Facility

ALICe

State-of-the-art advanced lightsheet imaging center

Lightsheet microscopes can reveal the 3D anatomy of entire small organs. They image brain tissue down to individual neurons and offer unprecedented maps of nervous system structure and function.

ALICe, the Advanced Lightsheet Imaging Center, integrates a series of innovative fluorescence microscopy tools in a single pipeline to image whole organs with custom-built microscopes at high spatial and temporal resolution.

Revealing 3D anatomy with lightsheet microscopy

3D spatial transcriptomics: Discovering new sub-cellular worlds in 3D brain samples

The multidisciplinary ALICe team unites expertise from physics, cell biology, neuroscience and engineering, and joins forces with research teams around the world to image and draw insights into the central and peripheral nervous systems, innervation of organs including the brain and heart as well as human brain samples. Another focus area is brain and spinal cord organization for researchers working to restore movement after paralysis or to investigate neuronal networks involved in cognition, pleasure and drug addiction.

Unlike traditional microscopy in which specimens are cut in slices with a blade before being viewed on a slide under a microscope, lightsheet microscopes optically slice samples with a sheet of light. This optical sectioning captures slivers of image without damaging the sample. The images are then combined to reconstruct a detailed three-dimensional image of a whole organ or specimen.

The ALICe pipeline

ALICe’s capabilities cover the entire pipeline, from sample preparation to image capture and post-processing. A variety of samples can be imaged from organoids to whole organs, labelled with genetically encoded fluorescent proteins or immuno-labeled with fluorescent antibodies.

A primer on lightsheet microscopy

The theoretical basis of lightsheet microscopy was established more than 100 years ago by R. A. Zsigmondy, an Austrian-Hungarian chemist^[1]. His idea was to direct light through a slit, creating a lightsheet to illuminate a sample.

In the early 2000s, Jan Huisken and collaborators demonstrated for the first time that this approach could be used to image biological samples at sub micrometer spatial resolution, with micrometer scale optical sectioning in millimeter thick tissue^[2]. Since that time and thanks to major advances in the field of tissue clarification, numerous types of lightsheet microscopes have emerged.

At one end of the spectrum is the family of high spatial resolution microscopes used either to image small transparent biological samples at high speed and extremely high spatial resolution (using multiplexed Bessel beams: Lattice lightsheet microscopy^[4]) or for centimeter-sized clarified tissues at submicronic in-plane resolution (CLARITY-optimized light-sheet microscopy (COLM)^[3]) or with sub-micronic isotropic resolution (Axially Swept Lightsheet Microscopy ctASLM^[5]).

At the other end of the spectrum, are the mesoscale lightsheet microscopes which can image centimeter-sized samples at high speed while preserving subcellular resolution (mesoscale selective plane illumination microscopy – mesoSPIM^[6]).

Here at the Advanced Lightsheet Imaging Center, we have implemented a high spatial resolution lightsheet microscope (COLM) which is complemented by a high speed mesoSPIM. A scientific advisory board constituted by Prof Lüscher, Prof. Courtine and Prof. Holtmaat followed and contributed for the development of ALICe’s project.

[1] H. Siedentopf and R. Zsigmondy, Ann. Phys. (Berlin), 10, 1–39 (1903).

[2] J. Huisken et al., Science, 305, 5686 (2004).

[3] R. Tomer et al., Cell, 163, 1796–1806 (2015).

[4] B-C. Chen et al., Science, 346 (2014)

[5] T. Chakraborty et al., Nat. Methods, 16 (2019)

[6] F.F. Voigt et al, Nat. Methods, 16 (2019)

1) Sample preparation: Tissue clearing for entire large samples

Lightsheet microscope samples are made completely transparent before they are imaged. This process, known as clearing, removes all opaque lipids including fats, present in brain tissue and renders the sample transparent.

ALICe tissue clearing is predominantly based on the CLARITY protocol, with additional experience in DISCO-based and any other published clearing protocols.

2) Custom-built lightsheet image acquisition: high spatial and temporal resolution imaging

ALICE is equipped with three lightsheet systems tailored for imaging large, cleared samples. The custom-built mesosSPIM microscope is optimized for large scale screening of multiple samples at cellular resolution with high speed in imaging performances.

Clarity Optimized Light-sheet Microscope (COLM) offers the unique opportunity to capture high spatial resolution (near diffraction-limited lateral resolution), whole brain fluorescence images without perturbing neuronal architecture.

Clearscope, from MBF is light sheet theta microscope designed to meet the unique challenges inherent to imaging at high spatial resolution and reconstruct entire human cleared organs. With its theta configuration, the clearscope is able to accommodate 15x10 cm wide samples.

Finally, our confocal systems are equipped with modules for cleared tissue section imaging.

3) Image and data analysis including 3D data-set exploration in virtual reality

The ALICe image analysis workstations are equipped with custom-software and industry-leading software solutions including Imaris, Arivis, Matlab, ImageJ and Terastitcher to enable efficient access, extraction and analysis of 3D data from lightsheet imaged samples.

The final step of the ALICe pipeline includes 3D data visualization and analysis in a Syglass virtual reality environment to allow interaction with samples in an immersive way, speeding up the analysis process.

Creating elastic brains

To image brain samples, fluorescent tags are used so that structures are visible with a microscope. To help these tags penetrate thick samples, the team is exploring a new method – ELAST (entangled link-augmented stretchable tissue-hydrogel). They embed the sample in an elastic polymer gel that allows it to deform and reshape. Then they stretch the sample over hundreds of cycles while it bathes in a solution of antibodies. The process makes labeling of thick samples more efficient.

Expansion microscopy

Chemical anchors in expanding gel attach to biomolecules, physically expanding the brain sample and allowing as much information as possible to be extracted from the tissue. Expansion microscopy deepens our understanding of disease mechanisms to accelerate development of therapies for neurological and psychiatric disorders, like Parkinson’s and Alzheimer’s diseases.

Prof. Holmaat, Unige

"Recent advances in microscopy have propelled the neurosciences into the era of connectomics, now allowing us to study single neurons with their synaptic connections in the whole brain."

Prof. Daniel Huber, Unige

"The work at ALICe allowed us to reveal the distribution and anatomy of sensory receptors with unprecedented resolution and specificity. I was stunned by the quality of the images."

Prof Fyhn - UiO

"Never before have we been able to visualise and quantify, in-depth, neuronal populations within the entire brain as we can now."

Whole CNS lightsheet imaging of motor circuit and 3D surface rendering (Asboth et al. 2018). Courtine Lab, EPFL

Available resources

COLM-SPED light-sheet

Clarity Optimized Light-sheet Microscope (COLM) - developed in collaboration with the original inventors of the technology - Raju Tomer, Columbia University and Karl Deisseroth, Stanford University - is optimized for large scale clarified tissues and allows a fast exchange of optics and samples. It offers the unique opportunity to capture high spatial resolution (near diffraction-limited lateral resolution), whole brain fluorescence images without perturbing neuronal architecture.

Objectives: Detection: 25x/1(CLARITY) WD 8mm ; 10x/0,6 (CLARITY) WD 8 mm; : 4x/0,28 WD 29 mm; Illumination: 4x/0,28 (d)
Filter cubes: 525/50; 609/50 680/42
Illumination: 488, 561, 647 nm
Detection: sCMOS: ORCA-Flash 3.0 CAMERALINK; Rolling top
Resolution: Up to 0.6 um in plane (10X magnification)
Stage: Motorized (xyz axes + rotation)
Software: Labview
Connection: Optical Fiber 10 Gb
Max size of sample: 5 X 5 X 5 (h) cm

mesoSPIM light-sheet

ALICE’s custom-built mesosSPIM microscope, developed in Fritjof Helmchen’s lab at the University of Zurich (UZH), is optimized for large scale screening of cleared samples at cellular resolution with high speed imaging performances.

Objectives: Detection: Macro-zoom system (Olympus MVX-10) Zoom factor from 0.63X to 4X;
Illumination: 405, 488, 561, 647 nm
Filter cubes: 530/43 – LP515 – LP561 – LP663 – 542/27 – 482/35 – 450/50
Detection: sCMOS: ORCA-Flash 4,0 CAMERALINK; Rolling top
Resolution: Max resolution: to 1.6 um in plane
Stage: Motorized (xyz axes + rotation)
Software: Custom – (Python)
Max size of sample 5 X 5 X 10 (h) cm

For more information on the mesoSPIM initiative, see mesoSPIM.org

Clearscope Light-sheet MBF

Clearscope, from MBF is light sheet theta microscope designed to meet the unique challenges inherent to imaging at high spatial resolution and allow an unprecedent travel range. This allows our researchers to work with specimens that are too large for traditional light sheet imaging, such as human, non-human primates, and tissue from expansion microscopy.

Objectives: 4X/0.28 WD 29 mm NA; 20X/1 NA WD 8 mm
Illumination : 4 lasers channel 405, 488, 561, 640 nm; LSTM with 2 oblique light-sheets; X-Cite LED for widefield detection
Filter cubes: filter cube with DAPI, TexasRed, FITC, Cy5 and 405/488/561/640 laser QUAD Band; Motorized filter wheel; Reflected light brightfield mirror cube
Detection: sCMOS: ORCA-Flash
Resolution: Max resolution: 300 nm in plane
Stage: Motorized - travel range 150mm x 100mm x 12mm

Olympus VS120 Automated Slide Scanner

The VS120 is a high-throughput slide scanner that is capable of automatically imaging and stitching up to 100 slides in color bright field mode as well as fluorescence mode. The online stitching allows a fast 2D reconstruction of large sections.

Objectives: 2X/0.08 WD=6.2, 4X/0.16, 10X/0.4 WD=3.1, 20X/0.75 WD=0.6, 40X/0.9 WD=0.18
Filter cubes: Ex: 392/23, 474/27, 554/23, 635/15, Em: 433/24, 520/35, 600/37 680/42 nm (motorized turret)
Illumination: Spectra-X LED
Detection: B&W CCD, color CCD
Resolution: Max resolution: 170 nm (40X)
Stage: Motorized
Software: VS-ASW
Slide holders: 100 slides 1” X 3”

TissueScope - Huron Digital Pathology

TissueScope is a customized automatic slide scanner system for serial 2D fluorescence/BF imaging of large section (scan up to 180x130 mm). Suitable for human brain 2D imaging.

Objectives: 20X/0.75 WD mm
Filter cubes: BFP, GFP, Cy3, Cy5
Illumination: Excelitas_X-Cite_NOVEM - Preview in darkfield
Detection: Color CMOS 6um pixel 4096 x 3072, Teledyne Photometrics – Kynetics (29.4 mm Field Of View)
Resolution: Max resolution: 0.3 um
Stage: Automatic scan and focus control
Software: Automatic or manual setup and scan, z-scan scan – image format JPEG , TIFF
Application: Serial and automatized fluorescence/BF microscopy (i.e mosaics of several large sections) - Human Brain
Slide holders: 120 slides 1” X 3”, 10 custom slide holders

Eclipse Ti2-U - Nikon

Inverted Widefield microscope DIC, equipped for Brightfield, phase contrast, fluorescence imaging. Excitation up to NIR wavelengths.

Microscope: Nikon Eclipse Ti2-U partially motorized
Objectives: Plan Fluor 4x/0.13; Plan Fluor 10x/ 0.3; Plan Fluor 20x/0.5; Plan Fluor 40x /0.75; manual nosepiece, manual condenser
Filter cubes: BFP, GFP, Cy3, Cy5, Cy7 (motorized turret)
Illumination: SOLA III V-nIR Light Engine
Detection: DS-Qi2 Mono Digital Camera
Stage: Motorized
Software: NIS Elements BR

Confocal LSM880 + Airyscan - Zeiss

Confocal system equipped with 6 laser lines, GaAsP-PMT detectors, Airy Scan detector for multi-channel detection at a resolution higher than traditional confocal. Suitable to any conventional confocal application, including sprectral un-mixing, ratiometric analysis and photobleaching studies. The systems also includes a module for imaging cleared tissue sections or organoids with a clarity optimized objective.

Objectives: 10x/0,45 (d) WD= 2.0; 20x/0.8 (d) WD= 0.55; 63x/1.4 (oil); 20x/1 (CLARITY), Corr WD= 5.6
Filter cubes: BFP, GFP, TexasRed
Illumination: Laser diode 405 nm, Laser Argon Multiline 458, 488, 514 nm; Laser HeNe 543; Laser HeNe 633 nm; Halogen Lamp
Detection: 1 T-PMT (Transm@ light); 3 ch spectral detection with 1 GaAsP-PMT and 2 PMTs. 1 Airyscan
Resolution: Max resolution: 140 nm laterally and 400 nm axially
Stage: Motorized
Software: Zen 2.3

Confocal - Spinning Disk CSU-W1

Spinning disk confocal microscopy utilizes a microlens array disk and a pinhole disk to allow a high-speed 3D imaging of thick sections. The system is optimized for live 3D imaging, thick section 3D and multi-tile imaging, spatial transcriptomics.

Microscope: Nikon Eclipse Ti2-E fully motorized
Objectives: Plan Apo Lambda D 10x; Plan Apo Lambda D 20x/0.8; Plan Apo Lambda D 40x /0.95; Plan Apo Lambda D 60x/1.42 Oil; Plan Apo Lambda S 20x/0.95 WI
Filter cubes: BFP, GFP, Cy3, Cy5, Cy7; (motorized)
Illumination: Diode laser 405/488/515/561/638/785 120/200/150/200mW with corresponding dichroic (405/488/561/640; 445/515/640; 785); pE-300 LED light source
Detection: 2X Hamamatsu Orca Fusion BT CoaXPress (22mm FOV, 95% QE, 6.5um pixel size, fast readout, low noise)
Spinning disk: Spinning Yokogawa CSU W1 - 50um pinhole single – 4000 RPM max speed
Stage: Motorized
Software: NIS Elements Advance Research, including JOBS

We are always looking for innovative collaborations to advance the techniques, hardware and software related to ALICe.

Director of Neuroimaging: Stéphane Pagès, PhD

Microscopy Facility Manager: Laura Batti, PhD

If you would like to find out more, please contact us: microscopy@wysscenter.ch

mesoSPIM

Christophe Lamy - UNIGE

Mackenzie Mathis - EPFL

Renier Lab - Paris Brain Institute

Michel Paques - Institut de la Vision

Nikita Vladimorov - University of Zurich

Daniel Huber - UNIGE

Valérie Dutoit, UNIGE, HUG

Luc Stoppini - HEPIA

Jérôme Extermann - HEPIA

Dotphoton

Yanik Mehmet Fatih Lab - ETH Zürich

G-LAB UPCOURTINE

A section of human cerebellum from the Lamylab at the University of Geneva imaged with the Wyss Center’s mesoSPIM lightsheet microscope. The image shows blood vessels labelled with the marker lectin. Blood supplies energy to the brain and is known to be disregulated in some diseases. Imaging brain vasculature with advanced microscopes helps drive research into neurodegenerative diseases and stroke. Credit: Tomàs Jordà – Lamylab, University of Geneva – Human Brain Mapping project

3D image of intestinal villi. Villi are controlled by the enteric nervous system (ENS) our gut’s own little brain. The team is using the three-dimensional microscopic imaging of the intestinal wall to better understand the involvement of enteric neurons in gut function and their connection with the brain. Image acquired with Clarity Optimised Lightsheet Microscope. Gut section cleared with iDisco+ protocol. Data are processed with a depth coding algorithm – the color represents the depth of the 3D image.

This image of virally labelled neurons in the brain helps reveal the cellular connectivity between the entorhinal cortex and hippocampus. These two regions are involved in memory processing and play a crucial role in the study of neurodegenerative disorders. Image courtesy of Noémie Mazaré, Giovanni Carriero, Roberta de Ceglia, Laura Solanelles, Bilian Xiong, Prof. Ludovic Telley and Prof. Andrea Volterra at the University of Lausanne.