Conventional imaging systems are capable of observing in 2D with relative high resolution (and even super resolution) with speeds that can vary from a few frames per second (fps) (confocal microscopy) to hundreds of fps (eg. LSFM). However, any interesting and fully useful dynamic biological process occurs in 3D (volumes).
Light Sheet Fluorescence Microscopy (LSFM) is a powerful technique for three-dimensional live imaging of biological samples that permits imaging large specimens over long periods of time. Typically, LSFM involves illuminating the sample with a beam, shaped into a thin sheet (the light sheet), in such a way that only a plane with thickness equivalent to that of the light sheet is excited. The light sheet is usually fixed and placed at the focal plane of the detection objective. The fluorescence light emitted from the fluorophores in this plane is collected by a perpendicular objective and registered by a camera as the sample is displaced through the light sheet, producing a three-dimensional image of the sample. Still, the need to physically move the sample entails that typical LSFM system are not fast enough to capture some of the fastest biological processes, such as neuronal calcium waves.
Wavefront Coding (WFC) is a technique that extends the depth of field (DoF) of the microscope by means of a phase mask, permitting the placement of the light sheet at different axial positions while still producing well-focused images. This way, it is possible to move the light-sheet through the sample (instead of the other way around), allowing recording speeds of tens of volumes per second.
However, WFC introduces a significant distortion on the observed image, which has to be corrected after acquisition by means of a deconvolution process. Traditionally, deconvolutions are made after the experiment has been completed, impeding the monitoring of the sample during it.
The aim of the MAFIn project is to develop a universally-compatible module capable of fast volumetric imaging under high resolution conditions, based on the combination of a LSFM with WFC techniques. In addition, the MAFIn module is also capable of correcting the optical aberrations suffered by the image by employing an Adaptive Optics (AO) system, which also provides the WFC capabilities of the system.
For this, a user friendly, real-time 3D deconvolution algorithm has been developed in order to allow monitoring of the sample during experiments. This algorithm is to be accompanied by an easy-to-use Graphical User Interface to control it along with the WFC and AO capabilities of the module.
