Image formation in structured illumination wide-field fluorescence microscopy

We present a theoretical analysis of the image formation in structured illumination wide-field fluorescence microscopy (SIWFFM). We show that the optically sectioned images obtained with this approach possess the optical sectioning strengths comparable to those obtained with the confocal microscope. We further show that the transfer function behaviour is directly comparable to that of the true confocal instrument. The theoretical considerations are compared with and confirmed by experimental results.     

Wide-field coherent anti-Stokes Raman scattering microscopy with non-phase-matching illumination

We have developed and tested a wide-field coherent anti-Stokes Raman scattering (CARS) microscopy technique, which provides the simultaneous imaging of an extended illuminated area without scanning. This method is based on the non-phase-matching illumination of a sample and imaging of a CARS signal with a CCD camera using conventional microscope optics. We have identified a set of conditions on the illumination and imaging optics, as well as on sample preparation. Imaging of test objects proved high spatial resolution and chemical selectivity of this technique.     

Coherent super-resolution microscopy via laterally structured illumination

Theory describing a super-resolution microscopy experiment using temporally and spatially coherent structured illumination was developed, and used to derive a method for processing experimental data. Numerical simulations were performed to verify that the method can, in principle, produce super-resolved images that are exactly equivalent to an image processed by a system with a much larger aperture (that is, the correct weighting between different regions of the image spectrum is maintained). The process was then demonstrated experimentally, showing a factor of two improvement in resolution over a diffraction-limited, coherently illuminated, microscope.     

Wide-field fluorescence sectioning with hybrid speckle and uniform-illumination microscopy

We describe a method of obtaining optical sectioning with a standard wide-field fluorescence microscope. The method involves acquiring two images, one with nonuniform illumination (in our case, speckle) and another with uniform illumination (in our case, randomized speckle). An evaluation of the local contrast in the speckle-illumination image provides an optically sectioned image with low resolution. This is complemented with high-resolution information obtained from the uniform-illumination image. A fusion of both images leads to a full resolution image that is optically sectioned across all spatial frequencies. This hybrid illumination method is fast, robust, and generalizable to a variety of illumination and imaging configurations.     

Single-exposure optical sectioning by color structured illumination microscopy

Structured illumination microscopy (SIM) is a wide-field technique that rivals confocal microscopy in optical sectioning ability at a small fraction of the acquisition time. For standard detectors such as a CCD camera, SIM requires a minimum of three sequential frame captures, limiting its usefulness to static objects. By using a color grid and camera, we surpass this limit and achieve optical sectioning with just a single image acquisition. The extended method is now applicable to moving objects and improves the speed of three-dimensional imaging of static objects by at least a factor of three.     

Wide-field imaging through scattering media by scattered light fluorescence microscopy

To obtain images through scattering media, scattered light fluorescence (SLF) microscopy that utilizes the optical memory effect has been developed. However, the small field of view (FOV) of SLF microscopy limits its application. In this paper, we have introduced a re-modulation method to achieve wide-field imaging through scattering media by SLF microscopy. In the re-modulation method, to raster scan the focus across the object plane, the incident wavefront is re-modulated via a spatial light modulator (SLM) in the updated phase compensation calculated using the optimized iterative algorithm. Compared with the conventional optical memory effect method, the re-modulation method can greatly increase the FOV of a SLF microscope. With the phase compensation theoretically calculated, the process of updating the phase compensation of a high speed SLM is fast. The re-modulation method does not increase the imaging time. The re-modulation method is, therefore, expected to make SLF microscopy have much wider applications in biology, medicine and physiology.     

Comment on "Wide-field coherent anti-Stokes Raman scattering microscopy with non-phase-matching illumination"

We comment on a Letter by Toytman et al. [Opt. Lett.32, 1941 (2007)] in which a novel setup for wide-field imaging in coherent anti-Stokes Raman scattering (CARS) microscopy is demonstrated. There the authors state that our phase-matching implementation of a wide-field CARS system [Appl. Phys. Lett.84, 816 (2004); New J. Phys.8, 36 (2006)] suffers from a strong background from the bulk medium. However, our results show quite the contrary, i.e., that our setup provides a very good signal contrast, due to an almost vanishing background level from the bulk solvent.     

Laser-tweezer-controlled solid immersion microscopy in microfluidic systems

We describe the creation and implementation of a near-field scanning solid immersion microscope that is specifically tailored for use in microfluidic systems. The microscope comprises a newly fabricated Weierstrass solid immersion lens (SIL), which is detached from its substrate and is free floating in the fluid, and a laser optical tweezer, which serves both as a trapping beam for alignment and positioning of the SIL and as a near-field scanning beam that images the sample through the SIL. A discussion of the SIL's fabrication method is presented along with experimental results that demonstrate the effectiveness of our microscope design.     

Resolution enhancement in digital holographic microscopy with structured illumination

We present a digital holographic microscope wherein the sample is illuminated by structured light to enable the capture of additional object spatial frequencies. Reconstructed images with increased spatial resolution are obtained by separating and synthesizing bandwidths of different frequency regions in the Fourier domain. The theoretical analysis and experimental results are presented.     

Full-wavelet approach for fluorescence diffuse optical tomography with structured illumination

We present a fast reconstruction method for fluorescence optical tomography with structured illumination. Our approach is based on the exploitation of the wavelet transform of the measurements acquired after wavelet-patterned illuminations. This method, validated on experimental data, enables us to significantly reduce the acquisition and computation times with respect to the classical scanning approach. Therefore, it could be particularly suited for in vivo applications.     

超半球形固体浸没透镜系统的亚表面显微

应用矢量衍射理论和薄膜光学方法,研究了有禁带存在时超半球形固体浸没透镜(SIL)系统的亚表面显微。模拟计算结果显示,光斑的强度,边瓣强度和光斑大小随禁带厚度发生振荡。光斑的大小和光斑的强度几乎是反相变化的。当禁带材料的折射率与固体浸没透镜(SIL)的折射率相匹配时,光斑的强度较大,分辨率较高。随着离开样品表面的距离的增加,光斑的强度不断减小,光斑的大小和边瓣强度逐渐增加。     

Theoretical assessment of optical resolution enhancement and background fluorescence reduction by three-dimensional nonlinear structured illumination microscopy using stimulated emission depletion

Three-dimensional structured illumination microscopy (SIM) enlarges frequency cutoff laterally and axially by a factor of two, compared with conventional microscopy. However, its optical resolution is still fundamentally limited. It is necessary to introduce nonlinearity to enlarge frequency cutoff further. We propose three-dimensional nonlinear structured illumination microscopy based on stimulated emission depletion (STED) effect, which has a structured excitation pattern and a structured STED pattern, and both three-dimensional illumination patterns have the same lateral pitch and orientation. Theoretical analysis showed that nonlinearity induced by STED effect, which causes harmonics and contributes to enlarging frequency cutoff, depends on the phase difference between two structured illuminations and that the phase difference of π is the most efficient to increase nonlinearity. We also found that undesirable background fluorescence, which degenerates the contrast of structured pattern and limits the ability of SIM, can be reduced by our method. These results revealed that optical resolution improvement and background fluorescence reduction would be compatible. The feasibility study showed that our method will be realized with commercially available laser, having 3.5 times larger frequency cutoff compared with conventional microscopy.     

ESPI with structured illumination

In practical ESPI applications in industry, the object under investigation often has low reflectance. From theoretical analysis, it is shown here that the implementation of phase shifting techniques will result in a smaller fraction of acceptable measurements for a given level of tolerable phase error. The use of higher power lasers may solve this problem; but it is an expensive solution. In this work, structured lighting using diffractive optical elements is proposed as a cost-effective solution. The approach is particularly useful when the deformation phase has to be obtained from only selected areas on the object. A simple experiment conducted verifies the workability of this approach.     

Three-dimensional superresolution in two-color excitation fluorescence microscopy using theta illumination method

In two-color excitation fluorescence (2CE) theta microscopy, the point spread function (PSF) of the system is the product of the two PSFs of both excitation beams and due to the ellipsoidal shape of the PSFs, the axial width of the PSF of one excitation beam is strongly reduced after multiplication with the lateral section of another PSF in nonconfocal theta microscopy. Taking the advantages of this setup, we propose using two leaky filters inserted in the two illumination arms, respectively, to compress its own lateral width of each PSF; it means, the lateral and axial superresolution (three-dimensional superresolution) of the whole system in 2CE theta microscope with two orthogonal illumination beams are realized.     

Simultaneous optically sectioned fluorescence and optical coherence microscopy with full-field illumination

Full-field optical coherence microscopy (FF-OCM) and optically sectioned fluorescence microscopy are two imaging techniques that are implemented here in a novel dual modality instrument. The two imaging modalities use a broad field illumination to acquire the entire field of view without raster scanning. Optical sectioning is achieved in both imaging modalities owing to the coherence gating property of light for FF-OCM, and a structured illumination setup for fluorescence microscopy. Complementary image data are provided by the dual modality instrument in the context of biological tissue screening. FF-OCM imaging modality shows the tissue microarchitecture, while fluorescence microscopy highlights specific tissue features with cellular-level resolution by using targeting contrast agents. Complementary tissue morphology and biochemical features could potentially improve the understanding of cellular functions and disease diagnosis.     

Lateral modulation boosts image quality in single plane illumination fluorescence microscopy

A new microscope combines optical sectioning by fluorophore excitation using a single light sheet with structured illumination. Several images with laterally intensity-modulated light sheets are recorded from scattering fluorescent specimens. By applying a simple data processing scheme, the nonmodulated volumes are identified. The blurred features become dark, and the resultant images are improved in terms of contrast and resolution. Hence, the instrument is capable of discriminating against contributions to the image that are induced by the optical properties of the specimen. The new microscope's capabilities are demonstrated by imaging the internals of the head of an adult Drosophila melanogaster (fruit fly) expressing green fluorescent protein-labeled polycomb proteins.     

Axial coding in full-field microscopy using three-dimensional structured illumination implemented with no moving parts

We report a simple optical setup to produce both axial and lateral structured illumination through a single objective lens. With a minimum of six full-field images obtained without moving either the sample or the microscope objective, 100 nm diameter fluorescent beads can be localized axially with an accuracy of 50 nm in a 1.76-microm-thick layer. We show that this axial localization improvement can easily be combined with classical lateral structured illumination, so that lateral resolution enhancement by a factor of 2 is maintained.     

Even fluorescence excitation by multidirectional selective plane illumination microscopy (mSPIM)

Multidirectional selective plane illumination microscopy (mSPIM) reduces absorption and scattering artifacts and provides an evenly illuminated focal plane. mSPIM solves two common problems in light-sheet-based imaging techniques: The shadowing in the excitation path due to absorption in the specimen is eliminated by pivoting the light sheet; the spread of the light sheet by scattering in the sample is compensated by illuminating the sample consecutively from opposing directions. The resulting two images are computationally fused yielding a superior image. The effective light sheet is thinner, and the axial resolution is increased by square root 2 over single-directional SPIM. The multidirectional illumination proves essential in biological specimens such as millimeter-sized embryos. The performance of mSPIM is demonstrated by the imaging of live zebrafish embryos.     

Structured illumination in total internal reflection fluorescence microscopy using a spatial light modulator

In wide-field fluorescence microscopy, illuminating the specimen with evanescent standing waves increases lateral resolution more than twofold. We report a versatile setup for standing-wave illumination in total internal reflection fluorescence microscopy. An adjustable diffraction grating written on a phase-only spatial light modulator controls the illumination field. Selecting appropriate diffraction orders and displaying a sheared (tilted) diffraction grating allows one to tune the penetration depth in very fine steps. The setup achieves 91 nm lateral resolution for green emission.