SPIM-DSLM

SPIM/DSLM/OPT

The SPIM/DSLM microscope can work in two different imaging regimes depending on the laser (L). The linear mode uses an Argon/Krypton laser providing excitation wavelengths of 488 nm, 568 nm and 647, while the non-linear mode uses a Ti:Sapphire laser with tunable excitation between 750 and 900 nm. The switch between the two imaging modes is performed manually through a flip mount mirror. In the case of the linear mode the excitation laser lines are selected using a filter wheel (FW1) with four different filters. The laser power is controlled using a varying neutral density filter. A shutter (S) is used to block the laser beam and control the light dosage applied to the sample for long time lapse recording when is only opened during image acquisition. In order to create the light sheet on the sample plane we can also choose two different modalities, DSLM, scanning in the vertical axis the laser beam using a galvanometric mirror (GM), or classical SPIM, stopping the galvanometer at 0 V and using a 50 mm focal length cylindrical lens (CL). In both cases the optical plane of the galvanometer is conjugated with the back focal aperture of the excitation objective (EO) lens using a 3.5x or 8x lens telescope (LT). In SPIM mode, the cylindrical lens is inserted in the optical path in such a way that the horizontal axis of the beam is focused on the back aperture of the excitation objective, while the vertical axis fills the aperture. In DSLM mode the cylindrical lens is replaced with a hollow tube lens to maintain the distance between optics. The light sheet created either by the cylindrical lens or the scanning galvo creates fluorescence at the focal plane of detection objective (DO), which is imaged at the camera (C) by a tube lens (TL). After the objective, the excitation wavelengths are rejected using different emission filters placed in infinity space before the camera. Those filters are mounted in a custom made motorized filter wheel (FW2). Different planes are collected by moving the sample using a translational stage (TS) through the light sheet. Multi-view images are obtained by rotating the sample with a stepper motor (SM). This easy and cheap solution allows up to 0.225 degrees steps and is controlled with an Arduino UNO board. Sample centering (X/Y axis) on the field of view of the camera is performed manually with two linear translation stages. The same system can be used for OPT microscopy. The OPT microscope uses the detection arm of the SPIM/DSLM system with frontal illumination performed with a LED system for transmitted light OPT and lateral illumination with a blue LED. Different chambers (SC) have been designed for water immersion and air detection objectives.