Speckle Illumination Research Articles

Near-Infrared Speckle-Illumination Imaging Based on a Multidimensionally Disordered Fiber Laser

Speckle illumination is extensively applied in various imaging scenarios due to its advantages in enhancing the performance of imaging. Generally, the existing speckle-illumination methods are mainly based on external spatial modulation of light through a rotating diffuser or spatial light modulator. Both the lighting efficiency and the system's compactness are limited for these conventional external modulation methods, due to their free-space light coupling and insertion or diffraction loss. Here, a multimode fiber laser with self-reconfigurable speckle illumination is proposed and realized, benefiting from the temporal-spectral randomness of a homemade coherent random-fiber laser and the spectral-sensitive output pattern of an out-coupling multimode fiber. Output characteristics in the spectral and spatial domain of the proposed laser are analyzed and show dynamically varying speckles within a moderate bandwidth. These features pave efficient ways for superresolution imaging through an opaque scattering medium as well as ghost imaging, which is realized and demonstrated based on the lighting of the proposed laser.

Demystifying speckle field interference microscopy

Dynamic speckle illumination (DSI) has recently attracted strong attention in the field of biomedical imaging as it pushes the limits of interference microscopy (IM) in terms of phase sensitivity, and spatial and temporal resolution compared to conventional light source illumination. To date, despite conspicuous advantages, it has not been extensively implemented in the field of phase imaging due to inadequate understanding of interference fringe formation, which is challenging to obtain in dynamic speckle illumination interference microscopy (DSI-IM). The present article provides the basic understanding of DSI through both simulation and experiments that is essential to build interference microscopy systems such as quantitative phase microscopy, digital holographic microscopy and optical coherence tomography. Using the developed understanding of DSI, we demonstrated its capabilities which enables the use of non-identical objective lenses in both arms of the interferometer and opens the flexibility to use user-defined microscope objective lens for scalable field of view and resolution phase imaging. It is contrary to the present understanding which forces us to use identical objective lenses in conventional IM system and limits the applicability of the system for fixed objective lens. In addition, it is also demonstrated that the interference fringes are not washed out over a large range of optical path difference (OPD) between the object and the reference arm providing competitive edge over low temporal coherence light source based IM system. The theory and explanation developed here would enable wider penetration of DSI-IM for applications in biology and material sciences.

Compressed imaging with focused light

Compressed sensing applied to optical microscopy enables imaging with a number of measurements below the Nyquist criterion. The illumination basis selected, often unstructured for randomness considerations, influences the performance of image reconstruction algorithms. Here, we show through modelling based on multimode fiber imaging that an illumination basis composed of a series of uniformly spaced foci provides improved robustness to noise, increased volumetric imaging performance, and greater resilience to external perturbation when compared to a speckle illumination basis. These observations have broad implications for computational super-resolution imaging, endo-microscopy, and post-processing of images acquired with any point-scanning imaging system.

Design and development of integrated TIRF and common-path quantitative phase microscopic health care system with high stability

We demonstrate a compact microscopy system that enables simultaneous cell imaging in both digital holographic and fluorescence mode thus providing valuable multi-modal information for biosciences applications. The digital holographic imaging system uses a speckle free illumination by employing a rotating diffuser. Further a compact shear plate based interferometer arrangement is used to significantly reduce the system size and increase its robustness when compared with Michelson or Mach-Zehnder interferometer configurations. The overall reduction in form factor of the system is achieved since we employ the shear plate configuration additionally as a beam-splitter for the fluorescence imaging part of the system. We have fabricated a compact total internal reflection fluorescence (TIRF) unit which is combined with the common path sheared interferometer for this purpose. Our experiments with this compact system show excellent temporal phase stability. The working of the system is illustrated with imaging of polystyrene beads and MG63 osteosarcoma cell line sample. The fluorescence image of MG63 cell shows high expression of vinculin protein around the nuclei when compared with the expression in the cell's filopodia. These proteins are among the major molecular markers for cell adhesion and play a vital role in sensing the mechanical stimulus of the microenvironment. Further the quantitative phase information about the cell also provides its optical thickness information that is spatially aligned with the fluorescence signal. The proposed system can find applications in bioscience laboratories as well as in diagnostics.

Wide-Field Super-Resolution Optical Fluctuation Imaging through Dynamic Near-Field Speckle Illumination.

Stochastic optical fluctuation imaging (SOFI) generates super-resolution fluorescence images by emphasizing the positions of fluorescent emitters via statistical analysis of their on-and-off blinking dynamics. In SOFI with speckle illumination (S-SOFI), the diffraction-limited grain size of the far-field speckles prevents independent blinking of closely located emitters, becoming a hurdle to realize the full super-resolution granted by SOFI processing. Here, we present a surface-sensitive super-resolution technique exploiting dynamic near-field speckle illumination to bring forth the full super-resolving power of SOFI without blinking fluorophores. With our near-field S-SOFI technique, up to 2.8- and 2.3-fold enhancements in lateral spatial resolution are demonstrated with computational and experimental fluorescent test targets labeled with conventional fluorophores, respectively. Fluorescent beads separated by 175 nm are also super-resolved by near-field speckles of 150 nm grain size, promising sub-100 nm resolution with speckle patterns of much smaller grain size.

Image Reconstruction Using Autofocus in Single-Lens System

To reconstruct the wavefront in a single-lens coherent diffraction imaging (CDI) system, we propose a closed-loop cascaded iterative engine (CIE) algorithm based on the known information of the imaging planes. The precision of diffraction distance is an important prerequisite for a perfect reconstruction of samples. For coherent diffraction imaging with a lens, autofocus is investigated to accurately determine the object distance and image distance. For the case of only the object distance being unknown, a diffuser is used to scatter the coherent beam for speckle illumination to improve the performance of autofocus. The optimal object distance is obtained stably and robustly by combing speckle imaging with clarity evaluation functions. SSIM and MSE, using the average pixel value of the reconstructed data set as a reference, are applied on two-unknown-distance autofocus. Simulation and experiment results are presented to prove the feasibility of the CIE and proposed auto-focusing method.

Resolution enhancement of digital holographic microscopy via synthetic aperture: a review

Digital holographic microscopy (DHM), which combines digital holography with optical microscopy, is a wide field, minimally invasive quantitative phase microscopy (QPM) approach for measuring the 3D shape or the inner structure of transparent and translucent samples. However, limited by diffraction, the spatial resolution of conventional DHM is relatively low and incompatible with a wide field of view (FOV) owing to the spatial bandwidth product (SBP) limit of the imaging systems. During the past decades, many efforts have been made to enhance the spatial resolution of DHM while preserving a large FOV by trading with unused degrees of freedom. Illumination modulation techniques, such as oblique illumination, structured illumination, and speckle illumination, can enhance the resolution by adding more high-frequency information to the recording system. Resolution enhancement is also achieved by extrapolation of a hologram or by synthesizing a larger hologram by scanning the sample, the camera, or inserting a diffraction grating between the sample and the camera. For on-chip DHM, spatial resolution is achieved using pixel super-resolution techniques. In this paper, we review various resolution enhancement approaches in DHM and discuss the advantages and disadvantages of these approaches. It is our hope that this review will contribute to advancements in DHM and its practical applications in many fields.

Slide-free histological imaging by microscopy with ultraviolet surface excitation using speckle illumination

Microscopy with ultraviolet surface excitation (MUSE) is a promising slide-free imaging technique to improve the time-consuming histopathology workflow. However, since the penetration depth of the excitation light is tissue dependent, the image contrast could be significantly degraded when the depth of field of the imaging system is shallower than the penetration depth. High-resolution cellular imaging normally comes with a shallow depth of field, which also restricts the tolerance of surface roughness in biological specimens. Here we propose the incorporation of MUSE with speckle illumination (termed MUSES), which can achieve sharp imaging on thick and rough specimens. Our experimental results demonstrate the potential of MUSES in providing histological images with ∼ 1 μm spatial resolution and improved contrast, within 10 minutes for a field of view of 1.7 mm × 1.2 mm . With the extended depth of field feature, MUSES also relieves the constraint of tissue flatness. Furthermore, with a color transformation assisted by deep learning, a virtually stained histological image can be generated without manual tuning, improving the applicability of MUSES in clinical settings.

Dynamic Speckle Illumination Digital Holographic Microscopy by Doubly Scattered System

The coherent noise always exists in digital holographic microscopy due to the laser source, degrading the image quality. A method of speckle suppression using the dynamic speckle illumination, produced by double-moving diffusers, is presented in digital holographic microscopy. The space–time correlation functions are theoretically analyzed from the statistics distribution in the doubly and singly scattered system, respectively. The configuration of double-moving diffusers is demonstrated to have better performance in speckle suppression compared with the single diffuser and moving-static double diffusers cases. The experiment results verify the feasibility of the approach. The presented approach only requires a single shot interferogram to realize the speckle reduction, accordingly it has the potential application in real-time measurement.

Phase-coded speckle illumination for laser Fourier ptychographic microscopy

It remains a tough task for designing a microscopy system to approach the diffraction limit over a wide field of view due to its restricted space bandwidth product (SBP). Fourier ptychographic microscopy (FPM) has been developed to broaden the SBP of a conventional microscope platform without major hardware modifications. In this paper, we propose a novel FPM system based on the phase-coded speckle illumination, termed PSI-FPM, which employs a laser source with a speckle illumination control optical system. A phase-only spatial light modulator (SLM) with a condenser lens is utilized in the PSI-FPM system to project the phase-coded speckle illumination patterns onto the sample. The convolution between the sample spectrum and the illumination spectrum in the Fourier domain extend the numerical aperture of the objective that acts as a low-pass filter with cut-off frequency. The SLM is introduced in the PSI-FPM system to produce the specific illumination patterns so that the number of the captured images used for reconstruction are greatly reduced. In addition, the generated pattern is scanned to different directions without mechanical movement, which improves the robustness of the system. The PSI-FPM system is validated against simulations and experiments, and as a result reconstructs high-resolution images once the phase retrieval algorithm is employed. We envision that the PSI-FPM system will be suitable for many other speckle imaging applications.

Wavelength-dependent speckle multiplexing for imaging through opacity

Memory-effect-based speckle-correlated imaging through opacity has been exploited widely in recent years. Generally, spatially incoherent light sources are required to provide effective illumination that can eliminate the interference noise. Here, the wavelength-dependent speckle multiplexing (WDSM) technique is first proposed and demonstrated as an effective method to suppress the interference noise, thereby facilitating the use of partially spatially coherent light as an alternative promising illuminating source. In contrast to the conventional decoherent approach which often employs a rapidly rotating diffuser to reduce the spatial coherence of a spatially coherent light, the WDSM shows greatly enhanced structural robustness by combining a tunable laser and the speckle illumination using a multimode fibre. The proposed WDSM not only expands the scope of candidate light sources for speckle-correlated imaging based on the classical phase-retrieval algorithm, but also enables the multi-channel detection determined by the speckle spectral decorrelation, which would further facilitate the waveband selective light-matter detection through opacity. This work also combines the advantages of fibre optics and laser technology and enriches the potential applications of speckle-correlated imaging.

Speckle illumination SFDI for projector-free optical property mapping.

Spatial frequency domain imaging can map tissue scattering and absorption properties over a wide field of view, making it useful for clinical applications such as wound assessment and surgical guidance. This technique has previously required the projection of fully characterized illumination patterns. Here, we show that random and unknown speckle illumination can be used to sample the modulation transfer function of tissues at known spatial frequencies, allowing the quantitative mapping of optical properties with simple laser diode illumination. We compute low- and high-spatial frequency response parameters from the local power spectral density for each pixel and use a lookup table to accurately estimate absorption and scattering coefficients in tissue phantoms, in vivo human hand, and ex vivo swine esophagus. Because speckle patterns can be generated over a large depth of field and field of view with simple coherent illumination, this approach may enable optical property mapping in new form-factors and applications, including endoscopy.

Ultimate resolution limits of speckle-based compressive imaging.

Compressive imaging using sparsity constraints is a very promising field of microscopy that provides a dramatic enhancement of the spatial resolution beyond the Abbe diffraction limit. Moreover, it simultaneously overcomes the Nyquist limit by reconstructing an N-pixel image from less than N single-point measurements. Here we present fundamental resolution limits of noiseless compressive imaging via sparsity constraints, speckle illumination and single-pixel detection. We addressed the experimental setup that uses randomly generated speckle patterns (in a scattering media or a multimode fiber). The optimal number of measurements, the ultimate spatial resolution limit and the surprisingly important role of discretization are demonstrated by the theoretical analysis and numerical simulations. We show that, in contrast to conventional microscopy, oversampling may decrease the resolution and reconstruction quality of compressive imaging.

Circumventing the optical diffraction limit with customized speckles

Speckle patterns have been used widely in imaging techniques such as ghost imaging, dynamic speckle illumination microscopy, structured illumination microscopy, and photoacoustic fluctuation imaging. Recent advances in the ability to control the statistical properties of speckles has enabled the customization of speckle patterns for specific imaging applications. In this work, we design and create special speckle patterns for parallelized nonlinear pattern-illumination microscopy based on fluorescence photoswitching. We present a proof-of-principle experimental demonstration where we obtain a spatial resolution three times higher than the diffraction limit of the illumination optics in our setup. Furthermore, we show that tailored speckles vastly outperform standard speckles. Our work establishes that customized speckles are a potent tool in parallelized super-resolution microscopy.

Influence of the sparsity of random speckle illumination on ghost imaging in a noise environment

The influence of the sparsity of random speckle illumination on traditional ghost imaging (GI) and GI via sparsity constraint (GISC) in a noise environment is investigated. The experiments demonstrate that both GI and GISC obtain their best imaging quality when the sparsity of random speckle illumination is 0.5, which is also explained by some parameters such as detection of the signal to noise ratio and mutual coherence of the measurement matrix.

Theoretical study of wide-field fluorescence microscopy based on dynamic speckle illumination

In order to obtain the internal fine structure of biological tissues and living cells, the microscopic imaging technology is required to be capable of microscopy. In the wide-field fluorescence microscopy with dynamic speckle illumination, a series of dynamically changing speckle patterns are used to illuminate a biological sample in the whole field. The fluorescence sectioning images of sample’s three-dimensional structural are obtained by extracting intensely changing fluorescence signals in the focal plane. In this paper, the process of obtaining fluorescence sectioning images by the fluorescence microscopy is studied by theoretical analysis and simulation. Two main factors affecting the imaging quality of fluorescence sectioning image are analyzed, which are the number of original fluorescence images recorded by CCD and granularity of diffuser. The simulation results indicates that the imaging quality of fluorescence sectioning images first increases and then tends to saturation with the number of original fluorescence images increasing. It first increases and then decreases with the graininess of diffusers increasing. Considering the imaging quality and imaging time, when the number of original fluorescence images is 60 that is used to extract fluorescence sectioning images, and the granularity of diffuser is about 1000, the high spatial resolution fluorescence sectioning images with contrast higher than 85% can be obtained. Theoretical analysis and simulation research provide a theoretical basis and guidance for designing the system structure, implementing and optimizing the wide-field fluorescence microscopy with dynamic speckle illumination.

Simultaneous multi-color optical sectioning fluorescence microscopy with wavelength-coded volume holographic gratings.

Optical sectioning fluorescence microscopy provides high contrast images of volumetric samples and has been widely used for many biological applications. However, simultaneously acquiring multi-color fluorescence images require additional optical elements and devices, which are bulky, wavelength specific, and not cost-effective. In this paper, wavelength-coded volume holographic gratings (WC-VHGs) based optical sectioning fluorescence microscopy is proposed to simultaneously offer multi-color fluorescence images with fine out-of-focus background rejection. Due to wavelength degeneracy, multiplexed WC-VHGs are capable of acquiring multi-wavelength fluorescence images in a single shot, and displaying the laterally separated multi-wavelength images onto CCD. In our system optical sectioning capability is achieved through speckle illumination and HiLo imaging method. To demonstrate imaging characteristics of our system, dual-wavelength fluorescence images of both standard fluorescent microspheres and ex vivo mT/mG mice cardiac tissue are presented. Current results may find important applications in hyperspectral imaging for biomedical research.

Design and development of laser speckle reduction device using waveguide diffuser and pyramidal cavity for projection imaging

We report the design and development of an efficient speckle reduction device for projection imaging. The device is composed of a combination of a rod-shaped waveguide diffuser and a reflective pyramidal cavity. The simulation results reveal that the pyramidal cavity with waveguide diffuser is a highly efficient light guide comparable to the other conventional light guides. Further, the device was developed and tested experimentally on the basis of speckle contrast and image quality for both the microscopic and macroscopic objects. An acrylic rod-shaped waveguide diffuser was developed and mounted at the middle of a pyramidal shaped cavity which was fabricated using 3D printer. Four highly reflecting mirrors were installed to make the cavity reflective. The system is highly effective for the speckle suppression due to the coherence reduction via multiple scattering from the rod-shaped waveguide diffuser and multiple reflections from the pyramidal cavity. In the system, a combined effect of spatial and angular diversity along with intensity averaging leads to an effective speckle free uniform illumination. Reduced speckle contrast using the proposed system was observed up to 2%, which is nearly equivalent to mechanically moving diffuser system. The device is able to reduce speckle contrast significantly without using any moving parts and in addition, it does not consume any electrical energy. The proposed system is compact, low cost, efficient, and very effective for low to high power laser sources in speckle reduction. Experimental results are compared with stationary and rotating diffuser system on the basis of computed speckle contrast and image quality.

Using fiber-bending-generated speckles for improved working distance and background rejection in lensless micro-endoscopy.

Lensless flexible fiber-bundle-based endoscopes allow imaging at depths beyond the reach of conventional microscopes with a minimal footprint. These multicore fibers provide a simple solution for wide-field fluorescent imaging when the target is adjacent to the fiber facet. However, they suffer from a very limited working distance and out-of-focus background. Here, we carefully study the dynamic speckle illumination patterns generated by bending a commercial fiber bundle and show that they can be exploited to allow extended working distance and background rejection, using a super-resolution fluctuations imaging analysis of multiple frames, without the addition of any optical elements.

Long working distance high resolution reflective sample imaging via structured embedded speckle illumination

Imaging beyond the diffraction limit at longer working distances using enhanced microscopic configurations has always been a challenge for biological and engineering samples. Even though multiple techniques have been widely used for sub-diffraction limit resolution imaging, the achievable resolution was relying on the use of objective lenses with a high numerical aperture (NA). In the case of engineering samples, in addition to sustaining higher resolutions at large working distances, improving the signal-to-noise ratio (SNR) is also critical. In this context, we propose and demonstrate a concept for high-resolution imaging at large working distances, termed as structured illumination embedded speckle microscopy. An imaging resolution of ~ 310 ± 5 nm was achieved with a microscope objective (0.55 NA; 50X) having 11 mm long working distance using a Siemen's star as the test sample. The demonstrated microscopy is therefore envisaged for engineering applications that demands high-resolution, high SNR imaging at long working distances.