Fast dual-beam alignment method for stimulated emission depletion microscopy using aggregation-induced emission dye resin

A stimulated emission depletion is capable of breaking the diffraction limit by exciting fluorescent molecules with a solid Gaussian beam and quenching the excited molecules with another donut beam through stimulated emission. The coincidence degree of these two beams in three dimensions will significantly influence the spatial resolution of the microscope.However, the conventional alignment approach based on raster scanning of gold nanoparticles by the two laser beams separately suffers from a ...     

Axial super-resolution by mirror-reflected stimulated emission depletion microscopy

In stimulated emission depletion (STED) microscopy, the lateral resolution is in the range of tens of nanometers depending on the sample and the instrument. The axial resolution, however, is in standard systems limited by diffraction to about 500 nm. We present an approach to three-dimensional diffraction-unlimited resolution by observing the sample at two optical angles. The system is realized by using an atomic force microscope (AFM) chip as a microreflector to deflect the STED beams near the region-of-interest (ROI), thus allowing observations at an angle ∠. Consequently, the superior lateral resolution can be utilized to resolve details in the axial direction of the main optical axis of the microscope. Here, fluorescent nanoparticles 90 nm apart and biological structures 80 nm apart along axial direction were distinguished by utilizing an off-the-shelf, commercial STED microscope, coupled with an AFM and an AFM chip micro-reflector.     

Quantitative phase retrieval in X‐ray Zernike phase contrast microscopy

In recent years, increasing attention has been devoted to X‐ray phase contrast imaging, since it can provide high‐contrast images by using phase variations. Among the different existing techniques, Zernike phase contrast microscopy is one of the most popular phase‐sensitive techniques for investigating the fine structure of the sample at high spatial resolution. In X‐ray Zernike phase contrast microscopy, the image contrast is indeed a mixture of absorption and phase contrast. Therefore, this technique just provides qualitative information on the object, which makes the interpretation of the image difficult. In this contribution, an approach is proposed for quantitative phase retrieval in X‐ray Zernike phase contrast microscopy. By shifting the phase of the direct light by π/2 and 3π/2, two images of the same object are measured successively. The phase information of the object can then be quantitatively retrieved by a proper combination of the measured images. Numerical experiments were carried out and the results confirmed the feasibility of the proposed method. It is expected that the proposed method will find widespread applications in biology, materials science and so on.     

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.     

Performance analysis of quantitative phase retrieval method in Zernike phase contrast X-ray microscopy

Since the invention of Zernike phase contrast method in 1930,it has been widely used in optical microscopy and more recently in X-ray microscopy.Considering the image contrast is a mixture of absorption and phase information,we recently have proposed and demonstrated a method for quantitative phase retrieval in Zernike phase contrast X-ray microscopy.In this contribution,we analyze the performance of this method at different photon energies.Intensity images of PMMA samples are simulated at 2.5 keV and 6.2 keV,respectively,and phase retrieval is performed using the proposed method.The results demonstrate that the proposed phase retrieval method is applicable over a wide energy range.For weakly absorbing features,the optimal photon energy is 2.5 keV,from the point of view of image contrast and accuracy of phase retrieval.On the other hand,in the case of strong absorption objects,a higher photon energy is preferred to reduce the error of phase retrieval.These results can be used as guidelines to perform quantitative phase retrieval in Zernike phase contrast X-ray microscopy with the proposed method.     

Quantitative optical phase microscopy

We present a new method for the extraction of quantitative phase data from microscopic phase samples by use of partially coherent illumination and an ordinary transmission microscope. The technique produces quantitative images of the phase profile of the sample without phase unwrapping. The technique is able to recover phase even in the presence of amplitude modulation, making it significantly more powerful than existing methods of phase microscopy. We demonstrate the technique by providing quantitatively correct phase images of well-characterized test samples and show that the results obtained for more-complex samples correlate with structures observed with Nomarski differential interference contrast techniques.     

Quantitative phase microscopy of biological samples using a portable interferometer

This Letter presents the τ interferometer, a portable and inexpensive device for obtaining spatial interferograms of microscopic biological samples without the strict stability and the highly coherent illumination that are usually required for interferometric microscopy setups. The device is built using off-the-shelf optical elements and can easily operate with low-coherence illumination, while being positioned in the output of a conventional inverted microscope. The interferograms are processed into the quantitative amplitude and phase profiles of the sample. Based on the phase profile, the optical-path-delay profile is obtained with temporal stability of 0.18 nm and spatial stability of 0.42 nm. Further experimental demonstration of using the τ interferometer for imaging the quantitative thickness profile of a live red blood cell is provided.     

Quantitative phase retrieval in dynamic laser speckle interferometry

The rapid progress of modern manufacturing and inspection technologies has posed stringent requirements on optical techniques for vibration characterization and dynamic testing. Due to its simplicity, accuracy and whole-field characters, laser speckle interferometry has served as one of the major techniques for dynamic measurement. In this paper, a two-step phase shifting method is developed for quantitative speckle phase measurement, which helps to eliminate the specklegrams needed for phase evaluation and facilitate dynamic measurement. Unlike previously reported two-step methods using fringe patterns with known phase shift of π/2, a small unknown phase shift is employed instead in the proposed method, which eliminates the need for phase shifting devices. Further investigation shows that small phase shifts are preferable over large phase shifts in this method. Shearographic experiments conducted have demonstrated the effectiveness of the proposed technique.     

Energy-resolved analysis of ferroelectric electron emission from TGS using emission electron microscopy

Ferroelectric electron emission arises when the spontaneous polarization of a ferroelectric is switched due to the application of an electric field. In order to study the origin of emission and the related emission mechanism, space-resolved emission electron microscopy has been employed. The integral energy distribution of the emitted electrons from triglycine-sulfate surfaces has been investigated using a cylindrical sector analyzer and an imaging retarding field analyzer. Space-resolved emission photography and energy distribution measurements were obtained, revealing the effect of ferroelectric switching on the electric field distribution and hence on the emission process. Evidence of secondary electron emission from the metal electrodes has been found.     

Application of phase-diverse phase retrieval to wavefront sensing in non-connected complicated pupil optics

Baseline algorithm, as a tool in wavefront sensing (WFS), incorporates the phase-diverse phase retrieval (PDPR) method with hybrid-unwrapping approach to ensure a unique pupil phase estimate with high WFS accuracy even in the case of high dynamic range aberration, as long as the pupil shape is of a convex set.However, for a complicated pupil, such as that in obstructed pupil optics, the said unwrapping approach would fail owing to the fake values at points located in obstructed areas of the pupil. Thus a modified unwrapping approach that can minimize the negative effects of the obstructed areas is proposed. Simulations have shown the validity of this unwrapping approach when it is embedded in Baseline algorithm.     

Numerical study of phase conjugation in stimulated backscatter with pump depletion

Two-dimensional numerical simulations are presented of phase conjugation in stimulated backscatter of a focused aberrated beam, including the effects of pump depletion. In all cases, pump depletion significantly improved the conjugation fidelity H. As expected, the far field distribution of light scattered back through the aberrator exhibited a prominent axial spike closely matching that of the incident beam; however, the near field distribution had large and rapid spatial inhomogeneities, even for H ≈ 90%. In spite of such structure, the backscatter was able to reproduce a rough image of large scale modulation in the incident beam.     

Experimental assessment of fluorescence microscopy signal enhancement by stimulated emission

The quantity of photons generated during fluorescence microscopy is principally determined by the quantum yield of the fluorescence dyes and the optical power of the excitation beam. However, even though low quantum yields can produce poor images, it is challenging to tune this parameter, while increasing the power of the excitation beam often results in photodamage. Here, we propose the use of stimulated emission (SE) as a means of enhancing both the signal intensity and signal-to-noise ratio during confocal fluorescence microscopy. This work experimentally confirmed that both these factors can be enhanced by SE radiation, through generating a greater number of photons than are associated with the standard fluorescence signal. We also propose the concept of stimulated emission enhancing fluorescence (SEEF) microscopy, which employs both the SE and fluorescence signals, and demonstrate that the intensity of an SEEF signal is greater than those of the individual SE and fluorescence signals.     

Quantitative surface analysis using ion-induced secondary ion and photon emission

The secondary ion and photon yield from a stainless steel target bombarded with Ar⁺ ions has been studied. The yields have been measured in a variety of oxygen background pressures. From the distribution of photon intensities, an effective arc temperature, T, for the bombarded surface has been derived. An empirical relationship has then been used to obtain the free electron density N_e, of the ion bombardment induced surface “plasma”. The derived values of (T, N_e) are then used in the Andersen-Hinthorne model, to calculate the changes in the Cr/Fe ratio as a function of the oxygen background pressure. The Cr/Fe ratio under the cleanest conditions available agrees very well with an electron microprobe measurement of the same ratio, while the changes in the Cr/Fe ratio as a function of oxygen pressure are consistent with electron spectroscopy studies of the changes in the Cr/Fe ratios in oxidised stainless steel.     

Quantitative analysis of magnetic excitations in Landau flux-closure structures using synchrotron-radiation microscopy

We have measured the excitation spectrum of six micron magnetic squares using x-ray magnetic circular dichroism. We observe all three excitations expected in a Landau flux-closure pattern. High temporal and spatial resolution allows quantitative analysis of the excitations. A short magnetic in plane pulse excites the magnetic element and we observe precessional motion of the magnetization within the domains as well as a domain wall mode and vortex motion. The vortex moves perpendicular to the excitation field and relaxes without showing a circulating orbit.     

Stimulated emission depletion microscopy with a supercontinuum source and fluorescence lifetime imaging

We demonstrate stimulated emission depletion (STED) microscopy implemented in a laser scanning confocal microscope using excitation light derived from supercontinuum generation in a microstructured optical fiber. Images with resolution improvement beyond the far-field diffraction limit in both the lateral and axial directions were acquired by scanning overlapped excitation and depletion beams in two dimensions using the flying spot scanner of a commercially available laser scanning confocal microscope. The spatial properties of the depletion beam were controlled holographically using a programmable spatial light modulator, which can rapidly change between different STED imaging modes and also compensate for aberrations in the optical path. STED fluorescence lifetime imaging microscopy is demonstrated through the use of time-correlated single photon counting.     

Photoacoustic analysis of stimulated emission in pulsed dye lasers

Stimulated emission in pulsed dye lasers was characterized in several experimental conditions by analyzing the changes in the acoustic signals generated in a dye solution, with the dye laser cavity either active or inhibited (i.e., by blocking the optical path or misaligning of the optical components). Pump energy threshold, optimum dye concentration, tuning range and maximum-emission wavelength of a rhodamine 6G dye laser were measured by this method. An approximate model for the photoacoustic signal generation consistent with the experiments is presented.Member of CONICET     

Two-dimensional phase retrieval using a window function

Two-dimensional phase retrieval by use of a window function is considered. First the uniqueness and the reconstruction of a two-dimensional signal from the Fourier intensities of the three signals, an original signal, the signal windowed by a window w(m, n) , and the signal windowed by its complementary window w(c)(m, n) = 1 - w(m, n) , are addressed. Then phase retrieval without a complementary window is considered. Conditions under which a signal can be uniquely specified from the Fourier intensities of the original signal and the windowed signal by w(m, n) are developed, and a reconstruction algorithm is presented.     

相位恢复算法用于光学面形检测的实验研究

 针对相位恢复测量中遇到的初始相位估计、传播因子和收敛判断等问题,以柱面透镜的光学面形为恢复对象,应用迭代相位恢复算法完成了对柱面透镜的光波波前的相位恢复实验,并对实验结果进行了分析。实验结果表明：根据被测对象的光波形态选择最接近的初始相位能够加快收敛速度,避免局部收敛,是保证结果准确与可靠的重要条件。通过对两个记录面上的光强之和做求比值运算计算传播因子 ,并应用到传播公式中,能够消除激光光束本身稳定性与探测器响应均匀性等因素对计算结果的影响。