Wide field-of-view, multi-region, two-photon imaging of neuronal activity in the mammalian brain

Two-photon calcium imaging provides an optical readout of neuronal activity in populations of neurons with subcellular resolution. However, conventional two-photon imaging systems are limited in their field of view to ~1 mm2, precluding the visualization of multiple cortical areas simultaneously. Here, we demonstrate a two-photon microscope with an expanded field of view (>9.5 mm2) for rapidly reconfigurable simultaneous scanning of widely separated populations of neurons. We custom designed and assembled an optimized scan engine, objective, and two independently positionable, temporally multiplexed excitation pathways. We used this new microscope to measure activity correlations between two cortical visual areas in mice during visual processing

Deployment and Analysis of Instance Segmentation Algorithm for In-field Grade Estimation of Sweetpotatoes

Shape estimation of sweetpotato (SP) storage roots is inherently challenging due to their varied size and shape characteristics. Even measuring "simple" metrics, such as length and width, requires significant time investments either directly in-field or afterward using automated graders. In this paper, we present the results of a model that can perform grading and provide yield estimates directly in the field quicker than manual measurements. Detectron2, a library consisting of deep-learning object detection algorithms, was used to implement Mask R-CNN, an instance segmentation model. This model was deployed for in-field grade estimation of SPs and evaluated against an optical sorter. Storage roots from various clones imaged with a cellphone during trials between 2019 and 2020, were used in the model's training and validation to fine-tune a model to detect SPs. Our results showed that the model could distinguish individual SPs in various environmental conditions including variations in lighting and soil characteristics. RMSE for length, width, and weight, from the model compared to a commercial optical sorter, were 0.66 cm, 1.22 cm, and 74.73 g, respectively, while the RMSE of root counts per plot was 5.27 roots, with r^2 = 0.8. This phenotyping strategy has the potential enable rapid yield estimates in the field without the need for sophisticated and costly optical sorters and may be more readily deployed in environments with limited access to these kinds of resources or facilities.Comment: 21 pages, 11 figure

Imaging linear and circular polarization features in leaves with complete Mueller matrix polarimetry

Spectropolarimetry of intact plant leaves allows to probe the molecular architecture of vegetation photosynthesis in a non-invasive and non-destructive way and, as such, can offer a wealth of physiological information. In addition to the molecular signals due to the photosynthetic machinery, the cell structure and its arrangement within a leaf can create and modify polarization signals. Using Mueller matrix polarimetry with rotating retarder modulation, we have visualized spatial variations in polarization in transmission around the chlorophyll a absorbance band from 650 nm to 710 nm. We show linear and circular polarization measurements of maple leaves and cultivated maize leaves and discuss the corresponding Mueller matrices and the Mueller matrix decompositions, which show distinct features in diattenuation, polarizance, retardance and depolarization. Importantly, while normal leaf tissue shows a typical split signal with both a negative and a positive peak in the induced fractional circular polarization and circular dichroism, the signals close to the veins only display a negative band. The results are similar to the negative band as reported earlier for single macrodomains. We discuss the possible role of the chloroplast orientation around the veins as a cause of this phenomenon. Systematic artefacts are ruled out as three independent measurements by different instruments gave similar results. These results provide better insight into circular polarization measurements on whole leaves and options for vegetation remote sensing using circular polarization

Infrared Stokes Polarimetry and Spectropolarimetry

In this work, three methods of measuring the polarization state of light in the thermal infrared (3-12 microns) are modeled, simulated, calibrated and experimentally verified in the laboratory. The first utilizes the method of channeled spectropolarimetry (CP) to encode the Stokes polarization parameters onto the optical power spectrum. This channeled spectral technique is implemented with the use of two Yttrium Vanadate (YVO4) crystal retarders. A basic mathematical model for the system is presented, showing that all the Stokes parameters are directly present in the interferogram. Theoretical results are compared with real data from the system, an improved model is provided to simulate the effects of absorption within the crystal, and a modified calibration technique is introduced to account for this absorption. Lastly, effects due to interferometer instabilities on the reconstructions, including nonuniform sampling and interferogram translations, are investigated and techniques are employed to mitigate them.Second is the method of prismatic imaging polarimetry (PIP), which can be envisioned as the monochromatic application of channeled spectropolarimetry. Unlike CP, PIP encodes the 2-dimensional Stokes parameters in a scene onto spatial carrier frequencies. However, the calibration techniques derived in the infrared for CP are extremely similar to that of the PIP. Consequently, the PIP technique is implemented with a set of four YVO4 crystal prisms. A mathematical model for the polarimeter is presented in which diattenuation due to Fresnel effects and dichroism in the crystal are included. An improved polarimetric calibration technique is introduced to remove the diattenuation effects, along with the relative radiometric calibration required for the BPIP operating with a thermal background and large detector offsets. Data demonstrating emission polarization are presented from various blackbodies, which are compared to data from our Fourier transform infrared spectropolarimeter. Additionally, limitations in the PIP technique with regards to the spectral bandwidth and F/# of the imaging system are analyzed. A model able to predict the carrier frequency's fringe visibility is produced and experimentally verified, further reinforcing the PIP's limitations.The last technique is significantly different from CP or PIP and involves the simulation and calibration of a thermal infrared division of amplitude imaging Stokes polarimeter. For the first time, application of microbolometer focal plane array (FPA) technology to polarimetry is demonstrated. The sensor utilizes a wire-grid beamsplitter with imaging systems positioned at each output to analyze two orthogonal linear polarization states simultaneously. Combined with a form birefringent wave plate, the system is capable of snapshot imaging polarimetry in any one Stokes vector (S1, S2 or S3). Radiometric and polarimetric calibration procedures for the instrument are provided and the reduction matrices from the calibration are compared to rigorous coupled wave analysis (RCWA) and raytracing simulations. The design and optimization of the sensor's wire-grid beam splitter and wave plate are presented, along with their corresponding prescriptions. Polarimetric calibration error due to the spectrally broadband nature of the instrument is also overviewed. Image registration techniques for the sensor are discussed and data from the instrument are presented, demonstrating a microbolometer's ability to measure the small intensity variations corresponding to polarized emission in natural environments

White-light channeled imaging polarimeter using broadband polarization gratings

A white-light snapshot channeled linear imaging (CLI) polarimeter is demonstrated by utilizing polarization gratings (PGs). The CLI polarimeter is capable of measuring the two-dimensional distribution of the linear Stokes polarization parameters by incorporating two identical PGs, in series, along the optical axis. In this configuration, the general optical shearing functionality of a uniaxial crystal-based Savart plate is realized. However, unlike a Savart plate, the diffractive nature of the PGs creates a linear dependence of the shear versus wavelength, thus providing broadband functionality. Consequently, by incorporating the PG-based Savart plate into a Savart plate channeled imaging polarimeter, white-light interference fringes can be generated. This enables polarimetric image data to be acquired at shorter exposure times in daylight conditions, making it more appealing over the quasi-monochromatic channeled imaging polarimeters previously described in the literature. Furthermore, the PG-based device offers significantly more compactness, field of view, optical simplicity, and vibration insensitivity than previously described white-light CLI polarimeters based on Sagnac interferometers. Included in this paper are theoretical descriptions of the linear (S0, S1, and S2) and complete (S0, S1, S2, and S3) channeled Stokes imaging polarimeters. Additionally, descriptions of our calibration procedures and our experimental proof of concept CLI system are provided. These are followed by laboratory and outdoor polarimetric measurements of S0, S1, and S2

Snapshot imaging Mueller matrix polarimeter using polarization gratings

A snapshot imaging Mueller matrix polarimeter (SIMMP) is theoretically described and empirically demonstrated through simulation. Spatial polarization fringes are localized onto a sample by incorporating polarization gratings (PGs) into a polarization generator module. These fringes modulate the Mueller matrix (MM) components of the sample, which are subsequently isolated with PGs in an analyzer module. The MM components are amplitude modulated onto spatial carrier frequencies which, due to the PGs, maintain high visibility in spectrally broadband illumination. An interference model of the SIMMP is provided, followed by methods of reconstruction and calibration. Lastly, a numerical simulation is used to demonstrate the system's performance in the presence of noise

Field of View Based Optimization of Aspheric Designed Geometric-Phase Doublet Lenses

Here we study aspheric phase pattern geometric-phase lenses through the design of doublet lenses in Zemax Optic Studio and in MATLAB using a simple ray tracing code. The solutions developed use variable spacer distances and are solved monochromatically (at 633 nm) and polychromatically (at 450, 532, and 633 nm). After selecting a Zemax generated polychromatically optimized doublet solution, we fabricated the lens utilizing liquid crystal films and direct writing. We characterized the liquid crystal alignment quality, efficiencies, and spot performance of this lens in comparison to a standard spherical singlet of the same back focal length and diameter (24.5 mm diameter and 40 mm back focal length at 633 nm). With this aspheric lens profile, we realize the development of novel focusing phase surfaces