Mechanical vibration compensation method for 3D+t multi-particle tracking in microscopic volumes

The acquisition and analysis of data in microscopic systems with spatiotemporal evolution is a very relevant topic. In this work, we describe a method to optimize an experimental setup for acquiring and processing spatiotemporal (3D+t) data in microscopic systems. The method is applied to a three-dimensional multi-tracking and analysis system of free-swimming sperm trajectories previously developed. The experimental set uses a piezoelectric device making oscillate a large focal-distance objective mounted on an inverted microscope (over its optical axis) to acquire stacks of images at a high frame rate over a depth on the order of 250 microns. A problem arise when the piezoelectric device oscillates, in such a way that a vibration is transmitted to the whole microscope, inducing undesirable 3D vibrations to the whole set. For this reason, as a first step, the biological preparation was isolated from the body of the microscope to avoid modifying the free swimming pattern of the microorganism due to the transmission of these vibrations. Nevertheless, as the image capturing device is mechanically attached to the “vibrating” microscope, the resulting acquired data are contaminated with an undesirable 3D movement that biases the original trajectory of these high speed moving cells. The proposed optimization method determines the functional form of these 3D oscillations to neutralize them from the original acquired data set. Given the spatial scale of the system, the added correction increases significantly the data accuracy. The optimized system may be very useful in a wide variety of 3D+t applications using moving optical devices.