SignificanceTo enable non-destructive longitudinal assessment of drug agents in intact tumor tissue without the use of disruptive probes, we have designed a label-free method to quantify the health of individual tumor cells in excised tumor tissue using multiphoton fluorescence lifetime imaging microscopy (MP-FLIM).AimUsing murine tumor fragments which preserve the native tumor microenvironment, we seek to demonstrate signals generated by the intrinsically fluorescent metabolic co-factors nicotinamide adenine dinucleotide phosphate [NAD(P)H] and flavin adenine dinucleotide (FAD) correlate with irreversible cascades leading to cell death.ApproachWe use MP-FLIM of NAD(P)H and FAD on tissues and confirm viability using standard apoptosis and live/dead (Caspase 3/7 and propidium iodide, respectively) assays.ResultsThrough a statistical approach, reproducible shifts in FLIM data, determined through phasor analysis, are shown to correlate with loss of cell viability. With this, we demonstrate that cell death achieved through either apoptosis/necrosis or necroptosis can be discriminated. In addition, specific responses to common chemotherapeutic treatment inducing cell death were detected.ConclusionsThese data demonstrate that MP-FLIM can detect and quantify cell viability without the use of potentially toxic dyes, thus enabling longitudinal multi-day studies assessing the effects of therapeutic agents on tumor fragments.
Significance: Three-photon excitation microscopy has double-to-triple the penetration depth in biological tissue over two-photon imaging and thus has the potential to revolutionize the visualization of biological processes in vivo. However, unlike the plug-and-play operation and performance of lasers used in two-photon imaging, three-photon microscopy presents new technological challenges that require a closer look at the fidelity of laser pulses.
Aim: We implemented state-of-the-art pulse measurements and developed innovative techniques for examining the performance of lasers used in three-photon microscopy. We then demonstrated how these techniques can be used to provide precise measurements of pulse shape, pulse energy, and pulse-to-pulse intensity variability, all of which ultimately impact imaging.
Approach: We built inexpensive tools, e.g., a second harmonic generation frequency-resolved optical gating (SHG-FROG) device and a deep-memory diode imaging (DMDI) apparatus to examine laser pulse fidelity.
Results: First, SHG-FROG revealed very large third-order dispersion (TOD). This extent of phase distortion prevents the efficient temporal compression of laser pulses to their theoretical limit. Furthermore, TOD cannot be quantified when using a conventional method of obtaining the laser pulse duration, e.g., when using an autocorrelator. Finally, DMDI showed the effectiveness of detecting pulse-to-pulse intensity fluctuations on timescales relevant to three-photon imaging, which were otherwise not captured using conventional instruments and statistics.
Conclusions: The distortion of individual laser pulses caused by TOD poses significant challenges to three-photon imaging by preventing effective compression of laser pulses and decreasing the efficiency of nonlinear excitation. Moreover, an acceptably low pulse-to-pulse amplitude variability should not be assumed. Particularly for low repetition rate laser sources used in three-photon microscopy, pulse-to-pulse variability also degrades image quality. If three-photon imaging is to become mainstream, our diagnostics may be used by laser manufacturers to improve system design and by end-users to validate the performance of their current and future imaging systems.
Optical coherence tomography provides volumetric reconstruction of brain structure with micrometer resolution. Gray matter and white matter can be highlighted using conventional and polarization-based contrasts; however, vasculature in ex-vivo fixed brain has not been investigated at large scale due to lack of intrinsic contrast. We present contrast enhancement to visualize the vasculature by perfusing titanium dioxide particles transcardially into the mouse vascular system. The brain, after dissection and fixation, is imaged by a serial optical coherence scanner. Accumulation of particles in blood vessels generates distinguishable optical signals. Among these, the cross-polarization images reveal the vasculature organization remarkably well. The conventional and polarization-based contrasts are still available for probing the gray matter and white matter structures. The segmentation and reconstruction of the vasculature are presented by using a deep learning algorithm. Axonal fiber pathways in the mouse brain are delineated by utilizing the retardance and optic axis orientation contrasts. This is a low-cost method that can be further developed to study neurovascular diseases and brain injury in animal models.
Spinocerebellar ataxia type 1 (SCA1) is a fatal inherited neurodegenerative disease. Post-mortem studies showed neurodegeneration involving white matter components in the cerebral lobes, the cerebellar peduncles and the more distal cranial nerves in human patients. However, the progression of SCA1 in the brain remains unclear. We present the study of white matter atrophy of SCA1 mouse models using serial optical coherence scanner (SOCS).
SOCS consists of a polarization sensitive optical coherence tomography and a tissue slicer (vibratome) with associated controls for serial imaging. The optical system has 5.5 µm axial resolution and utilizes a scan lens or a water-immersion microscope objective to provide 10 µm or 4 µm lateral resolution, respectively. Brain imaging with SOCS showed that the reflectivity contrast portrays morphology, and the polarization contrasts primarily probe myelinated nerve fibers in the white matter. In the cerebellum, the cerebellar cortical layers and white matter are distinguished by using intrinsic optical contrasts. We use SOCS to image the cerebellums of SCA1 mouse models. Data have been acquired from multiple sections at different age groups.
The label-free contrasts show the pathological changes in molecular layer in SCA1 mouse models. White matter size in midline section was quantified at different time points to show white matter degeneration. Moment analysis for retardance contrast and distribution of axis orientation contrast reveal white matter atrophy. High-resolution (4 µm) SOCS visualizes the atrophy of fine features in midline sagittal cerebellum sections as well.
Glioblastoma is a primary malignant brain tumor characterized by highly migratory glioma cells capable of invading into surrounding healthy tissue. The mechanism and the physical paths by which glioma cells are capable of invading healthy brain are not well understood. Using human glioblastoma cell line U251 plated on healthy mouse brain slices, glioma cell migration behavior and dynamics are investigated by multimodality imaging.
Polarization-sensitive optical coherence tomography (PS-OCT) is used to delineate nerve fiber tracts. PS-OCT is capable of generating depth-resolved images of reflectivity, phase retardance and optic axis orientation. Because of the birefringence property of myelin sheath, nerve fiber tracts as small as a few tens of micrometers can be resolved from phase retardance images. Swept field confocal imaging system is used to image U251 cells expressing GFP-actin and brain vasculature stained by Isolectin B4. Cell migration is acquired by time-lapse imaging and then correlated with brain vasculature and nerve fiber tracts after fine registration of the two modalities.
We found out U251 cells preferentially adhere to and migrate along blood vessels. Our data do not suggest a strong correlation between U251 cell migration and white matter tracts distinguished by tissue birefringence. In addition, U251 cell motility is higher in gray matter compared with white matter. Finally, using higher temporal resolution and high magnification, we are able to observe short time-scale dynamic of U251 cells and the ability of U251 cells to exert forces as they deform blood vessels.
KEYWORDS: Brain mapping, Cerebellum, System on a chip, Coherence (optics), Scanners, Optical coherence tomography, Polarization, Vibration control, Control systems, Visualization
We present the serial optical coherence scanner (SOCS), which consists of a polarization sensitive optical coherence tomography and a vibratome with associated controls for serial imaging, to visualize the cerebellum and adjacent brainstem of mouse. The cerebellar cortical layers and white matter are distinguished by using intrinsic optical contrasts. Images from serial scans reveal the large-scale anatomy in detail and map the nerve fiber pathways in the cerebellum and adjacent brainstem. The optical system, which has ~5.5 μm axial resolution, utilizes a scan lens or a water-immersion microscope objective resulting in 10 μm or 4 μm lateral resolution, respectively. The large-scale brain imaging at high resolution requires an efficient way to collect large datasets. It is important to improve the SOCS system to deal with large-scale and large number of samples in a reasonable time. The imaging and slicing procedure for a section took about 4 minutes due to a low speed of the vibratome blade to maintain slicing quality. SOCS has potential to investigate pathological changes and monitor the effects of therapeutic drugs in cerebellar diseases such as spinocerebellar ataxia 1 (SCA1). The SCA1 is a neurodegenerative disease characterized by atrophy and eventual loss of Purkinje cells from the cerebellar cortex, and the optical contrasts provided by SOCS is being evaluated for biomarkers of the disease.
We present the visualization of the mouse cerebellum and adjacent brainstem using a serial optical coherence scanner, which integrates a vibratome slicer and polarization-sensitive optical coherence tomography for ex vivo imaging. The scanner provides intrinsic optical contrasts to distinguish the cerebellar cortical layers and white matter. Images from serial scans reveal the large-scale anatomy in detail and map the nerve fiber pathways in the cerebellum and brainstem. By incorporating a water-immersion microscope objective, we also present high-resolution tiled images that delineate fine structures in the cerebellum and brainstem.
The optic axis of birefringent samples indicates the direction of optical anisotropy, which should be described in three-dimensional (3-D) space. We present a method to quantify the complete 3-D optic axis orientation calculated from in-plane optic axis measurements from a polarization-sensitive optical coherence tomography system. The in-plane axis orientations with different illumination angles allow the calculation of the necessary polar angle. The method then provides the information to produce the actual birefringence. The method and results from a biological sample are presented.
The optic axis of birefringent tissues indicates the direction of structural anisotropy. Polarization-sensitive Optical Coherence Tomography (PS-OCT) can provide reflectivity contrast as well as retardance and optic axis orientation contrasts that originate from tissue birefringence. We introduce imaging 3D tissue anisotropy by using a single-camera and polarization-maintaining fiber (PMF) based PS-OCT, which utilizes normal and angled illuminations.
Because environmental factors such as the movement of PMF and temperature fluctuations induce arbitrary phase changes, the optic axis orientation measurement has a time-varying offset. In order to measure the absolute axis orientation, we add a calibration path which dynamically provides the arbitrary offset to be subtracted from the relative axis orientation values.
The axis orientation on the normal plane is the 2D projection of the fiber direction in 3D space. We propose to characterize the axis orientation in different planes (xy, xy’ and x’y planes) by using normal and angled illuminations. This allows calculation of the polar angle that completes the orientation information in 3D. Polarization-based optical systems relying on one illumination angle measure the “apparent birefringence” that light encounters rather than the “true birefringence”. Birefringence as a measure of anisotropy is quantified with the orientation information in 3D. The method and validation with a biological tissue are presented. The study can facilitate imaging and mapping the structural connections in anisotropic tissues including the brain.
Target acquisition is of great importance for ship borne range-gated night vision system which can achieve target finding,
target tracing and ranging. A digital image processing algorithm is developed for the mentioned night vision equipment
above. Target contour is extracted using Canny edge detection algorithm based on self-adapted Otsu threshold
segmentation. Furthermore, edge thinning, edge connection and morphologic methods are implemented to ameliorate the
acquired contour. Pixels inside the contour are collected utilizing horizontal-vertical traverse. After ship targets from
range-gated equipment being all tested, target contour and inner pixels can both be acquired through this algorithm.
How to simulate the decay pattern is crucial during lifetime inversion while utilizing intensity images acquired at
increasing delays in time gated fluorescence lifetime imaging microscopy (FLIM) method. A relatively novel
understanding of fluorescence decay pattern theory and stimulation algorithms of time gated FLIM method have been
analyzed in this paper comprehensively. Main lifetime computing algorithms can be classified as exponential pattern
retrieve and polynomial fitting procedure. Especially, a novel lifetime computing method based on bi-exponential decay
has been discussed. In experiment, we have validated the proposed algorithms utilizing synthetic images. Performances
like calculating precision and computing speed of the algorithms above have also been compared.
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