Imaging biological specimens with the confocal scanning laser microscope/macroscope

A new confocal scanning laser microscope/macroscope (cslm/M) has recently been developed. It combines in one instrument the high resolution capability of a confocal scanning beam microscope for imaging small specimens, with good resolution confocal imaging of macroscopic specimens. Some of its main features include: (a) 0.25 μm lateral resolution in the microscope mode and 5 μm lateral resolution in the macroscope mode; (b) a field of view that can vary from 25 μm × 25 μm to 75,000 μm × 75,000 μm; (c) capability for acquiring large data sets from 512 × 512 pixels to 2048 × 2048 pixels; (d) 0.5 μm depth resolution in the microscope mode and 200 μm depth resolution in the macroscope mode.

In this work the cslm/M was used to image whole biological specimens (> 5 m diameter), including insects which are ideal specimens for the macroscope. Specimens require no preparation, unlike scanning electron microscope (SEM) specimens which require a conductive coating. The specimens described in this paper are too large to be imaged in their entirety by a scanning beam laser microscope, however they can be imaged by slower scanning stage microscopes. In the macroscope mode the cslm/M was used to acquire a large number (e.g. 20–40) of confocal image slices which were then used to reconstruct a three-dimensional image of the specimen. High resolution images were collected by the cslm/M by switching to the microscope mode where high numerical aperture (NA) objectives were used to image a small area of interest. Reflected-light and fluorescence images of plant and insect specimens are presented which demonstrate the morphological details obtained in various imaging modes. A process for three-dimensional visualization of the data is described and images are shown.     

Complex cylindrical vector beam excludes the orientation dependence of the intensity of scanning fluorescence images of single molecules

A polarization inhomogeneous vector beam, which is the complex superposition of radially and azimuthally polarized beams, has been studied computationally and theoretically in application to scanning fluorescence microscopy of single molecules. It has been shown that its application makes it possible to almost exclude the dependence of the intensity maximum of the scanning fluorescence image of a single molecule on its orientation. It has been demonstrated that the scanning of one horizontal plane of a solid matrix containing impurity luminescent molecules provides images of all molecules located in a 1-μm-thick layer, replacing three-dimensional scanning. Recommendations on the technical implementation of the proposed beam have been given.     

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     

Ground-state-depletion fluorscence microscopy: A concept for breaking the diffraction resolution limit

We introduce and study a novel concept in farfield fluorescence microscopy fundamentally overcoming the classical diffraction resolution limit. This is accomlished by reducing the spatial extent of the effective focus of a scanning fluorescence microscope. The reduction is achieved by depleting the ground-state energy of the molecules located in the outer region of the focus. Our theoretical study shows that ground-state-depletion fluorescence microscopy has the potential of increasing the resolution of far-field fluorescence microscopy by an order of magnitude which is equivalent to a lateral resolution of 15 NM.     

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.     

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.     

二次谐波共焦成像的分辨率

通过与双光子荧光共焦成像过程比较,研究了非线性二次谐波的强度脉冲响应函数并对它进行频域分析,获得了横向、纵向截止频率和通频带.研究结果表明：二次谐波共焦成像具有更高的分辨率.     

Quantitative atomic force microscopy with carbon monoxide terminated tips

Noncontact atomic force microscopy (AFM) has recently progressed tremendously in achieving atomic resolution imaging through the use of small oscillation amplitudes and well-defined modification of the tip apex. In particular, it has been shown that picking up simple inorganic molecules (such as CO) by the AFM tip leads to a well-defined tip apex and to enhanced image resolution. Here, we use the same approach to study the three-dimensional intermolecular interaction potential between two molecules and focus on the implications of using molecule-modified AFM tips for microscopy and force spectroscopy experiments. The flexibility of the CO at the tip apex complicates the measurement of the intermolecular interaction energy between two CO molecules. Our work establishes the physical limits of measuring intermolecular interactions with scanning probes.     

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.     

Near-field scanning optical microscopy of single molecules by femtosecond two-photon excitation

We present a demonstration of near-field scanning optical microscopy of single molecules based on ultrafast two-photon-induced fluorescence. Measurements were performed by use of 100-fs pulses at 800 nm from a Ti:sapphire laser to excite the two-photon transition in Rhodamine B molecules. Although near-field probes are normally metal coated to achieve superresolution, we used uncoated tips to achieve sufficiently high optical powers to generate acceptable fluorescence emission rates. Images of single molecules demonstrate a resolution of ~175nm(< lambda/4) on a topographically smooth surface, which surpasses the apparent lambda/2 resolution limit for uncoated tips operating in the linear response regime.     

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.     

Nonlinear fluorescence through intermolecular energy transfer and resolution increase in fluorescence microscopy

We investigate a novel concept to efficiently generate multiphoton induced fluorescence from organic molecules. The concept is based on frustrating the energy transfer between a fluorescent donor and one or more acceptors in conjugated molecules. The nonlinearity is not based on higher order molecular susceptibilities but entirely on their linear properties. Therefore, in contrast to nonresonant multiphoton absorption, this method does not require high local intensities. Likewise, the production of visible fluorescence does not require an infrared excitation wavelength. Hence, when applied to scanning microscopy this property is predicted to increase spatial resolution. Instead of the ∼10 GW/cm² required in non-resonant multiphoton excitation, focal intensities ∼10 MW/cm² are expected to produce an equally strong nonlinear signal. The predicted resolution is up to 30% greater than that of an ideal confocal microscope operating at the same fluorescence wavelength. The resolution improvement over non-resonant two-photon absorption microscopes is about two-fold in all directions.     

High-resolution imaging of C60 molecules using tuning-fork-based non-contact atomic force microscopy

Recent advances in non-contact atomic force microscopy (nc-AFM) have led to the possibility of achieving unprecedented resolution within molecular structures, accomplished by probing short-range repulsive interaction forces. Here we investigate C(60) molecules adsorbed on KBr(111) and Cu(111) by tuning-fork-based nc-AFM. First, measurements of C(60) deposited on KBr(001) were conducted in cryogenic conditions revealing highly resolved nc-AFM images of the self-assembly. Using constant-frequency shift mode as well as three-dimensional spectroscopic measurements, we observe that the relatively weak molecule-substrate interaction generally leads to the disruption of molecular assembled structures when the tip is probing the short-range force regime. This particular issue hindered us in resolving the chemical structure of this molecule on the KBr surface. To obtain a better anchoring of C(60) molecules, nc-AFM measurements were performed on Cu(111). Sub-molecular resolutions within the molecules was achieved which allowed a direct and unambiguous visualization of their orientations on the supporting substrate. Furthermore, three-dimensional spectroscopic measurements of simultaneous force and current have been performed above the single molecules giving information of the C(60) molecular orientation as well as its local conductivity. We further discuss the different imaging modes in nc-AFM such as constant-frequency shift nc-AFM, constant-height nc-AFM and constant-current nc-AFM as well as three-dimensional spectroscopic measurement (3D-DFS) employed to achieve such resolution at the sub-molecular scale.     

10 nm Spatial Resolution Fluorescence Imaging of Single Molecules by Near-Field Scanning Optical Microscopy Using a Tiny Aperture Probe

We demonstrate high-resolution fluorescence imaging of single molecules using near-field scanning optical microscopy (NSOM) with a tiny aperture probe for two different wavelengths in visible range in the illumination mode of operation. The spatial resolutions obtained at both excitation wavelengths were almost the same and the highest resolution realized was about 10 nm. To discuss the achievable resolution in aperture NSOM, we also employed a computer simulation by the finite-difference time-domain method for various aperture sizes and wavelengths. The resolution of 10 nm is predicted to be contributed by the single peak of localized near-field light around the rim of the aperture.     

Real-space observation of nanojet-induced modes in a chain of microspheres

The three-dimensional real-space observation of photonic nanojet-induced modes in a chain of microspheres with different diameters is reported. The optical transmission properties of a chain of microspheres are studied by using high resolution finite-difference time-domain calculation. The photonic nanojet-induced modes in different chains of microspheres are measured by using a scanning optical microscope system with an optical-fiber probe. We observe the photonic nanojet-induced modes from optical microscope images for chains of 3 μm, 5 μm, and 8 μm microspheres deposited on a patterned silicon substrate. The incident beam can be periodically reproduced in chains of dielectric microspheres giving rise to lossless periodically optical focusing with period of two diameters. Detailed theoretical and experimental data on the transmission, scattering loss, and field-of-view are presented. This waveguide technique can be used in biomedical microscopy, ultra-precise laser process, microfluidics, and nanophotonic circuits.     

Adsorbate-substrate vibrational modes of benzene on Ag(110) resolved with scanning tunneling spectroscopy

Using a low temperature scanning tunneling microscope, we have detected low energy adsorbate-substrate (external or frustrated) vibrational modes of benzene molecules adsorbed on a Ag(110) surface. We demonstrate that such vibrations represent a fingerprint of the molecules' chemical state and environment; two different vibrational spectra are measured on molecules populating two different adsorption states. We also find that the distortion of the adsorption geometry of the molecules may give rise to the excitation of additional (initially hidden) modes. Important differences in the spatial distribution of the inelastic signal are also observed for these external modes.     

Three-dimensional mapping of cholinergic molecules by confocal laser scanning microscopy in sea urchin larvae.

Confocal laser scanning microscopy (CLSM) was used to examine molecules related to the cholinergic neurotransmission system and detected at all the larval stages of Paracentrotus lividus, by histochemical and immunohistochemical methods. CLSM, providing spatial resolution of the cells located both at the larval surface and at depth, allows a complete mapping in a three-dimensional volumetric frame. At early larval stages acetylcholinesterase- as well as choline acetyltransferase-like molecules were found mainly in the gut wall cells, and along the ciliary bands of the arms, together with muscarinic acetylcholine receptors. At perimetamorphic stages, cholinergic molecules were present in the ciliate strands along the arms, in the larval body and in the rudiment. At metamorphosis, positivity to cholinergic molecules translocated to the juvenile, where a high frequency of mAChR- and ChAT-like positive cells was found.     

随机扫描多光子荧光显微成像系统

多光子荧光显微成像是生物学研究的有力手段,但目前的成像速度难以满足神经成像中快事件检测的需要。针对这一问题,提出了一套随机扫描快速多光子荧光显微成像系统。系统采用二维声光偏转器快速扫描飞秒激发光束,能够以每点10μs的速度对特定的感兴趣区域进行跳跃式扫描,即随机扫描,使得有效的扫描速度大为提高。引入单棱镜补偿方法解决应用声光偏转器带来的色散问题。以170 nm荧光小球为样品,测得系统的横向分辨力为0.3μm,纵向分辨力为1.3μm。给出了随机扫描系统和商品化多光子荧光显微镜对同一个荧光细胞的成像结果,证明了新系统的成像能力。     

Two-photon near- and far-field fluorescence microscopy with continuous-wave excitation

We report on scanning far- and near-field two-photon microscopy of cell nuclei stained with DAPI and bisbenzimidazole Hoechst 33342 (BBI-342) with the 647-nm laser line of a cw ArKr mixed-gas laser. Two-photon-excited fluorescence images are obtained for 50-200 mW of average power at the sample. A nearly quadratic dependence of fluorescence intensity on laser power confirmed the two-photon effect. The nonlinearity was further supported by evidence of three-dimensional sectioning in a scanning far-field microscope. We find that the cw two-photon irradiation sufficient for imaging within typically 5 s does not significantly impair cell cycling of BBI-342-labeled live cells. Finally, high-resolution imaging in scanning near-field microscopy with good contrast is demonstrated.     

Subdiffraction resolution in total internal reflection fluorescence microscopy with a grating substrate

We propose a fluorescence surface imaging system that presents a power of resolution beyond that of the diffraction limit without resorting to saturation effects or probe scanning. This is achieved by depositing the sample on an optimized periodically nanostructured substrate in a standard total internal reflection fluorescence microscope. The grating generates a high-spatial-frequency light grid that can be moved throughout the sample by changing the incident angle. An appropriate reconstruction procedure permits one to recover the fluorescence amplitude from the images obtained for various incidences. Simulations of this imaging system show that the resolution is not limited by diffraction but by the period of the grating.