High-resolution confocal microscopy by saturated excitation of fluorescence

We demonstrate the use of saturated excitation in confocal fluorescence microscopy to improve the spatial resolution. In the proposed technique, we modulate the excitation intensity temporally and detect the harmonic modulation of the fluorescence signal which is caused by the saturated excitation in the center of the laser focus. Theoretical and experimental investigations show that the demodulated fluorescence signal is nonlinearly proportional to the excitation intensity and contributes to improve the spatial resolution in three dimensions beyond the diffraction limit of light.     

Subdiffraction resolution in far-field fluorescence microscopy

We overcame the resolution limit of scanning far-field fluorescence microscopy by disabling the fluorescence from the outer part of the focal spot. Whereas a near-UV pulse generates a diffraction-limited distribution of excited molecules, a spatially offset pulse quenches the excited molecules from the outer part of the focus through stimulated emission. This results in a subdiffraction-sized effective point-spread function. For a 1.4 aperture and a 388-nm excitation wavelength spatial resolution is increased from 150 +/- 8 nm to 106 +/- 8 nm with a single offset beam. Superior lateral resolution is demonstrated by separation of adjacent Pyridine 2 nanocrystals that are otherwise indiscernible.     

Saturated structured confocal microscopy with theoretically unlimited resolution

The resolution of fluorescence microscopes is limited by diffraction, which determines the extension of their point spread functions. We propose and study numerically a simple method, based on a combination of subtraction microscopy with regular and annular excitation beams, which permits to double the resolution compared to wide field microscopy. When combined with the fluorescence saturation phenomenon, this approach would be able to deliver a resolution of a few tens of nanometers.     

多变视场的多焦点多光子激发荧光显微技术

发展了一种新型的多焦点多光子激发荧光显微技术,通过软件控制空间光调制器,在所需要的成像区域产生相应的激发光点阵,通过扫描入射角实现激发点阵快速并行扫描激发,通过CCD并行记录所产生的荧光信号,获得任意视场图像。分别实现了10×10和50×50点阵激发下的全视场双光子荧光图像,并且实现了同时多个区域寻址的多焦点激发成像。对比其他多焦点显微技术,该技术具有明显的灵活性,不但保持了多焦点激发的快速成像的优点,而且还增加了任意数量成像区域同时寻址和阵列点密度变化,所有这些变化只需要通过软件加载相应的相位图案给空间光调制器,而不需要任何硬件更改。     

Far-field fluorescence microscopy with repetitive excitation

We introduce the concept of repeatedly exciting an excited state of a photostable fluorescent entity to generate a nonlinear fluorescence signal which is solely based on the linear susceptibility of the molecule. The excitation cycle between the fluorescent state and another state prolongs the average lifetime of , with emphasis on those molecules that are in the center of the focus. The photons emitted by the long-lived molecules in the center are recorded by a temporal filter and constitute fluorescence that depends nonlinearly on the excitation intensity. Theoretical analysis reveals that this concept can provide three-dimensional imaging and improve the spatial resolution in far-field fluorescence microscopy. We show that despite the presence of diffraction the effective focal waist can in principle be narrowed down to the molecular scale at the expense of signal. Received: 3 December 1998     

Breaking the diffraction barrier in fluorescence microscopy by optical shelving

We report the breaking of the diffraction resolution barrier in far-field fluorescence microscopy by transiently shelving the fluorophore in a metastable dark state. Using a relatively modest light intensity of several kW/cm(2) in a focal distribution featuring a local zero, we confine the fluorescence emission to a spot whose diameter is a fraction of the wavelength of light. Nanoscale far-field optical resolution down to 50 nm is demonstrated by imaging microtubules in a mammalian cell and proteins on the plasma membrane of a neuron. The presence of dark states in virtually any fluorescent molecule opens up a new venue for far-field microscopy with resolution that is no longer limited by diffraction.     

Near-Field Fluorescence and Topography Characterization of a Single Nanometre Fluorophore by Apertureless Tip-Enhanced Scanning Near-Field Microscopy

Tip-enhanced near-field fluorescence and topography characterization of a single nanometre fluorophore is conducted by using an apertureless scanning near-field microscopy system. A fluorophore with size 80hm is mapped with a spatial resolution of 10hm. The corresponding near-field fluorescence data shows significant signal enhancement due to the apertureless tip-enhanced effect. With the nanometre spatial resolution capability and nanometre local tip-enhanced effect, the apertureless tip-enhanced scanning near-field microscopy may be further used to characterize a single molecule by realizing the local near-field spectrum assignment corresponding to topography at nanometre scale.     

Lateral resolution of 28 nm (<Emphasis Type="BoldItalic">λ</Emphasis> /25) in far-field fluorescence microscopy

We demonstrate sub-diffraction lateral resolution of 28±2 nm in far-field fluorescence microscopy through stimulated emission depletion effected by an amplified laser diode. Measurement of the optical transfer function in the focal plane reveals a 6-fold enlargement of the spatial bandwith over the diffraction limit. The resolution is established by imaging individual fluorescent molecules on a surface. Corresponding to 1/25 of the responsible wavelength, the attained resolution represents a new benchmark in far-field microscopy and underscores the viability of fluorescence nanoscopy with visible light, conventional optics and compact laser systems . PACS 32.50.+d; 42.30.-d; 78.45.+h; 87.57.Ce     

Resolution improvement by nonconfocal theta microscopy

We present a novel scanning fluorescence microscopy technique, nonconfocal theta microscopy (NCTM), that provides almost isotropic resolution. In NCTM, multiphoton absorption from two orthogonal illumination directions is used to induce fluorescence emission. Therefore the point-spread function of the microscope is described by the product of illumination point-spread functions with reduced spatial overlap, which provides the resolution improvement and the more isotropic observation volume. We discuss the technical details of this new method.     

Hybrid Rayleigh,Raman and two‐photon excited fluorescence spectral confocal microscopy of living cells

A hybrid fluorescence–Raman confocal microscopy platform is presented, which integrates low‐wavenumber‐resolution Raman imaging, Rayleigh scatter imaging and two‐photon fluorescence (TPE) spectral imaging, fast ‘amplitude‐only’ TPE‐fluorescence imaging and high‐spectral‐resolution Raman imaging. This multi‐dimensional fluorescence–Raman microscopy platform enables rapid imaging along the fluorescence emission and/or Rayleigh scatter dimensions. It is shown that optical contrast in these images can be used to select an area of interest prior to subsequent investigation with high spatially and spectrally resolved Raman imaging. This new microscopy platform combines the strengths of Raman ‘chemical’ imaging with light scattering microscopy and fluorescence microscopy and provides new modes of correlative light microscopy. Simultaneous acquisition of TPE hyperspectral fluorescence imaging and Raman imaging illustrates spatial relationships of fluorophores, water, lipid and protein in cells. The fluorescence–Raman microscope is demonstrated in an application to living human bone marrow stromal stem cells. Copyright © 2009 John Wiley & Sons, Ltd.     

Scanning near-field optical microscopy-based study of local dynamics of receptor-ligand interactions at the single molecule level

A scanning near-field optical microscope (SNOM)—based modification of the method to study the dynamics of single molecule receptor—ligand interactions exploiting the fluorescence imaging by total internal reflection fluorescence microscopy is introduced. The main advantage of this approach consists in the possibility to study the single molecule interaction dynamics with a subwavelength spatial resolution and a submillisecond time resolution. Additionally, due to the much smaller irradiation area and some other technical features, such a modification enables to enlarge the scope of the receptor—ligand pairs to be investigated and to improve the temporal resolution. We briefly discuss corresponding experimental set up with a special accent on the SNOM operation in liquid and present some preliminary results of related investigations.     

基于电动可调焦透镜的大范围快速光片显微成像

搭建了一种基于电动可调焦透镜(electrically tunable lens)的大范围快速光片荧光显微成像系统.通过引入电动可调焦透镜与一维振镜以实现成像物平面和光片位置的快速移动,再结合高速s CMOS完成快速光片荧光显微成像.另外实验中通过改善光路与提升动态成像质量,实现了大范围扫描并减少了伪像.最终对成像性能进行测试,本系统的纵向分辨率和横向分辨率分别达到约5.5μm和约0.7μm,单幅图像稳定成像的速度约为275 frames/s,成像深度可超过138μm,能满足对具有一定尺寸的生物样本进行实时清晰成像的需求.     

Lateral resolution improvement in two-photon excitation microscopy by aperture engineering

A technique for resolution improvement in two-photon excitation (2PE) fluorescence microscopy based on radially-symmetric annular binary filter (consist of central circular aperture and a concentric peripheral annulus) is proposed. Resolution improvement is achieved by engineering the aperture of the objective lens in a way so as to enhance high spatial frequencies. The structure of the electromagnetic field in the regions of focus and nearby regions are determined. The central lobe of the time-averaged electric energy density is considerably reduced for both linearly- and circularly-polarized illuminated light. An impressive combined comparative percentage improvement of 40% and 53.71% both at low (α = 30) and high (α = 60) aperture angle is obtained for linearly-polarized light. Proposed aperture engineering technique complements conventional, confocal, two-photon fluorescence microscopy, and may facilitate working at low-to-medium magnifications and large free-working distances.     

25 nm resolution single molecular fluorescence imaging by scanning near-field optical/atomic force microscopy

High-resolution single molecular near-field fluorescence images were observed by scanning near-field optical/atomic force microscopy (SNOM/AFM). We modified the SNOM/AFM for both high-resolution fluorescence imaging and high-resolution topographic imaging. The imaged fluorophore, Alexa 532, is prepared with a poly-methyl-methacrylate (PMMA) film coating. A fluorescence resolution of 25 nm was obtained with a simultaneous topographic image of a flat surface. A sample prepared with a lower PMMA concentration exhibited a rough surface in the micro area. The results for the flat surface indicated that the fluorescence resolution is worst in the rough surface sample, that the maximum fluorescence intensities for the individual fluorophore are similar, and that the decay rate is faster. Thus, we concluded that the morphological effect is an important factor in fluorescence image resolution and the apparent lifetimes of the fluorescence molecules.     

超分辨率成像荧光探针材料应用进展

为了进一步认知复杂环境中的细胞生物学过程,研究人员发展了各种各样的生物成像技术。在这些技术中,生物荧光成像因简单的成像条件以及对生物样品的相容性而得到了广泛的发展。然而,传统的荧光成像技术受到了光学衍射极限的限制,无法分辨低于200 nm的空间结构,阻碍了对亚细胞结构的生物学过程研究。超分辨荧光显微镜技术突破了传统光学衍射对成像分辨率的限制,能够获取纳米尺度的细胞动态过程。除了对传统的宽场荧光显微镜框架的改进及升级改造之外,目前典型的超分辨成像显微镜技术通常依赖于荧光探针材料的光物理性质。常用的荧光探针材料包括荧光蛋白、有机荧光分子和纳米荧光材料等。本文介绍了几种主流的超分辨荧光显微成像技术并总结了已经成功应用到超分辨生物荧光成像中的荧光探针材料的应用进展。     

Application of metal nanoparticles in confocal laser scanning microscopy: improved resolution by optical field enhancement

We describe experiments in which the field enhancement by metal nanoparticles that accompanies surface plasmon excitation has been exploited in confocal laser scanning microscopy. The objective was to make use of the rapid decay of the enhanced light field with distance from the nanoparticles to increase the longitudinal resolution, by confining the emission of fluorescence light to the immediate vicinity of the contact area between the substrate and the objects under study. We demonstrate that the increased longitudinal resolution is accompanied by a better lateral resolution if the imaged objects have a curved surface.     

不同荧光波长的双光子共焦成像分析

研究了双光子共焦显微镜中不同荧光波长对成像特性的影响,导出了不同荧光波长的三维脉冲响应函数和三维光学传递函数并进行数值计算.研究结果表明：不同荧光波长对双光子共焦显微镜的三维光学传递函数、三维脉冲响应函数和空间截止频率产生明显的影响,随着荧光波长的增大,分辨率明显下降,但不会出现单光子共焦显微镜中的失锥现象,选取适当的荧光波长进行成像,有利于进一步改善图像分辨率和成像质量.     

Kindling molecules: a new way to ‘break’ the Abbe limit

Fluorescence microscopy is a key tool for biological investigations. However, compared to other techniques like electron microscopy, the achievable resolution is still limited. Tremendous efforts have been devoted to improve the resolution of far-field optical microscopy. Several techniques do exist; however their adoption by biologists has been slowed by several technical limitations. We propose a new method based on a recently discovered family of optically switchable fluorescent molecules. Kindling proteins open the way to very high resolution in far-field fluorescence 3-D microscopy with relatively simple techniques. To cite this article: O. Haeberlé, C. R. Physique 5 (2004).     

Extended resolution wide-field optical imaging: objective-launched standing-wave total internal reflection fluorescence microscopy

Standing-wave total-internal-reflection fluorescence (SW-TIRF) microscopy uses a super-diffraction-limited standing evanescent wave to extract the high-spatial-frequency content of an object through a diffraction-limited optical imaging system. The effective point-spread function is better than a quarter of the emission wavelength. With a 1.45 numerical aperture objective and 532 nm excitation wavelength, a Rayleigh resolution of approximately 100 nm can be achieved, which is better than twice the resolution of conventional TIRF microscopy. This first experimental realization of SW-TIRF in an objective-launched geometry demonstrates the potential for extended resolution imaging at high speed by using wide-field 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.