M32 – Zeiss Z1 Lightsheet Microscope

System Description

Lightsheet microscopy is the method of choice for delicate living samples and for large cleared samples. A sheet-shaped light beam is used for exciting a single plane of the sample. The fluorescent light from the excited plane is imaged by perpendicular, camera based detection. This results in optical sectioning with low-dosage excitation and next to no phototoxic stress for living samples. Only in-focus volumes that are actually imaged, are illuminated. Using long-distance objectives and fast z-scanning, large cleared samples (up to 1x1x1 cm) can be acquired in short time.

The M32 Zeiss Z1https://www.zeiss.com/microscopy/int/light/lightsheet-z-1.html lightsheet microscope allows for bidirectional excitation, sample rotation, tile scanning, z-scanning and time-lapse imaging. Dual cameras provide fast acquisition for two channels, although with intermittent filter changes, more colors can be acquired easily. An optical zoom ranges from 0.36x to 2.5 x, although reasonable beam shapes (edges not thicker than two times the beam waist thickness) require a zoom of at least 0.71. Samples are mounted on a vertical, hanging support, dipped into the medium and illuminated and imaged horizontally. Scanning perpendicular to the light sheet plane is done by moving the sample through the light sheet. 3D scans are limited to a maximum speed of one stack per second.

The best achievable resolving power along the z-axis of the detection objective is about 2µm (20x/1.0 objective at optical zoom 2.5), providing cellular and sometimes sub-cellular resolution. For cleared samples, proper refractive index matching of embedding medium and imaging medium to the objective is of paramount importance. See the list of available objectives below.

The system is able to run multidimensional experiments including any combination of the following:

·         multiple positions

·         multiple wavelengths

·         z-Stacks, time-lapse

·         multiple rotational views

·         dual side illumination

·         pivot scanning

·         incubation (temperature, CO2 bubbling) with 5x dry objective or 10x / 20x water objectives only

Illumination

 White Light LED

 

 

Laser for Lightsheet

405nm 20mW

445nm 25mW

488nm 50mW

514nm 20mW

561nm 50mW

638nm 60mW

 

Beamsplitter & Filters

The 5 Beamsplitters divide the emission light into two channels, each of which is detected by a PCO edge sCMOS camera. The beamsplitters are associated with fixed emission filters, for camera one and camera two, respectively.

Set1 - Standard

Beamsplitter 1 (LP 490): cam1 BP [420-470] nm / cam2 BP 505-545 nm
Beamsplitter 2 (LP 510): cam1 BP [420-470] nm / cam2 BP 575-615 nm
Beamsplitter 3 (LP 560): cam1 BP [505-545] nm / cam2 BP 575-615 nm
Beamsplitter 4 (LP 560): cam1 BP [505-545] nm / cam2 LP 585 nm

Beamsplitter 5 (LP 560): cam1 BP [505-545] nm / cam2 LP 660 nm

 

Set2 - on request

The 5 Beamsplitters divide the emission light into two channels, each of which is detected by a PCO edge sCMOS camera. The beamsplitters are associated with fixed emission filters, for camera one and camera two, respectively.

Beamsplitter 1 (mirror):  cam1 none                      / cam2 none
Beamsplitter 2 (LP 510): cam1 BP [420-470] nm / cam2 BP 525-545 nm
Beamsplitter 3 (LP 510): cam1 BP [460-500] nm / cam2 BP 525-565 nm
Beamsplitter 4 (LP 510): cam1 BP [460-500] nm / cam2 BP 575-615 nm
Beamsplitter 5 (LP 510): cam1 BP [460-500] nm / cam2 LP 585 nm
Beamsplitter 6 (LP 560): cam1 BP [505-545] nm / cam2 LP 660 nm

Excitation Objectives

5x/0.1 (for use with 5x or 10x detection objective)

10x/0.2 (for use with 20x detection objectives)

5x/0.1foc (for use with 5x or 10x detection objective)

10x/0.2foc (for use with 20x detection objectives)

Detection Objectives

5x/0.16: EC-Plan Neofluar 5x/0.16: 5x/0.16 nd=1 WD= 5.1 mm

5x/0.16: foc EC-Plan Neofluar 5x/0.16: 5x/0.16 nd=1 WD= 10.5 mm

10x/0.5: W Plan-Apochromat 10x/0,5 nd=1,33 WD=3.7 mm

20x/1.0: W Plan-Apochromat 20x/1.0 Corr DIC; nd=1.33 WD=2.4 mm

20x/1.0: Clr Plan-Apochromat 20x/1.0 Corr nd=1.38 WD=5.6 mm

20x/1.0: Clr Plan-Neofluar 20x/1.0 Corr nd=1.45 WD=5.6 mm

20x/1.53: Clr Plan-Neofluar 20x/1.0 Corr DIC; nd=1.53 WD=  6.4mm

Imaging Chambers

The following chambers are available:

Water chamber (n = 1.33) to be used with 5×, 10×, 20× and 40× water immersion lenses

20× Clearing chamber (n = 1.35 – 1.58) to be used with Clr 20× immersion lenses

Large sample chamber (n = 1.33 – 1.58) to be used with 5× foc objective

Cameras

PCO Edge 5.5m sCMOS cameras (2x): 6,5 µm/pixel, 1920 x 1920 pixels

Additional Devices

Incubation unit with heating (by a peltier element) and CO2 (bubbling in the imaging medium)

Peristaltic pump for media perfusion