

LIGHT SHEET THICKNESS

0.4µm at 50µm length

 

RESOLUTION

230 x 230 x 370nm (Dither) @ 1.1NA, 150 x 230 x 280nm (SIM) @ 1.1NA

 

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 500mW, 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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Phone: +1 (303)-607-9429 x1
Email: sales@intelligent-imaging.com

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Phone: +1 (303)-607-9429 x2
Email: support@intelligent-imaging.com

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