KEYWORDS: Optogenetics, 3D modeling, Monte Carlo methods, Photon transport, Tissue optics, Optical fibers, Brain, Fiber optics, Scattering, Diffusion, Chemical elements
Configuring the light power emitted from the optical fiber is an essential first step in planning in-vivo optogenetic
experiments. However, diffusion theory, which was adopted for optogenetic research, precluded accurate estimates of
light intensity in the semi-diffusive region where the primary locus of the stimulation is located. We present a 3D Monte
Carlo model that provides an accurate and direct solution for light distribution in this region. Our method directly records
the photon trajectory in the separate volumetric grid planes for the near-source recording efficiency gain, and it
incorporates a 3D brain mesh to support both homogeneous and heterogeneous brain tissue. We investigated the light
emitted from optical fibers in brain tissue in 3D, and we applied the results to design optimal light delivery parameters
for precise optogenetic manipulation by considering the fiber output power, wavelength, fiber-to-target distance, and the
area of neural tissue activation.
Two-photon microscopy is a very attractive tool for the study of the three-dimensional (3D) and dynamic processes in
cells and tissues. One of the feasible constructions of two-photon microscopy is the combination a confocal laser
scanning microscope and a mode-locked Ti:sapphire laser. Even though this approach is the simplest and fastest
implementation, this system is highly cost-intensive and considerably difficult in modification. Many researcher
therefore decide to build a more cost-effective and flexible system with a self-developed software for operation and data
acquisition. We present a custom-built two-photon microscope based on a mode-locked Yb3+ doped fiber laser and
demonstrate two-photon fluorescence imaging of biological specimens. The mode-locked fiber laser at 1060 nm delivers
320 fs laser pulses at a frequency of 36 MHz up to average power of 80 mW. The excitation at 1060 nm can be more
suitable in thick, turbid samples for 3D image construction as well as cell viability. The system can simply accomplish
confocal and two-photon mode by an additional optical coupler that allows conventional laser source to transfer to the
scanning head. The normal frame rate is 1 frames/s for 400 x 400 pixel images. The measured full width at half
maximum resolutions were about 0.44 μm laterally and 1.34 μm axially. A multi-color stained convallaria, rat basophilic
leukemia cells and a rat brain tissue were observed by two-photon fluorescence imaging in our system.
KEYWORDS: Heart, Video, Tissues, 3D image processing, Two photon excitation microscopy, Mirrors, Quantitative analysis, Data acquisition, Imaging systems, Medicine
Cardiac architecture is inherently three-dimensional, yet most characterizations rely on two-dimensional histological slices or dissociated cells, which remove the native geometry of the heart. We previously developed a method for labeling intact heart sections without dissociation and imaging large volumes while preserving their three-dimensional structure. We further refine this method to permit quantitative analysis of imaged sections. After data acquisition, these sections are assembled using image-processing tools, and qualitative and quantitative information is extracted. By examining the reconstructed cardiac blocks, one can observe end-to-end adjacent cardiac myocytes (cardiac strands) changing cross-sectional geometries, merging and separating from other strands. Quantitatively, representative cross-sectional areas typically used for determining hypertrophy omit the three-dimensional component; we show that taking orientation into account can significantly alter the analysis. Using fast-Fourier transform analysis, we analyze the gross organization of cardiac strands in three dimensions. By characterizing cardiac structure in three dimensions, we are able to determine that the crystallin mutation leads to hypertrophy with cross-sectional area increases, but not necessarily via changes in fiber orientation distribution.
The tongue consists of a complex, multiscale array of myofibers that comprise the anatomical underpinning of lingual mechanical function. 3-D myoarchitecture was imaged in mouse tongues with diffusion spectrum magnetic resonance imaging (DSI) at 9.4 T (bmax 7000 s/mm, 150-µm isotropic voxels), a method that derives the preferential diffusion of water/voxel, and high-throughput (10 fps) two-photon microscope (TPM). Net fiber alignment was represented for each method in terms of the local maxima of an orientational distribution function (ODF) derived from the local diffusion (DSI) and 3-D structural autocorrelation (TPM), respectively. Mesoscale myofiber tracts were generated by alignment of the principal orientation vectors of the ODFs. These data revealed a consistent relationship between the properties of the respective ODFs and the virtual superimposition of the distributed mesoscale myofiber tracts. The identification of a mesoscale anatomical construct, which specifically links the microscopic and macroscopic spatial scales, provides a method for relating the orientation and distribution of cells and subcellular components with overall tissue morphology, thus contributing to the development of multiscale methods for mechanical analysis.
The ability to apply quantifiable mechanical stresses at the microscopic scale is critical for studying cellular responses to mechanical forces. This necessitates the use of force transducers that can apply precisely controlled forces to cells while monitoring the responses non- invasively. This paper describes the development of a micro manipulation workstation integrating two-photon, 3-D imaging with a high-force, uniform-gradient, magnetic manipulator. The uniform-gradient magnetic field applies nearly equal forces to a large cell population, permitting statistical quantification of select molecular responses to mechanical stresses. The magnetic transducer design is capable of exerting over 200 pN of force on 4.5 micrometers diameter paramagnetic particles and over 800 pN on 5.0 micrometers ferromagnetic particles. These forces vary less than 10% over an area 200 x 200 micrometers 2. The compatibility with the use of high numerical aperture (approximately equals 1.0) objectives is an integral part of the workstation design allowing sub- micron resolution 3-D two-photon imaging. Three dimensional maps of cellular deformation under localized mechanical strain are reported. These measurements indicate that the response of cells to large focal stresses is not always a local deformation.
We present the design of a magnetic tweezers microscope for cellular manipulation. Our design allows versatile and significant 3D stress application over a large sample region. For linear force application, forces up to 250 pN per 4.5 micrometers magnetic bead can be applied. Finite element analysis shows that variance in force level is around 10 percent within an area of 300 X 300 micrometers 2. Our eight-pole design potentially allows 3D liner force application and exertion of torsional stress. Furthermore, our design allows high resolution imaging using high numerical aperture objective. Both finite element analysis of magnetic field distribution and force calibration of our design are presented. As a feasibility study, we incubated fibronectin coated 4.5 micrometers polystyrene beads with Swiss 3T3 mouse fibroblast cells. Under application around 250 pN of force per magnetic particle, we observed relative movement between attached magnetic and polystyrene beads to be on the order of 1 micrometers . Elastic, viscoelastic, and creeping responses of cell surfaces were observed. Our results are consistent with previous observations using similar magnetic techniques.
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