EasySPIM: An Easy Light Sheet Microscope
Optimizing Light Sheet Microscopy at an Imaging Facility
- Fig. 1: The EasySPIM system. (a) Overview of the setup: laser beam is in blue, the two detection channels in green and red. On the bottom, the game controller used to move the stages and other devices. (b) Our software solution: a C# home-made software controlling both hardware and Metamorph (Molecular Devices) software.
- Fig. 2: Sample preparation and holder. (a) 3D view of the sample chamber attached to the detection objective. The removable parts are shown, allowing easy replacement of coverslip or cleaning of the chamber. (b) Preparation of phytagel cylinder and sample embedding protocol. c) Closer view of the sample embedded in the phytagel cylinder inside a syringe. (d) View of the complete sample holding system with the syringe in place in the chamber and held by the moving stages.
- Fig. 3: Examples of biological applications. (a) Time-lapse (25 ms time interval during 4 min) image sequence of an EGFP-histone labelled zebrafish embryo showing the heart beats. (coll. F. Verweij) (b) Image sequence of an mCherry-Lectin labelled brain mouse (c)Three angle view of a 3D cell culture (spheroid) using two cell type (Green: GFP fibroblast, red: mTomato cancer cells) (coll. F. Bertillot).
Presented here, is a light sheet microscopy setup, developed at the Curie Institute Imaging Facility, optimized for fast, multicolor (4 laser lines from 405 to 638 nm, 2 cameras) 3D imaging. Careful choice of all hardware and software components led to an easy to use system that merges smoothly with the facility. A 3D printed sample holding system has been developed along with software to particularly ensure both modularity and simplicity. The system can be used for various biological applications.
Single Plane Illumination Microscopy has undergone fast developments over the last years. The increasing need to be able to image cells in a 3D environment (spheroids, organoids) or in vivo has pushed the expansion of this technology. Indeed, the fact that the sample is illuminated from the side using a thin sheet of light, preserves the parts of the sample that are not imaged, highly reducing any photo damage. Furthermore, each plane can be captured in a few milliseconds, allowing fast 3D imaging. Very promising techniques are emerging and relevant biological applications are growing, but since most systems are home-made, their usage needs either dedicated people to operate them or lots of training. Commercial systems, on the other hand, are made to be easy to use, but are usually closed to any further modification and adaptability. Moreover, the tendency to build a home-made system is still favored in the community for cost reasons.
At the Curie Institute Imaging Facility (PICT-IBiSA part of the INBS-France Bio-Imaging), the aim of the EasySPIM was to provide core facility users with a fully integrated, reliable and easy to use system with optimized image quality, but also with a modular design that allows future technological evolution.
How to Setup a Light-sheet Microscope on a Multi-user Open Imaging Facility?
Bringing new technology to a facility means making relevant choices as early as the conception of the instrument. Wanting to bring the users an easy-to-use but still efficient and upgradable system several issues had to be faced:
• How to attenuate the usual shadowing effect?
• How to ensure the long-term biocompatibility of the sample holder and its adaptabil-ity for various samples and experimental conditions?
• Which software solution would be suitable on an open facility?
• How to design an instrument which can evolve for future applications/technology?
To answer those questions, it was decided to:
• Use scanning mirrors to move the laser beam to another range of angles in order to average and attenuate the shadowing effect
• Use 3D printing technology with biocompatible materials, giving the possibility to adapt the sample holder to various sample sizes and other biological constraints.
• Design a home-made software for integration into a commonly used software plat-form (already in use in more than 20 microscopes in our facility), for fast user ac-cessibility.
• Build an adaptable system, to give the freedom of choice on the different parts and consequently ready for modification and implementation of new modalities.
The basis of the EasySPIM system, shown in figure 1 (a), consists of a single illumination setup with a cylindrical lens in conjugation with a low magnification objective (10x NA 0.3) to shape the laser beam as a sheet of light.
Lasers are fiber-coupled into the system and expanded using an a-focal system. A detection module composed of a water-dipping objective (16x 0.8 NA or 40x 0.8 NA) is used, along with a dichroic mirror to split fluorescence into two channels. Each of the channels are equipped with motorized filter wheels and a sCMOS camera, allowing fast simultaneous two-color imaging or sequential four-color imaging.
To achieve the previously set goals, specific hardware and software were implemented:
• Scanning mirrors (Errol).
• An adjustable slit that allows the removal of unwanted interferences or diffraction patterns.
• Custom white light bright-field illumination (Errol) controlled by our software.
A 3D printed sample chamber, an incubator, a CO2 control system, and a culture medium circulation system using a peristaltic pump were designed in order to carry out the long-term observation of living samples in the best conditions.
A software solution was developed alongside the hardware (fig, 1b). Particular emphasis was placed on keeping it easy-to-use so that users can be trained with minimal effort. To make the system even more user-friendly, a game controller was introduced which allows the user to control hardware parts (lasers shutters, translational stage motion, detection objective position and live acquisition).
During conception of the Easy-SPIM system, specific parts were designed using two 3D printing technologies (PolyJet and FFF: Fused Filament Fabrication), particularly to print the sample chamber and a few opto-mechanical parts, ensuring a tailored flexibility of the setup.
Sample Preparation and Holding
For sample preparation, which is a crucial aspect in light-sheet microscopy, tools that are available in any biology wet lab are used, (fig. 2 (b, d)).
The holder is made of a semi-rigid phytagel gel cylinder with a central hole whose size can be adapted depending on the sample (up to 2 mm). It is then filled with low-melting agarose gel, water or any other suitable embedding medium. This cylinder is then immersed in the culture medium contained in the sample chamber as shown in figure 2 (b). More details about the preparation are shown in figure 2 (b, c).
The integrated system (from sample preparation to image acquisition) opened up several biological applications from facility users, from cell dynamics in 3D culture to fixed and clarified tissues.
A first example was the high-frame rate observation in a single plane of a living zebrafish embryo (36HPF), the goal was to follow the heart beats. It was imaged using a detection objective 16x NA 0.8, at 27°C to respect the environmental conditions for embryo development.
The setup was also used to follow cell dynamics in spheroids using 3D imaging. Spheroids were formed by two cell types, EGFP Fibroblast and mTomato cancer cells. Figure 3 (b) shows maximum intensity projections of a stack obtained from a multi-angle 3D acquisition (120 planes, 2 µm step, 3 angles from 0° to 360° with 120° step) using a 40x 0.8 NA objective
The most common option for installing a new module in an imaging facility is to buy a commercial setup. However, cost and need for versatility often impair the acquisition of the required technology, whatever the consistency regarding biological applications of users. On the other hand, home-made systems, as alternatives, are developed within teams that focus on restricted applications and often lack an open community spirit. Here it has been shown that a home-made solution, when designed with “easy-to-use” and “service driven” goals in mind, is valid. It requires full integration of the experimental workflow, from hardware parts through to software, including easy handling protocols of samples by multiple users. The goal of bringing this technology to non-expert users has been reached.
The authors thank the CellTisPhyBio Labex (N° ANR-10-LBX-0038) part of the IDEX PSL (N°ANR-10-IDEX-0001-02 PSL) for funding.
Ludovic Leconte1, François Waharte1, Jean Salamero1
1 Institut Curie, Cell & Tissue Imaging, Photonics, Paris, France
Dr. Ludovic Leconte
Cell & Tissue Imaging, Photonics