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.
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.
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
Mouse Embryonic Fibroblasts expressing SiR-Actin (642nm)
High resolution 1.1NA water immersion objective with depthof field perfectly
matched to the light sheet thickness, generating excellent optical sectioning.
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
|Technique||Peak Light Intensity||Total Light Dose||Axial Resolution||Temporal Resolution|
|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|
INDEPENDENT EPI-FLUORESCENCE LIGHT PATH
MOTORIZED SAMPLE CHAMBER
MOTORIZED ANNULAR MASK
|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|
User controls all hardware necessary to perform daily alignment and acquire standard PSFs. Allows for the capture of dithered or SIM PSFs.
User controls all hardware necessary to perform acquisition of biological samples. Allows for the capture of dithered or SIM data.
|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.|
0.4µm at 50µm length
230 x 230 x 370nm (Dither) @ 1.1NA, 150 x 230 x 280nm (SIM) @ 1.1NA
1.1NA water objective, 2.0mm WD, 62.5x total magnification
0.71NA water objective, 3.7mm WD
405 350mW, 445 100mW, 488 500mW, 515 150mW, 560 500mW, 592 500mW, 642 500mW
Hamamatsu ORCA-Flash 4.0 v3 sCMOS
Single sCMOS, Dual sCMOS direct 1x projection, Dual EMCCD relayed 2.5x projection
Medical grade stainless steel with TEC temperature control and perfusion capabilities
Standard, horizontally-oriented 5mm round coverslip
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
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
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