No abstract

Antibiotic-resistant pathogens are a major health concern in everyday clinical practice. Because their detection by conventional microbial techniques requires minimally 24 h, some of us have recently introduced a nanomechanical sensor, which can reveal motion at the nanoscale. By monitoring the fluctuations of the sensor, this technique can evidence the presence of bacteria and their susceptibility to antibiotics in less than 1 h. Their amplitude correlates to the metabolism of the bacteria and is a powerful tool to characterize these microorganisms at low densities. This technique is new and calls for an effort to optimize its protocol and determine its limits. Indeed, many questions remain unanswered, such as the detection limits or the correlation between the bacterial distribution on the sensor and the detection's output. In this work, we couple fluorescence microscopy to the nanomotion investigation to determine the optimal experimental protocols and to highlight the effect of the different bacterial distributions on the sensor.

The application of 2-photon laser scanning microscopy (TPLSM) techniques to measure the dynamics of cellular calcium signals in populations of neurons is an extremely powerful technique for characterizing neural activity within the central nervous system. The use of TPLSM on awake and behaving subjects promises new insights into how neural circuit elements cooperatively interact to form sensory perceptions and generate behavior. A major challenge in imaging such preparations is unavoidable animal and tissue movement, which leads to shifts in the imaging location (jitter). The presence of image motion can lead to artifacts, especially since quantification of TPLSM images involves analysis of fluctuations in fluorescence intensities for each neuron, determined from small regions of interest (ROIs). Here, we validate a new motion correction approach to compensate for motion of TPLSM images in the superficial layers of auditory cortex of awake mice. We use a nominally uniform fluorescent signal as a secondary signal to complement the dynamic signals from genetically encoded calcium indicators. We tested motion correction for single plane time lapse imaging as well as multiplane (i.e., volume) time lapse imaging of cortical tissue. Our procedure of motion correction relies on locating the brightest neurons and tracking their positions over time using established techniques of particle finding and tracking. We show that our tracking based approach provides subpixel resolution without compromising speed. Unlike most established methods, our algorithm also captures deformations of the field of view and thus can compensate e.g., for rotations. Object tracking based motion correction thus offers an alternative approach for motion correction, one that is well suited for real time spike inference analysis and feedback control, and for correcting for tissue distortions.

For their capacity to shape optical wavefronts in real time into any desired illumination pattern, phase-only Spatial Light Modulators (SLM) have proven to be powerful tools for optical trapping and micromanipulation applications. SLMs are also becoming increasingly utilized in selective photo-stimulation of groups of neurons in the brain. However, conventional SLM based wavefront modulation introduces artifacts that are particularly detrimental for photo-stimulation applications. The primary issue is the unmodulated light that travels along the 0th order of diffraction. This portion of light is commonly blocked at the center of the object plane, which prevents photo-stimulation in the blocked region. We demonstrate a virtual lens configuration that moves the 1st order diffraction with the desired illumination pattern into the Fourier plane of the 0th order light. This virtual lens setup makes the whole field of view accessible for photo-stimulation and eliminates the need for removing the 0th order light in two-photon applications. Furthermore, in an example application to reconstruct a pattern consisting of an array of points, the virtual lens configuration increases the uniformity of the intensities these points. Moreover, diffraction-induced artifacts are also significantly reduced within the target plane. Therefore, our proposed high fidelity configuration yields target points with high signal to noise ratio.

The application of 2-photon laser scanning microscopy (TPLSM) techniques to measure the dynamics of cellular calcium signals in populations of neurons is an extremely powerful technique for characterizing neural activity within the central nervous system. The use of TPLSM on awake and behaving subjects promises new insights into how neural circuit elements cooperatively interact to form sensory perceptions and generate behavior. A major challenge in imaging such preparations is animal and tissue movement, which leads to shifts in the imaging location (jitter). Although there are surgical and technical approaches to minimize brain motion under these conditions, it is generally unavoidable. The presence of image motion can lead to artifacts, especially since quantification of TPLSM images involves analysis of fluctuations in fluorescence intensities for each neuron, determined from small regions of interest (ROIs).Here, we validated a new motion correction approach to compensate for motion of TPLSM images in the superficial layers of auditory cortex of awake mice. We use a nominally uniform fluorescent signal as a secondary signal to complement the dynamic signals from genetically encoded calcium indicators. We tested motion correction for single plane time lapse imaging as well as multiplane (i.e. volume) time lapse imaging of cortical tissue. Our procedure of motion compensation relies on locating the brightest neurons and tracking their positions over time using established techniques of particle finding and tracking. The performance of our technique both for 2D (single plane) and 3D (volume) image sequences is comparable to established techniques in its ability to suppress false neural signals. Object tracking based motion compensation thus offers an alternative approach for motion compensation, one that is well suited for real time feedback control and for analysis of tissue distortions.