In point-scanning microscopy, such as confocal laser scanning microscopy (CLSM), the imaging resolution is limited by the size of the focal spot. Breaking through the diffraction limit by inserting a modulation phase mask in the objective pupil plane is a compact, high efficiency and adaptable scheme. Here, we propose a feedback phase design method based on genetic algorithm to control the 3D shape of the focal spot. The lateral and axial size can be jointly designed for various usages. Simulation and experimental results present the feasibility of this method. The axial size can be decreased by 50% while the lateral size is decreased by 15% simultaneously. This method of 3D super-diffraction limit focusing may potentially be used in CLSM for 3D super resolution imaging.
Optical microscopy is an indispensable imaging tool in life science. However, light cannot be focused owing to high scattering, and hence, the imaging depth and spatial resolution are restricted. Here, we propose an imaging method that combines wavefront shaping and image scanning microscopy. The reflected signal is used as feedback to acquire an optimal phase that can refocus the scattered light behind the scattering media. The experimental results show that the proposed method works in multilayer scattering media and can improve both the resolution and imaging depth of optical microscopy.
The Bessel beam belongs to a typical class of non-diffractive optical fields that are characterized by their invariant focal profiles along the propagation direction. However, ideal Bessel beams only rigorously exist in theory; Bessel beams generated in the lab are quasi-Bessel beams with finite focal extensions and varying intensity profiles along the propagation axis. The ability to engineer the on-axis intensity profile to the desired shape is essential for many applications. Here we demonstrate an iterative optimization-based approach to engineering the on-axis intensity of Bessel beams. The genetic algorithm is used to demonstrate this approach. Starting with a traditional axicon phase mask, in the design process, the computed on-axis beam profile is fed into a feedback tuning loop of an iterative optimization process, which searches for an optimal radial phase distribution that can generate a generalized Bessel beam with the desired onaxis intensity profile. The experimental implementation involves a fine-tuning process that adjusts the originally targeted profile so that the optimization process can optimize the phase mask to yield an improved on-axis profile. Our proposed method has been demonstrated in engineering several zeroth-order Bessel beams with customized on-axis profiles. High accuracy and high energy throughput merit its use in many applications.
Bessel beams have been used in many applications due to their unique optical properties of maintaining their intensity profiles unchanged during propagation. In imaging applications, Bessel beams have been successfully used to provide extended focuses for volumetric imaging and uniformed illumination plane in light-sheet microscopy. Coupled with two-photon excitation, Bessel beams have been successfully used in realizing fluorescence projected volumetric imaging. We demonstrated previously a stereoscopic solution–two-photon fluorescence stereomicroscopy (TPFSM)–for recovering the depth information in volumetric imaging with Bessel beams. In TPFSM, tilted Bessel beams were used to generate stereoscopic images on a laser scanning two-photon fluorescence microscope; upon post image processing we could successfully provide 3D perception of acquired volume images by wearing anaglyph 3D glasses. However, tilted Bessel beams were generated by shifting either an axicon or an objective laterally; the slow imaging speed and severe aberrations made it hard to use in real-time volume imaging. In this article, we report recent improvements of TPFSM with newly designed scanner and imaging software, which allows 3D stereoscopic imaging without moving any of the optical components on the setup. This improvement has dramatically improved focusing qualities and imaging speed so that the TPFSM can be performed potentially in real-time to provide 3D visualization in scattering media without post image processing.
Structured-illumination microscopy (SIM) is an efficacious tool to decrease the contribution of the out-of-focus light to images of specimens. However, in SIM, the frequency of the spatial modulation applied to specimens should be adjustable according to the optical properties of the specimens to reach the optimal contrasts. Hence, a common theme in SIM is how the flexibility and quality of modulations at different frequencies can be improved. Digital scanned laser light-sheet microscopy with structured illumination (DSLM-SI) has been the most flexible means for generating modulation and optical sectioning. The complexity of synchronization between the temporal modulation and the beam scanning makes it hard to use and less stable; it also takes more time to acquire images for one plane than selective plane illumination microscopy (SPIM). In this report, we present a recent effort to use a spatial light modulator (SLM) to provide spatial modulation in SPIM. With the SLM, both of the frequency and phase of lateral modulation can be changed rapidly; moreover, this SLM-based SPIM can achieve fast imaging without mechanical moving parts.
Bessel beams have been proved to have the ability to extend the depth of focus in fluorescence microscopy. But the depth discrimination was not investigated thoroughly. Following our previous work2, we investigated focal fields of Bessel-Gauss beams at different scanning angles. We found that the central focusing lines were tilted differently at different scanning angles. This effect manifests the ability of the true perspective view in the fluorescence stereomicroscopy.
Three dimensional distributions of cells can be usually acquired by optical sectioning methods, such as multiphoton
excitation and confocal fluorescence laser scanning microscopy. Though the lateral scan rates can reach up to
several kHz, the relatively slow axial scan comprises the speed of real-time imaging of a volume. Here we propose a
three dimensional imaging method that uses Bessel beams as excitation in multiphoton fluorescence microscopy.
The extended focus of the Bessel beam allows recording a volume of cells without scanning the depth. The depth
information can be retrieved by recording a pair of parallax views of the same volume. We have demonstrated the
stereoscope capability on a homebuilt two-photon fluorescence microscope.
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