Pulse compression in two-photon excitation fluorescence microscopy

Liang, Xiaobao; Hu, Wenyan; Fu, Ling

doi:10.1364/oe.18.014893

Cited by 17 publications

(9 citation statements)

References 35 publications

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“…We applied a long-pass filter cut-on at 750 nm and a short-pass filter cut-off at 850 nm to enable a spectrum centered around 824 nm for Alexa Fluor 546 excitation ( Mütze et al., 2012 ), and the same long-pass filter with a short-pass filter cut-off at 1,000 nm to allow a spectrum centered around 919 nm for Thy1-GFP excitation ( Drobizhev et al., 2011 ). A grating-pair for pulse pre-chirping ( Liang et al., 2010 ) was finally utilized to make pulse durations at the focal point of the objective lens shorter than 60 fs in each case.…”

Section: Methodsmentioning

confidence: 99%

Nyquist-exceeding high voxel rate acquisition in mesoscopic multiphoton microscopy for full-field submicron resolution resolvability

et al. 2021

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Summary The Nyquist-Shannon criterion has never been realized in a laser-scanning mesoscopic multiphoton microscope (MPM) with a large field-of-view (FOV)-resolution ratio, especially when employing a high-frequency resonant-raster-scanning. With a high optical resolution nature, a current mesoscopic-MPM either neglects the criterion and degrades the digital resolution to twice the pixel size, or reduces the FOV and/or the raster-scanning speed to avoid aliasing . We introduce a Nyquist figure-of-merit (NFOM) parameter to characterize a laser-scanning MPM in terms of its optical-resolution retrieving ability. Based on NFOM, we define the maximum aliasing-free FOV, and subsequently, a cross-over excitation wavelength, below which the FOV becomes NFOM-constrained irrespective of an optimized optical design. We validate our idea in a custom-built mesoscopic-MPM with millimeter-scale FOV yielding an ultra-high FOV-resolution ratio of >3,000, while securing up-to a 1.6 mm Nyquist-satisfied aliasing-free FOV, a ∼400 nm lateral resolution, and a 70 M/s effective voxel-sampling rate, all at the same time.

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Section: Methodsmentioning

confidence: 99%

Nyquist-exceeding high voxel rate acquisition in mesoscopic multiphoton microscopy for full-field submicron resolution resolvability

et al. 2021

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“…Long-pass and short-pass filters with cut-on and cut-off at 750 and 1000 nm, respectively, were used to ensure a spectrum centered around 919 nm. A pulse duration of <60 fs was ensured after the objective lens by means of pulse pre-chirping (Liang et al, 2010) with a grating-pair.…”

Section: Assessment and Comparison Of Time Complexity And Validation ...mentioning

confidence: 99%

A rapid denoised contrast enhancement method digitally mimicking an adaptive illumination in submicron-resolution neuronal imaging

Borah

Sun

2022

iScience

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Optical neuronal imaging often shows ultrafine structures, such as a nerve fiber, coexisting with ultrabright structures, such as a soma with a substantially higher fluorescence-protein concentration. Owing to experimental and environmental factors, a laser-scanning multiphoton optical microscope (MPM) often encounters a high-frequency background noise that might contaminate such weak-intensity ultrafine neuronal structures. A straightforward contrast enhancement often leads to the saturation of the brighter ones, and might further amplify the high-frequency background noise. We report a digital approach called rapid denoised contrast enhancement (DCE), which digitally mimics a hardware-based adaptive/controlled illumination technique by means of digitally optimizing the signal strengths and hence the visibility of such weak-intensity structures while mostly preventing the saturation of the brightest ones. With large field-of-view (FOV) two-photon excitation fluorescence (TPEF) neuronal imaging, we validate the effectiveness of DCE over state-ofthe-art digital image processing algorithms. With compute-unified-device-architecture (CUDA)-acceleration, a real-time DCE is further enabled with a reduced time complexity.

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“…Long-pass and short-pass filters with cut-on and cut-off at 750 nm and 1000 nm, respectively, were used to ensure a spectrum centered around 919 nm. A pulse duration of <60 fs was ensured after the objective lens by means of pulse pre-chirping [67] with a grating-pair.…”

Section: Author Contributionsmentioning

confidence: 99%

Real-time Noise-suppressed Wide-Dynamic-Range Compression in Ultrahigh-Resolution Neuronal Imaging

Sun

2021

Preprint

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With a limited dynamic range of an imaging system, there are always regions with signal intensities comparable to the noise level, if the signal intensity distribution is close to or even wider than the available dynamic range. Optical brain/neuronal imaging is such a case where weak-intensity ultrafine structures, such as, nerve fibers, dendrites and dendritic spines, often coexist with ultrabright structures, such as, somas. A high fluorescence-protein concentration makes the soma order-of-magnitude brighter than the adjacent ultrafine structures resulting in an ultra-wide dynamic range. A straightforward enhancement of the weak-intensity structures often leads to saturation of the brighter ones, and might further result in amplification of high-frequency background noises. An adaptive illumination strategy to real-time-compress the dynamic range demands a dedicated hardware to operate and owing to electronic limitations, might encounter a poor effective bandwidth especially when each digitized pixel is required to be illumination optimized. Furthermore, such a method is often not immune to noise-amplification while locally enhancing a weak-intensity structure. We report a dedicated-hardware-free method for rapid noise-suppressed wide-dynamic-range compression so as to enhance visibility of such weak-intensity structures in terms of both contrast-ratio and signal-to-noise ratio while minimizing saturation of the brightest ones. With large-FOV aliasing-free two-photon fluorescence neuronal imaging, we validate its effectiveness by retrieving weak-intensity ultrafine structures amidst a strong noisy background. With compute-unified-device-architecture (CUDA)-acceleration, a time-complexity of <3 ms for a 1000x1000-sized 16-bit data-set is secured, enabling a real-time applicability of the same.

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Pulse compression in two-photon excitation fluorescence microscopy

Cited by 17 publications

References 35 publications

Nyquist-exceeding high voxel rate acquisition in mesoscopic multiphoton microscopy for full-field submicron resolution resolvability

Nyquist-exceeding high voxel rate acquisition in mesoscopic multiphoton microscopy for full-field submicron resolution resolvability

A rapid denoised contrast enhancement method digitally mimicking an adaptive illumination in submicron-resolution neuronal imaging

Real-time Noise-suppressed Wide-Dynamic-Range Compression in Ultrahigh-Resolution Neuronal Imaging

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