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1.
Appl Opt ; 59(34): 10673-10679, 2020 Dec 01.
Artículo en Inglés | MEDLINE | ID: mdl-33361885

RESUMEN

A liquid crystal variable retarder (LCVR) enables fast, automated control of retardance that can be used as a variable waveplate in polarimetric instruments. However, precise control of the polarization state requires calibration of the LCVR. A manufacturer calibration curve is typically supplied for a single specific wavelength and temperature, but for applications under different conditions, additional calibration is needed. Calibration is typically performed with crossed polarizers to generate an intensity curve that is converted to retardance, but this method is prone to noise when retardance is close to zero. Here, we demonstrate a simple common-path Sagnac interferometer to measure retardance and provide open source software for automated generation of calibration curves for retardance as a function of wavelength and voltage. We also provide a curve fitting method and closed-form functional representation that outputs the voltage needed to achieve a desired retardance given a specified wavelength.

2.
Biomed Opt Express ; 15(9): 5053-5066, 2024 Sep 01.
Artículo en Inglés | MEDLINE | ID: mdl-39296386

RESUMEN

We present a novel approach for deep vascular imaging in rodent cortex at excitation wavelengths susceptible to water absorption using two-photon microscopy with photons of dissimilar wavelengths. We demonstrate that non-degenerate two-photon excitation (ND-2PE) enables imaging in the water absorption window from 1400-1550 nm using two excitation sources with temporally overlapped pulses at 1300 nm and 1600 nm that straddle the absorption window. We explore the brightness spectra of indocyanine green (ICG) and assess its suitability for imaging in the water absorption window. Further, we demonstrate in vivo imaging of the rodent cortex vascular structure up to 1.2 mm using ND-2PE. Lastly, a comparative analysis of ND-2PE at 1435 nm and single-wavelength, two-photon imaging at 1300 nm and 1435 nm is presented. Our work extends the excitation range for fluorescent dyes to include water absorption regimes and underscores the feasibility of deep two-photon imaging at these wavelengths.

3.
bioRxiv ; 2024 Jan 16.
Artículo en Inglés | MEDLINE | ID: mdl-38293101

RESUMEN

We present a novel approach for deep vascular imaging in rodent cortex at excitation wavelengths susceptible to water absorption using two-photon microscopy with photons of dissimilar wavelengths. We demonstrate that non-degenerate two-photon excitation (ND-2PE) enables imaging in the water absorption window from 1400-1550 nm using two synchronized excitation sources at 1300 nm and 1600 nm that straddle the absorption window. We explore the brightness spectra of indocyanine green (ICG) and assess its suitability for imaging in the water absorption window. Further, we demonstrate in vivo imaging of the rodent cortex vascular structure up to 1.2 mm using ND-2PE. Lastly, a comparative analysis of ND-2PE at 1435 nm and single-wavelength, two-photon imaging at 1300 nm and 1435 nm is presented. Our work extends the excitation range for fluorescent dyes to include water absorption regimes and underscores the feasibility of deep two-photon imaging at these wavelengths.

4.
J Cereb Blood Flow Metab ; : 271678X241270465, 2024 Aug 07.
Artículo en Inglés | MEDLINE | ID: mdl-39113424

RESUMEN

This manuscript quantitatively investigates remodeling dynamics of the cortical microvascular network (thousands of connected capillaries) following photothrombotic ischemia (cubic millimeter volume, imaged weekly) using a novel in vivo two-photon angiography and high throughput vascular vectorization method. The results suggest distinct temporal patterns of cerebrovascular plasticity, with acute remodeling peaking at one week post-stroke. The network architecture then gradually stabilizes, returning to a new steady state after four weeks. These findings align with previous literature on neuronal plasticity, highlighting the correlation between neuronal and neurovascular remodeling. Quantitative analysis of neurovascular networks using length- and strand-based statistical measures reveals intricate changes in network anatomy and topology. The distance and strand-length statistics show significant alterations, with a peak of plasticity observed at one week post-stroke, followed by a gradual return to baseline. The orientation statistic plasticity peaks at two weeks, gradually approaching the (conserved across subjects) stroke signature. The underlying mechanism of the vascular response (angiogenesis vs. tissue deformation), however, is yet unexplored. Overall, the combination of chronic two-photon angiography, vascular vectorization, reconstruction/visualization, and statistical analysis enables both qualitative and quantitative assessments of neurovascular remodeling dynamics, demonstrating a method for investigating cortical microvascular network disorders and the therapeutic modes of action thereof.

5.
Neurophotonics ; 10(4): 045006, 2023 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-37937198

RESUMEN

Significance: Two-photon microscopy is used routinely for in vivo imaging of neural and vascular structures and functions in rodents with a high resolution. Image quality, however, often degrades in deeper portions of the cerebral cortex. Strategies to improve deep imaging are therefore needed. We introduce such a strategy using the gating of high repetition rate ultrafast pulse trains to increase the signal level. Aim: We investigate how the signal generation, signal-to-noise ratio (SNR), and signal-to-background ratio (SBR) improve with pulse gating while imaging in vivo mouse cerebral vasculature. Approach: An electro-optic modulator with a high-power (6 W) 80 MHz repetition rate ytterbium fiber amplifier is used to create gates of pulses at a 1 MHz repetition rate. We first measure signal generation from a Texas Red solution in a cuvette to characterize the system with no gating and at a 50%, 25%, and 12.5% duty cycle. We then compare the signal generation, SNR, and SBR when imaging Texas Red-labeled vasculature using these conditions. Results: We find up to a 6.73-fold increase in fluorescent signal from a cuvette when using a 12.5% duty cycle pulse gating excitation pattern as opposed to a constant 80 MHz pulse train at the same average power. We verify similar increases for in vivo imaging to that observed in cuvette testing. For deep imaging, we find that pulse gating results in a 2.95-fold increase in the SNR and a 1.37-fold increase in the SBR on average when imaging mouse cortical vasculature at depths ranging from 950 to 1050 µm. Conclusions: We demonstrate that a pulse gating strategy can either be used to limit heating when imaging superficial brain regions or used to increase signal generation in deep regions. These findings should encourage others to adopt similar pulse gating excitation schemes for imaging neural structures through two-photon microscopy.

6.
bioRxiv ; 2023 Apr 03.
Artículo en Inglés | MEDLINE | ID: mdl-37066310

RESUMEN

Significance: Two-photon microscopy is used routinely for in vivo imaging of neural and vascular structure and function in rodents with a high resolution. Image quality, however, often degrades in deeper portions of the cerebral cortex. Strategies to improve deep imaging are therefore needed. We introduce such a strategy using gates of high repetition rate ultrafast pulse trains to increase signal level. Aim: We investigate how signal generation, signal-to-noise ratio (SNR), and signal-to-background ratio (SBR) improve with pulse gating while imaging in vivo mouse cerebral vasculature. Approach: An electro-optic modulator is used with a high-power (6 W) 80 MHz repetition rate ytterbium fiber amplifier to create gates of pulses at a 1 MHz repetition rate. We first measure signal generation from a Texas Red solution in a cuvette to characterize the system with no gating and at a 50%, 25%, and 12.5% duty cycle. We then compare signal generation, SNR, and SBR when imaging Texas Red-labeled vasculature using these conditions. Results: We find up to a 6.73-fold increase in fluorescent signal from a cuvette when using a 12.5% duty cycle pulse gating excitation pattern as opposed to a constant 80 MHz pulse train. We verify similar increases for in vivo imaging to that observed in cuvette testing. For deep imaging we find pulse gating to result in a 2.95-fold increase in SNR and a 1.37-fold increase in SBR on average when imaging mouse cortical vasculature at depths ranging from 950 µm to 1050 µm. Conclusions: We demonstrate that a pulse gating strategy can either be used to limit heating when imaging superficial brain regions or used to increase signal generation in deep regions. These findings should encourage others to adopt similar pulse gating excitation schemes for imaging neural structure through two-photon microscopy.

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