Content area
This dissertation presents the design and development of VitaSlice, a software developed to control a custom-built Light Sheet Fluorescence Microscope (LSFM). The software was developed at the Advanced Light Microscopy (ALM) scientific platform of i3S – Institute for Research and Innovation in Health - and is optimized for use in a shared facility environment. Specifically, it is tailored to provide accessibility of a custom-built system to users without optical or technical expertise.
While commercial LSFM systems offer robust performance and ease of use, their cost can exceed six figures, often putting them beyond the financial reach of some research institutions. In contrast, the development of a custom system significantly reduces the cost by employing individual hardware devices and open-source software. For this reason, this type of microscope is highly suitable for institutions that intend to provide access to advanced imaging techniques within budget constraints.
The microscope is based on the Digital Scanned Laser Light Sheet Microscope (DSLM) architecture. Its volumetric imaging is done using a movable sample and a scanned laser beam to simulate a planar light sheet. The microscope’s optical system is composed of four distinct lasers, one-sided illumination, dual-camera detection, filter wheels, translation and rotation stages, and galvanometric scanning mirrors. Time synchronicity is achieved using a ScanLab RTC5 control board as a master, which sends TTL signals to the scanning system, cameras, and lasers. The optical system was aligned with careful attention to beam collinearity and proper focal plane positioning of the beams, ensuring appropriate sample illumination and image acquisition, enhancing the resulting quality.
To ensure hardware coordination between the devices, a custom-made communication box was designed and implemented. It was built to handle the electrical redistribution of the signals from the RTC5 board to all four lasers’ digital modulation inputs and the cameras’ external trigger inputs. The internal circuit was built using a Darlington transistor array integrated circuit for current amplification and voltage-dividing resistors for signal adaptation. The box provides five BNC output terminals for lasers and camera triggering. Its success was validated through direct signal measurement using an oscilloscope, as well as the successful activation of laser emission and camera exposure triggering as a response to the emitted signals.
A Python-based software was developed to control the microscope. It was built from scratch, communicating with each microscope device through high-level Application Programming Interfaces (APIs), and employing a modular, widget-based graphical user interface built with the PySide6 library. The embedding of napari enables real-time image display, and the use of the OME-Zarr format for the image files ensures storage in compliance with modern bioimaging standards, while also employing built-in metadata.
The software supports real-time imaging, multi-channel volumetric imaging, and timelapse acquisition. Its technical success was validated through a series of imaging experiments, including the acquisition of relevant biological samples, such as microglia and oligodendrocyte primary cells encapsulated in hydrogels, and zebrafish larvae. The system demonstrated high throughput of image data, with approximately 470 frames per minute in volumetric acquisitions, and high stability over long acquisition periods. Its usability was also approved by the facility users’ feedback.
VitaSlice, by allowing control of a complex microscopy system through an intuitive approach, showcases the potential of modern, custom-built tools and precise engineering in granting access to advanced microscopy techniques. In this way, the work presented in this dissertation makes a positive contribution to the field of accessible scientific instrumentation.