SPIM-Fluid

Concept

Schemes

Multiple imaging options

We have explored new designs for light sheet fluorescence microscopy (LSFM) based 3D imaging combined with flow cytometry and fluidics approaches providing high-throughput (HT) information as well as high spatial resolution comparable with confocal microscopy.

Using the framework of OpenSpinMicroscopy platform we have extended the functionalities of the systems adding a sample aspiration and positioning system, including motorized syringes and multi-well plate loading . In order to facilitate the automated sample loading essential for HT screenings we have designed special chambers for different type of objective.

We have created specialized chambers in acrylic plastic for different types of detection objectives, air and water dipping, filled with water. In both cases the FEP tubes of different diameter (0.5, 0.8, 1 µm) depending on the sample size , which refractive index is close to the water one, crosses the chamber with an angle of nearly 45º respect the illumination axis at a focal position of the detection objective. The FEP tube is positioned at the intersection of the illumination and detection focal plane, transports the samples, that can be aspired and push back and forward with an Arduino controlled stepper motor attached to a syringe or injector (Eppendorf CellTram). The controller allows performing micro-steps up to 0.225º that translate on samples steps of 2 microns (FEP tube of 1mm inner diameter) and to detect sample passage using photodiodes in order to automatize the process (see Sub-Systems).

In that way sample mounting procedure for some specific samples, as zebrafish embryos and larvae or 3D-cell cultures, gets less aggressive and extremely facilitated compared with agarose embedding methods.

The illumination block consists in a home-made laser combiner consisting in three lasers lines (L): 473 nm (DPSSL MBL-III-473-50), 561 nm (Coherent OBIS 561-50 LS) and 642 nm (Vortran Stradus 165 mW) and two dichroic beam splitters (DM): 505DCLP ans FF593-Di03-25x36 (Semrock). Excitation laser lines are selected using a Arduino controlled filter wheel (FW1) with three different filters (Semrock 473/10, 561/10 and 640/8). The laser illumination is controlled using a shutter (S) (Uniblitz electronics LS3T2) and a varying neutral density filter.

The laser scanning is carried in the vertical axis using a galvanometric mirror (GM) (6210H Cambridge Technologies) which optical plane is conjugated with the back focal aperture of an objective lens (Nikon Plan Fluor 4× 0.13 WD17.4 mm) using a 3.5× telescope system consisting in a 50 mm and a 180 mm achromatic lenses (Thorlabs).

For detection, an air objective (Nikon 10x 0.3NA WD 16.7 mm), placed perpendicularly to the excitation plane, is used to collect fluorescence emission. Excitation light is rejected using emission filters placed in infinity space before the camera, with filters mounted in a second automatic filter wheel (FW2), consisting of the following: HQ 535/70m-2p, HQ 580/25m-2p, HQ 620/90m-2p, and ET 700/75. Finally 200 mm tube lens creates the image on the chip of the sCMOS cameras (Hamamatsu Orca-Flash4).

We have also designed a motorized loading platform in order to fully automatize the sample management for large scale experiment. Multi-well plates are mounted in a XY stage consisting in two stepper motor stages (Thorlabs LTS150/M). A third DC motor (Thorlabs MTS25/M-Z8 with TDC001 controller) moves the FEP tube tip vertically at every position, in order to load the sample. In order to speed up the automated mounting system we use a photo-detection system to control sample positioning.

The upgrade also permits to control a secondary camera, which can be placed at different locations, thus increasing the flexibility of the system.

To the previous imaging modes (SPIM/DSLM and OPT) we added two more modes: F-SPIM and V-SPIM. F-SPIM provides low resolution high speed imaging of the samples as they flow through the FEP tube, crossing a static light-sheet plane, pushed by a controlled syringe pump. V-SPIM uses a camera placed vertically above the sample chamber, while the detection objective is mounted on a translational stage that moves in coordination with the galvo mirror, in order to refocus the selected plane. This allows obtaining high-resolution 3D images, while the aspiration system allows large sample compositions by sticking.

The configuration here proposed allows to image perform large scale experiments in two complementary ways. The flow detection arm, siting horizontally on the optical bench, allow to continuously image the sample plane, recoding images sequentially as the sample travels through this plane. Using the F-SPIM mode the controller provides small rotation angles up to (0.225º) synchronized with the camera for precise control of the sample flow imaging.

The second detection arm, placed vertically to the sample plane (V-SPIM mode), consists in a water dipping objective (Olympus x10 NA0.3 WD 3.5 mm) mounted on a DC motor (Thorlabs MTS25/M-Z8 with TDC001 controller), allowing to focus at different depths. Using a 50mm cylindrical lens (CL) (Thorlabs) (or alternatively a secondary galvo), we create a plane of illumination that can be scanned using the primary galvo mirror in coordination with the motorized focusing system. Both, the galvo and the filter wheels are controlled using open source hardware solutions, namely Arduino boards, as described in Sub-systems.

The flux diagram followed by those different modes: