Let's talk Lattice Lightsheet!

Did you miss the ZEISS Lattice Lightsheet 7 launch event stream? Watch the on-demand version here. We are excited to show you a breakthrough for lattice light sheet microscopy.

Enjoy the event, let's talk lattice lightsheet!

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Speakers

Prof. Dr. Eric Betzig

Guest

Prof. Dr. Eric Betzig

Professor of Physics and Molecular and Cell Biology, Howard Hughes Medical Institute Investigator, University of California, Berkeley

Prof. Dr. Lukas Kapitein

Guest

Prof. Dr. Lukas Kapitein

Professor of Molecular and Cellular Biophysics, Department of Biology, Utrecht University

Dr. Kirstin Elgass

ZEISS Microscopy

Dr. Kirstin Elgass

Application Development Engineer, Special 3D Solutions, ZEISS Microscopy

Dr. Peter O'Toole

Host

Dr. Peter O'Toole

Head of Imaging and Cytometry, Department of Biology, University of York


ZEISS Lattice Lightsheet 7 | Questions and Answers

During the launch event you asked many interesting questions – thank you for your great interest in ZEISS Lattice Lightsheet 7! Please find a collection of your questions answered below.


What are the resolution specifications of the system?

The maximum resolution you can achieve with Lattice Lightsheet 7 is 330 nm x 330 nm x 550 nm. By applying deconvolution, the resolution can be improved to 290 nm x 290 nm x 450 nm.

Find more technical specifications on the ZEISS Lattice Lightsheet 7 product website.
 

What is the maximum imaging depth or working distance?

Depending on the sample and the chosen light sheet length, Lattice Lightsheet 7 allows for a maximum imaging depth of 200 µm above the coverslip bottom.

Find more technical specifications on the ZEISS Lattice Lightsheet 7 product website.
 

Is the instrument compatible with live mouse embryo culture?

Yes, it is to a cell stadium not exceeding 200 µm. You can culture in a 96 well plate which is in small volumes often required for early mouse embryo stages (1 - 16 cells). As Lattice Lightsheet 7 is extreme low in phototoxicity, it is very much suited for such early embryonic stages.
 

Is the imaging depth suitable for tissue slices imaging?

If slices are thinner than the maximum imaging depth of 200 µm, you will be able to image them completely.
 

Can we also image 3D organoids or samples thicker than 100 µm?

Yes, since the maximum imaging depth is 200 µm, you can image organoids within that range.
 

Can I observe fluorescent bacteria in the intestine of zebrafish larvae?

Certainly, you will be able to observe bacteria in zebrafish larvae up to an imaging depth of 200 µm.
 

Is perfusion/microfluidics applicable?

The incubation tray is prepared for perfusion and microfluidics. The only prerequisite is a chamber that comes with a No. 1.5 glass bottom.
 

How many objectives are included in the instrument?

To achieve maximum imaging stability and user friendliness, we decided to equip the system with only one illumination and one detection objective. The magnification and NA of the detection objective are well suited to cover subcellular resolution and a large field of view.
 

What are the specifications of the detection objective?

48x / NA 1.0

Find more technical specifications on the ZEISS Lattice Lightsheet 7 product website.
 

How many channels simultaneous or sequential?

Lattice Lightsheet 7 provides three fast sequential, virtually simultaneous color channels.

Find more technical specifications on the ZEISS Lattice Lightsheet 7 product website.
 

Is it using a white light laser or multiple lasers combined? How many?

Three diode lasers are available: 488 nm, 561 nm, and 640 nm.

Find more technical specifications on the ZEISS Lattice Lightsheet 7 product website.
 

Can you please comment on the longevity and durability of all of the delicate (high precision) components?

Due to its integrated and robust design with almost no need for manual adjustments or interventions, the longevity and robustness even in core facility and multi-user environments is assured. Some of the early customer systems have already proven their robustness in multi-user environments during the First User Program. Cleaning strokes are implemented to prevent the stage from wear and to maintain its high precision. Warranty can of course be extended by service contracts.
 

How much alignment is required for every sample? How would you do these alignments?

The required alignments – tilt in X and Y, the focus position of the light sheet in regard to the detection objective, the waist of the light sheet in regard to the optical axis of the detection objectives, the aberration correction for the cover glass – can be performed automatically or manually.
 

What is the maximum field of view and will different FOV sizes affect resolution?

The maximum field of view is X = 300 µm. The Y dimension is defined by the scan range (1000+ µm). The field of view does not affect resolution.

Find more technical specifications on the ZEISS Lattice Lightsheet 7 product website.
 

What lattice mode is used to achieve isotropic resolution?

Lattice Lightsheet 7 provides near-isotropic resolution (z/x = 1.52) with the 15 µm x 500 nm light sheet.

Find more technical specifications on the ZEISS Lattice Lightsheet 7 product website.
 

Is there a fixed annulus or can the user choose different annuli for different sheet thicknesses?

You can choose from six light sheet presets with different lengths and thicknesses by just a click. Annulus change is not needed. A specifically designed fixed aperture blocks the 0th order from the Spatial Light Modulator for each light sheet pattern. Additional SLM programming allows for flexible light sheet design.

Find more technical specifications on the ZEISS Lattice Lightsheet 7 product website.
 

How do you deal with the huge data sets after imaging for 3 days?

Data acquisition is supported by the same familiar storage system known from our Lightsheet 7 instrument. Acquired data can be streamed directly to the storage system. Processing tasks can be automated with powerful Direct Processing and Batch Processing modules. If more space is needed, the Lattice Lightsheet system can be connected to your local server structure, or a scalable storage device like the HIVE from Acquifer, via the 10 GB data transfer lines.

Find more technical specifications on the ZEISS Lattice Lightsheet 7 product website.
 

How does a tilted stage not introduce issues for the optical path?

The tilt is used to level the cover glass to exactly avoid these issues. Consumable-type sample carriers have a variation of their cover glass levelness. The tilt option of the stage compensates for those deviations to optically level your sample.

Find more technical specifications on the ZEISS Lattice Lightsheet 7 product website.
 

How does the system co-op with multi-well imaging and the stage aligning in long term multi point live imaging?

The overall tilt of a multi-well plate can be corrected in X and Y. However, if the glass bottom of the multi-well plate is under tension and shows a wave-shaped deformation, only a focus map would help.
 

How much of what you showed during the launch event has been acquired on the ZEISS system?

Except the examples shown in Dr. Eric Betzig’s presentation, all application examples have been acquired with ZEISS Lattice Lightsheet 7, either by ZEISS or by customers participating in the First User Program.

Find more technical specifications on the ZEISS Lattice Lightsheet 7 product website.
 

Why aren't single molecule applications shown? What is the challenge in going down to the single molecule level?

Due to the challenging COVID-19 conditions with limited lab time and access to suitable samples, we simply have not yet tried to image single molecules. We look forward to seeing first examples from our customers.
 

What's the difference of your system compared to the 3i product?

Lattice Lightsheet 7 is an integrated turn-key solution. Its inverted layout allows to use standard sample carriers and cell culture dishes. It provides additional transmitted light, a larger field of view, and a small footprint of only 0.26 m².
 

Is the dithered mode the only option or can the stepwise mode with superresolution reconstruction also be used?

A dithered light sheet is the only useful mode. Stepwise mode with superresolution does not lead to an increase in resolution since illumination objectives have lower NA which prevents resolution enhancement.
 

How do you deal with the deskew process? Do you also rotate to "coverslip" orientation and does that cause resolution loss?

Deskewing is just a shearing of the image stack to a physically relevant stack. Cover glass transformation is also available. Both deskewing and cover glass conversion do not result in an overall resolution loss.
 

How much laser power can be delivered to the sample? Does the system require 1W+ lasers for live cell imaging?

Depending on the light sheet, the maximum power in the sample is 2mW corresponding to a maximum intensity of around 700 W/cm². Typical imaging is performed at less than 1% of the available laser power with intensities of less than 10W/cm². Since the beam shaping has a high efficiency, 1W+ lasers are not necessary.
 

Is the SLM binary or greyscale? If greyscale how do you handle wavefront calibration?

The SLM in Lattice Lightsheet 7 is based on nematic liquid crystals which allows to display greyscale phase patterns. The SLM is calibrated for a certain wavelength. The remaining dependencies of the phase shift on the wavelength of the used lasers are considered in the phase pattern of the respective light sheet. Aberrations due to oblique illumination and detection are taken care of by free-form optics in the illumination and detection objective.