Microcirculation imaging is crucial in understanding the function and health of various tissues and organs. However, conventional imaging methods suffer from fluorescence label dependency, lack of depth resolution, and quantification inaccuracy. Here, we report a light-sheet dynamic light-scattering imaging (LSH-DSI) system to overcome these shortcomings. LSH-DSI utilizes selected plane illumination for an optical sectioning, while a time-frequency analysis method retrieves blood flow velocity estimates from dynamic changes in the detected light intensity. We have performed imaging experiments with zebrafish embryos to obtain angiographs from the trunk and head regions. The results show that LSH-DSI can capture label-free tomographic images of microvasculature and three-dimensional quantitative maps of local blood flow velocities.
Structured illumination microscopy (SIM) is an established optical superresolution imaging technique. However, conventional SIM based on wide-field image acquisition is generally limited to visualizing thin cellular samples. We propose combining one-dimensional image rescan and structured illumination in the orthogonal direction to achieve superresolution without the need to rotate the illumination pattern. The image acquisition speed is consequently improved threefold, which is also beneficial for minimizing photobleaching and phototoxicity. Optical sectioning in thick biological tissue is enhanced by including a confocal slit in the system to significantly suppress the out-of-focus background and the associated noise. With all the technical improvements, our method captures three-dimensional superresolved image stacks of neuronal structures in mouse brain tissue samples for a depth range of more than 200 μm.
Laser speckle imaging has been an indispensable tool for in vivo imaging of blood flow in biological tissues. Here we report a novel design of laser speckle imaging system, which combines confocal illumination and detection with various speckle analysis methods. An illumination line is formed using a cylindrical lens and a 1-D scanning mirror is used to rapidly scan the line across the sample surface. The backscattered light is detected with a line camera at the confocal position. The acquired line speckle patterns can be analyzed with different methods, including temporal autocorrelation and spatial evaluation of speckle contrast, to retrieve the maps of correlation time and flow velocity. The line-scan configuration enables fast image acquisition, while confocal detection helps reject out-of-focus light and define a small focal volume. In vivo image experiments with chick embryos have demonstrated the excellent imaging performance, including depth selectivity, high spatial resolution for visualizing blood flow in the microvasculature, and high temporal resolution for dynamic flow quantification.
Two-photon microscopy (TPM) is one of the most important imaging techniques in biological imaging since it was invented in 1990s. Due to its unique capabilities, this technique enables noninvasive study of scattering biological specimens in three dimensions with submicrometer resolution and penetration depth up to a few hundred micrometers. Focal modulation microscopy (FMM) provides sub-cellular spatial resolution at large penetration depths in tissue samples by rejecting out-of-focus signal. Combined with focal modulation techniques, this paper proposes two-photon focal modulation microscopy (TPFMM) to further enhance penetration depth by bringing a spatiotemporal phase modulator (STPM) in the TPM. The STPM is equivalent to a time-dependent phase-only pupil filter that alternates between a homogeneous filter and an inhomogeneous filter. When the STPM is homogeneous, the excitation beam is properly focused into the focal volume by the objective lens. The inhomogeneous filter is so designed that it leads to redistribution of the excitation beam and minimize the focal intensity, which can be binary phase and continuous phase distribution. Using the vectorial diffraction theory, we have theoretically demonstrated that TPFMM with the designed STPM can significantly suppress the background contribution from out-of-focus ballistic excitation and achieve almost the same resolution as TPM. The improved background rejection of this imaging modality, enabled by focal modulation, are quantified with three dimensional imaging data obtained from fluorescent beads and fixed tissue samples using a home-made TPFMM. These investigations have its potential to extend the penetration depth of nonlinear microscopy in imaging multiple-scattering biological tissues, such as mouse brain.
A spatiotemporal phase modulator (STPM) is theoretically investigated using the vectorial diffraction theory. The STPM is equivalent to a time-dependent phase-only pupil filter that alternates between a homogeneous filter and a stripe-shaped filter with a sinusoidal phase distribution. It is found that two-photon focal modulation microscopy (TPFMM) using this STPM can significantly suppress the background contribution from out-of-focus ballistic excitation and achieve almost the same resolution as two-photon microscopy. The modulation depth is also evaluated and a compromise exists between the signal-to-background ratio and signal-to-noise ratio. The theoretical investigations provide important insights into future implementations of TPFMM and its potential to further extend the penetration depth of nonlinear microscopy in imaging multiple-scattering biological tissues.
KEYWORDS: Modulation, Microscopy, Line scan image sensors, Microsoft Foundation Class Library, Luminescence, Image filtering, Optical filters, Tissues, Geometrical optics, Scattering
Line-scan focal modulation microscopy (LSFMM) is an emerging imaging technique that affords high imaging speed and good optical sectioning at the same time. We present a systematic investigation into optimal design of the pupil filter for LSFMM in an attempt to achieve the best performance in terms of spatial resolutions, optical sectioning, and modulation depth. Scalar diffraction theory was used to compute light propagation and distribution in the system and theoretical predictions on system performance, which were then compared with experimental results.
As a non-invasive technique, optical imaging has become a widely used tool in both biological research and clinical diagnostics to investigate biological tissues. A key parameter to consider is the penetration depth of optical imaging in the tissues. Several techniques have been developed to enhance the penetration depth of optical imaging within scattering biological tissues, such as optical coherence microscopy (OCM) and multi-photon microscopy (MPM). Recently, focal modulation microscopy (FMM) has been developed and an imaging depth comparable to these techniques has been achieved. Here, combined with focal modulation techniques, two-photon focal modulation microscopy (TPFMM) is demonstrated theoretically and experimentally. First, TPFMM in turbid media using a novel spatiotemporal phase modulator (STPM) is theoretically investigated using the vector diffraction theory. At the destructive stage during the excitation beam modulation, this STPM is equivalent to a strip-shaped pupil filter with a sinusoidal phase distribution. Compared to the previous filter patterns with sharp phase transitions, the contribution of out-of-focus ballistic excitation to the background is largely reduced using the continuous phase filters. In addition, this new STPM has been designed and integrated into TPFMM to achieve high performance imaging of the biological tissues. It is found that TPFMM using this new STPM can significantly suppress scattered excitation and reduce out-of-focus ballistic excitation with acceptable modulation depth and resolution. Therefore, TPFMM with some new STPMs has the great potential to further extend the penetration depth in imaging the scattering biological tissues.
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