Wide-field intensity fluctuation imaging.

The temporal intensity fluctuations contain important information about the light source and light-medium interaction and are typically characterized by the intensity autocorrelation function, g2(τ). The measurement of g2(τ) is a central topic in many optical sensing applications, ranging from stellar intensity interferometer in astrophysics, to fluorescence correlation spectroscopy in biomedical sciences and blood flow measurement with dynamic light scattering. Currently, g2(τ) at a single point is readily accessible through high-frequency sampling of the intensity signal. However, two-dimensional wide-field imaging of g2(τ) is still limited by the cameras' frame rate. We propose and demonstrate a 2-pulse within-exposure modulation approach to break through the camera frame rate limit and obtain the quasi g2(τ) map in wide field with cameras of only ordinary frame rates.

Non-Degenerate Two-Photon Imaging of Deep Rodent Cortex using Indocyanine Green in the water absorption window.

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.

A Deep Learning Approach for Improving Two-Photon Vascular Imaging Speeds.

A potential method for tracking neurovascular disease progression over time in preclinical models is multiphoton fluorescence microscopy (MPM), which can image cerebral vasculature with capillary-level resolution. However, obtaining high-quality, three-dimensional images with traditional point scanning MPM is time-consuming and limits sample sizes for chronic studies. Here, we present a convolutional neural network-based (PSSR Res-U-Net architecture) algorithm for fast upscaling of low-resolution or sparsely sampled images and combine it with a segmentation-less vectorization process for 3D reconstruction and statistical analysis of vascular network structure. In doing so, we also demonstrate that the use of semi-synthetic training data can replace the expensive and arduous process of acquiring low- and high-resolution training pairs without compromising vectorization outcomes, and thus open the possibility of utilizing such approaches for other MPM tasks where collecting training data is challenging. We applied our approach to images with large fields of view from a mouse model and show that our method generalizes across imaging depths, disease states and other differences in neurovasculature. Our pretrained models and lightweight architecture can be used to reduce MPM imaging time by up to fourfold without any changes in underlying hardware, thereby enabling deployability across a range of settings.

Wide-field Intensity Fluctuation Imaging.

The temporal intensity fluctuations contain important information about the light source and light-medium interaction and are typically characterized by the intensity autocorrelation function, g2(τ). The measurement of g2(τ) is a central topic in many optical sensing applications, ranging from stellar intensity interferometer in astrophysics, to fluorescence correlation spectroscopy in biomedical sciences and blood flow measurement with dynamic light scattering. Currently, g2(τ) at a single point is readily accessible through high-frequency sampling of the intensity signal. However, two-dimensional wide-field measurement of g2(τ) is still limited by camera frame rates. We propose and demonstrate a 2-pulse within-exposure modulation approach to break through the camera frame rate limit and obtain the quasi g2(τ) map in wide field with cameras of only ordinary frame rates.

Pulse train gating to improve signal generation for in vivo two-photon fluorescence microscopy.

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.

Pulse train gating to improve signal generation for in vivo two-photon fluorescence microscopy.

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.

Evaluation of resonant scanning as a high-speed imaging technique for two-photon imaging of cortical vasculature.

We demonstrate a simple, low-cost two-photon microscope design with both galvo-galvo and resonant-galvo scanning capabilities. We quantify and compare the signal-to-noise ratios and imaging speeds of the galvo-galvo and resonant-galvo scanning modes when used for murine neurovascular imaging. The two scanning modes perform as expected under shot-noise limited detection and are found to achieve comparable signal-to-noise ratios. Resonant-galvo scanning is capable of reaching desired signal-to-noise ratios using less acquisition time when higher excitation power can be used. Given equal excitation power and total pixel dwell time between the two methods, galvo-galvo scanning outperforms resonant-galvo scanning in image quality when detection deviates from being shot-noise limited.

Diamond Raman laser and Yb fiber amplifier for in vivo multiphoton fluorescence microscopy.

Here we introduce a fiber amplifier and a diamond Raman laser that output high powers (6.5 W, 1.3 W) at valuable wavelengths (1060 nm, 1250 nm) for two-photon excitation of red-shifted fluorophores. These custom excitation sources are both simple to construct and cost-efficient in comparison to similar custom and commercial alternatives. Furthermore, they operate at a repetition rate (80 MHz) that allows fast image acquisition using resonant scanners. With our system we demonstrate compatibility with fast resonant scanning, the ability to acquire neuronal images, and the capability to image vasculature at deep locations (>1 mm) within the mouse cerebral cortex.