Influence of fluorescence time characteristics on the spatial resolution of CW-stimulated emission depletion microscopy

As one of the most important realizations of stimulated emission depletion(STED) microscopy, the continuouswave(CW) STED system, constructed by using CW lasers as the excitation and STED beams, has been investigated and developed for nearly a decade. However, a theoretical model of the suppression factors in CW STED has not been well established. In this investigation, the factors that affect the spatial resolution of a CW STED system are theoretically and numerically studied. The full-width-at-half-maximum(FWHM) of a CW STED with a doughnut-shaped STED beam is also reanalyzed. It is found that the suppression function is dominated by the ratio of the local STED and excitation beam intensities. In addition, the FWHM is highly sensitive to both the fluorescence rate(inverse of fluoresce lifetime) and the quenching rate, but insensitive to the rate of vibrational relaxation. For comparison, the suppression function in picosecond STED is only determined by the distribution of the STED beam intensity scaled with the saturation intensity. Our model is highly consistent with published experimental data for evaluating the spatial resolution. This investigation is important in guiding the development of new CW STED systems.     

Theoretical investigation of optical patterning of monolayers with subwavelength resolution

We formulate a model of monolayer patterning via optically-controlled chemical reactions, with the goal of beating the diffraction limit in photolithography. We consider the use of the proven technique of STimulated Emission Depletion (STED) to selectively place a handful of molecules in a reactive excited state. We show that repeated optical excitation has a greater effect on pattern formation than increasing the reaction rate, auguring well for experimental work. We also consider optically-controlled deposition of a soluble species via STED, and show that even for very large concentrations and excited state lifetimes the full width at half maximum of the features formed is robust against the effects of diffusion and saturation.     

Advantages of STED-Inspired 3D Direct Laser Writing for Fabrication of Hybrid Nanostructures

We present a new method of additive laser technology referred to as STED nanolithography technique. This technique provides a means for fabrication of 3D dielectric and plasmonic composite nanostructures. The new technology is of the utmost interest for the electronics manufacturing industry, in particular, for formation of specific hybrid (metal–dye) nanostructures, which can be utilized as luminescent markers in biology, medicine, criminalistics, and the trade industry. In the present study, we demonstrate the advantages of STED-inspired nanolithography for fabrication of metallic and hybrid nanostructures. The 3D-scanning setup implemented offers the possibility to form both periodic and aperiodic nanostructured arrays. We show the possibility to decrease substantially the lateral size of the lines formed with the use of STED nanolithography as compared to the direct laser writing (DLW) method. The STED nanolithography technique proposed provides a means for synthesizing metallic nanoparticles in the specified points of the volume of the studied object in vivo. In addition, we demonstrate the synthesis of metallic lines by means of STED nanolithography. Moreover, nanometer spatial precision of positioning of the synthesized nanoobjects is achieved. Therefore, it is possible to obtain significant local enhancement of the emission of luminescent markers (surface enhanced luminescence) at any desired point or area of the sample due to plasmonic enhancement of the electromagnetic fields near the surface of metallic nanostructures.     

Stimulated-emission-depletion microscopy with a multicolor stimulated-Raman-scattering light source

We describe a subdiffraction-resolution far-field fluorescence microscope employing stimulated emission depletion (STED) with a light source consisting of a microchip laser coupled into a standard single-mode fiber, which, via stimulated Raman scattering (SRS), yields a comb-like spectrum of seven discrete peaks extending from the fundamental wavelength at 532 nm to 620 nm. Each of the spectral peaks can be used as STED light for overcoming the diffraction barrier. This SRS light source enables the simple implementation of multicolor STED and provides a spectral output with multiple available wavelengths from green to red with potential for further expansion.     

Super‐resolution deep imaging with hollow Bessel beam STED microscopy

Stimulated emission depletion (STED) microscopy has become a powerful imaging and localized excitation method, breaking the diffraction barrier for improved spatial resolution in cellular imaging, lithography, etc. Because of specimen‐induced aberrations and scattering distortion, it is a great challenge for STED to maintain consistent lateral resolution deep inside specimens. Here we report on deep imaging STED microscopy using a Gaussian beam for excitation and a hollow Bessel beam for depletion (GB‐STED). The proposed scheme shows an improved imaging depth of up to about 155 μm in a solid agarose sample, 115 μm in polydimethylsiloxane, and 100 μm in a phantom of gray matter in brain tissue with consistent super resolution, while standard STED microscopy shows a significantly reduced lateral resolution at the same imaging depth. The results indicate the excellent imaging penetration capability of GB‐STED, paving the way for deep tissue super‐resolution imaging and three‐dimensional precise laser fabrication.

     

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.     

New possibilities for materials science with STED microscopy

Up to now, STED microscopy has been mainly used to study biological systems. However with the development of the technique, many benefits are expected in materials science for imaging 1D, 2D or 3D nanomaterials. We review here the use of STED microscopy in materials science, its challenges, and opportunities.     

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.     

Self-aligned fiber-based dual-beam source for STED nanolithography

A fiber-based source that can be exploited in a stimulated emission depletion(STED) inspired nanolithography setup is presented. Such a source maintains the excitation beam pulse, generates a ring-shaped depletion beam, and automatically realizes dual-beam coaxial alignment that is critical for two beam nanolithography. The mode conversion of the depletion beam is realized by using a customized vortex fiber, which converts the Gaussian beam into a donut-shaped azimuthally polarized beam. The pulse width and repetition frequency of the excitation beam remain unchanged, and its polarization states can be controlled. According to the simulated point spread function of each beam in the focal region, the full width at half-maximum of the effective spot size in STED nanofabrication could decrease to less than 28.6 nm.     

Pump-probe spectroscopy on photoinitiators for stimulated-emission-depletion optical lithography

We report on femtosecond pump-probe experiments on two different photoinitiators in solution. These two molecules have recently appeared as attractive candidates for far-field optical lithography based on stimulated-emission-depletion (STED) inspired approaches aiming at beating Abbe's diffraction limit. For the case of 7-diethylamino-3-thenoylcoumarin (DETC), we find that stimulated emission clearly dominates over excited-state absorption, whereas the opposite holds true for the case of isopropylthioxanthone. We argue that it is desirable that stimulated emission dominates over excited-state absorption as depletion mechanism in STED photoresists. Thus, DETC is an attractive corresponding photoinitiator.     

Manipulation of doughnut focal spot by image inverting interferometry

Based on image inverting interference combined with phase modulation, we theoretically demonstrate that the doughnut focal spot can readily be manipulated, and either shrinkage or expansion of size of the central dark spot is possible in a large scale (peak-to-peak value: 0.555λ-0.830λ, or 93.3%-140.8% compared with the standard one). As the interference and phase modulation can both be achieved by a double Porro prism, it is feasible to introduce this approach into optical tweezers to improve their performance. As much as 33.9% intensity of stimulated emission depletion (STED) beam can be reduced if the further optimized configuration is utilized in STED microscopy.     

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.     

Saturated Förster resonance energy transfer microscopy with a stimulated emission depletion beam: a pathway toward single-molecule resolution in far-field bioimaging

We theoretically demonstrate that the spatial resolution of stimulated emission depletion (STED) microscopy can be substantially enhanced without increasing the intensity of the STED beam. In our scheme, tiny nanobeads codoped with donor and acceptor molecules are used as fluorescent probes, in which F?rster resonance energy transfer (FRET) can occur with an ~100% efficiency between the donors and acceptors. Enhancement of the depletion of acceptors in the nanobeads with the doughnut-shaped depletion beam can lead to an increase of FRET efficiency in the outer area of the excitation spot, which itself is used for deexciting donor molecules and, consequently, enhancing the optical resolution.     

Generation of tightly focused twin Bessel beams using circular Dammann gratings under radial polarization incidence

Generation of longitudinally polarized focusing twin Bessel beams in focal region of a high numerical aperture (NA) objective is described based on circular Dammann gratings for radially polarized Bessel–Gauss input fields. Numerical simulations show that, under focusing of an objective of NA=0.95, the depth of focus (DOF) of the focused twin Bessel beams can reach as long as tens or even ～10² of wavelengths while its average transverse spot over the whole range of the DOF is kept subdiffration-limited. At the same time, the longitudinal polarization purity in focus volume is higher than 90% for the central lobe. Therefore, this tightly focused non-diffracting field should be of great interest for applications in numerous areas, such as particle acceleration and manipulation, micromachining, second-harmonic generation, Raman spectroscopy, etc.     

波前编码显微物镜景深扩展的研究

为了解决传统显微物镜景深与分辨率的矛盾,采用波前编码的方法,设计研究了波前编码的10倍显微物镜,结合传统光学设计软件,采用基于MTF一致性的相位板参数的优化方法,实现了在像面附近一定范围内系统的点扩散函数一致性;此外,还设计了扩展三次相位编码板的10倍显微物镜,比较了采用两种不同相位板系统的焦深扩展的效果,结果显示三次相位板的焦深扩展效果较好,可以将传统10倍显微物镜的焦深扩展15倍。成像模拟仿真结果显示滤波后的编码像在±15倍焦深范围内成像清晰,从而扩大了系统的景深。     

Improved pinhole spatial filter

If the pinhole placed at the focus of a microscope objective to spatially filter a laser beam is replaced by an annular phase mask, it is possible to obtain a clean beam providing very nearly uniform illumination over an extended area. We now show that even better results can be obtained by adding an absorbing layer to the mask.     

STED microscopy based on axially symmetric polarized vortex beams

A stimulated emission depletion(STED) microscopy scheme using axially symmetric polarized vortex beams is proposed based on unique focusing properties of such kinds of beams. The concept of axially symmetric polarized vortex beams is first introduced, and the basic principle about the scheme is described. Simulation results for several typical beams are then shown, including radially polarized vortex beams, azimuthally polarized vortex beams, and high-order axially symmetric polarized vortex beams. The results indicate that sharper doughnut spots and thus higher resolutions can be achieved, showing more flexibility than previous schemes based on flexible modulation of both phase and polarization for incident beams.     

Compression of fiber supercontinuum pulses to the Fourier-limit in a high-numerical-aperture focus

A multiphoton intrapulse interference phase scan (MIIPS) adaptively and automatically compensates the combined phase distortion from a fiber supercontinuum source, a spatial light modulator pulse shaper, and a high-NA microscope objective, allowing Fourier-transform-limited compression of the supercontinuum pulses at the focus of the objective. A second-harmonic-generation-based method is employed to independently validate the transform-limited compression. The compressed pulses at the focus of the objective have a tunable duration of 10.8-38.9 fs (FWHM), a central wavelength of ~1020 nm, an average power of 18-70 mW, and a repetition rate of 76 MHz, permitting the application of this source to nonlinear optical microscopy and coherently controlled microspectroscopy.     

Two-photon fluorescence scanning near-field microscopy based on a focused evanescent field under total internal reflection

We present two-photon fluorescence near-field microscopy based on an evanescent field focus produced by a ring beam under total internal reflection. The evanescent field produced by this method is focused by a high-numerical-aperture objective, producing a tightly confined volume that can effectively induce two-photon excitation. The imaging system is characterized by the two-photon-excited images of the nanocrystals, which show that the focused evanescent field is split into two lobes because of the enhancement of the longitudinal polarization component at the focus. This feature is confirmed by the theoretical prediction. Unlike other two-photon near-field probes, this method does not have the heating effect and requires no control mechanism of the distance between a sample and the probe.     

Automatic compensation of laser beam focusing parameters for flying optics

Flying optics technologies are used for a range of applications such as a large workpiece processing and laser texturing. It is essential to compensate the variations of laser beam focus parameters while the focus head is moving. A flying optics automatic compensation approach is proposed to achieve invariable laser focal size, focus depth and focus position through computer controlled objective lens and focus lens position. A simple mechanical control method is also presented for the realization of constant beam parameters for flying optics. Numerical simulation is illustrated for a CO₂ laser texturing application. The flying optics parameters compensation is simple and easy to control.