Continuous wave excitation two-photon fluorescence microscopy exemplified with the 647-nm ArKr laser line

We report on efficient two-photon fluorescence imaging in beam scanning microscopy by exciting UV dyes at the 647-nm line of a continuous wave ArKr mixed gas laser. For a numerical aperture of 1.4 (oil), we used an illumination power of up to 210 mW at the sample. High-resolution images were obtained for DAPI-labelled cell nuclei within 4–60 s. Our method is a simple two-photon alternative to UV confocal imaging with the potential of becoming a very useful feature of laser scanning microscopy.     

Fast scanning STED and two-photon fluorescence excitation microscopy with continuous wave beam

In this paper we report stimulated emission depletion (STED) and two-photon excitation (2PE) fluorescence microscopy with continuous wave (CW) laser beam using a new generation laser scanning confocal microscope equipped for STED-CW (TCS STED-CW, Leica Microsystems, Mannheim, Germany). We show the possibility to achieve CW-2PE with the very same beam used for STED-CW. This feature extends the performance of the microscope allowing multimodal imaging (CW-2PE, STED-CW, confocal).     

Antecedents of two-photon excitation laser scanning microscopy

In 1931, Maria G?ppert-Mayer published her doctoral dissertation on the theory of two-photon quantum transitions (two-photon absorption and emission) in atoms. This report describes and analyzes the theoretical and experimental work on nonlinear optics, in particular two-photon excitation processes, that occurred between 1931 and the experimental implementation of two-photon excitation microscopy by the group of Webb in 1990. In addition to Maria G?ppert-Mayer's theoretical work, the invention of the laser has a key role in the development of two-photon microscopy. Nonlinear effects were previously observed in different frequency domains (low-frequency electric and magnetic fields and magnetization), but the high electric field strength afforded by lasers was necessary to demonstrate many nonlinear effects in the optical frequency range. In 1978, the first high-resolution nonlinear microscope with depth resolution was described by the Oxford group. Sheppard and Kompfner published a study in Applied Optics describing microscopic imaging based on second-harmonic generation. In their report, they further proposed that other nonlinear optical effects, such as two-photon fluorescence, could also be applied. However, the developments in the field of nonlinear optical stalled due to a lack of a suitable laser source. This obstacle was removed with the advent of femtosecond lasers in the 1980s. In 1990, the seminal study of Denk, Strickler, and Webb on two-photon laser scanning fluorescence microscopy was published in Science. Their paper clearly demonstrated the capability of two-photon excitation microscopy for biology, and it served to convince a wide audience of scientists of the potential capability of the technique.     

An evaluation of two-photon excitation versus confocal and digital deconvolution fluorescence microscopy imaging in Xenopus morphogenesis.

The ability to visualize cell motility occurring deep in the context of opaque tissues will allow many currently intractable issues in developmental biology and organogenesis to be addressed. In this study, we compare two-photon excitation with laser scanning confocal and conventional digital deconvolution fluorescence microscopy, using the same optical configuration, for their ability to resolve cell shape deep in Xenopus gastrula and neurula tissues. The two-photon microscope offers better depth penetration and less autofluorescence compared to confocal and conventional deconvolution imaging. Both two-photon excitation and confocal microscopy also provide improved rejection of "out-of-focus" noise and better lateral and axial resolution than conventional digital deconvolution microscopy. Deep Xenopus cells are best resolved by applying the digital deconvolution method on the two-photon images. We have also found that the two-photon has better depth penetration without any degradation in the image quality of interior sections compared to the other two techniques. Also, we have demonstrated that the quality of the image changes at different depths for various excitation powers.     

Two-photon scanning microphotolysis for three-dimensional data storage and biological transport measurements

Scanning microphotolysis is a method that permits the user to select, within the scanning field of a confocal microscope, areas of arbitrary geometry for photobleaching or photoactivation. Two-photon absorption, by contrast, confers on laser scanning microscopy a true spatial selectivity by restricting excitation to very small focal volumes. In the present study the two methods were combined by complementing a laser scanning microscope with both a fast programmable optical switch and a titan sapphire laser. The efficiency and accuracy of fluorescence photobleaching induced by two-photon absorption were determined using fluorescein-containing polyacrylamide gels. At optimal conditions a single scan was sufficient to reduce the gel fluorescence by ≈40%. Under these conditions the spatial accuracy of photobleaching was 0.5±0.1 μm in the lateral ( x y ) and 3.5±0.5 μm in the axial ( z ) direction, without deconvolution accounting for the optical resolution. Deconvolution improved the accuracy values by ≈30%. The method was applied to write complex three-dimensional patterns into thick gels by successively scanning many closely spaced layers, each according to an individual image mask. Membrane transport was studied in a model tissue consisting of human erythrocyte ghosts carrying large transmembrane pores and packed into three-dimensional arrays. Upon equilibration with a fluorescent transport substrate single ghosts could be selectively photobleached and the influx of fresh transport substrate be monitored. The results suggest that two-photon scanning microphotolysis provides new possibilities for the optical analysis and manipulation of both technical and biological microsystems.     

Multiphoton confocal microscopy using a femtosecond Cr:forsterite laser

With its output wavelength covering the infrared penetrating window of most biological tissues at 1,200-1,250 nm, the femtosecond Cr:forsterite laser shows high potential to serve as an excellent excitation source for the multiphoton fluorescence microscope. Its high output power, short optical pulse width, high stability, and low dispersion in fibers make it a perfect replacement for the currently widely used Ti:sapphire laser. In this paper, we study the capability of using a femtosecond Cr:forsterite laser in multiphoton scanning microscopy. We have performed the multiphoton excited photoluminescence spectrum measurement on several commonly used bioprobes using the 1,230 nm femtosecond pulses from a Cr:forsterite laser. Efficient fluorescence can be easily observed in these bioprobes through two-photon or three-photon excitation processes. These results will assist in the selection of dichroic beam splitter and band pass filters in a multiphoton microscopic system. We have also performed the autofluorescence spectrum measurement from chlorophylls in live leaves of the plant Arabidopsis thaliana excited by 1,230 nm femtosecond pulses from the Cr:forsterite laser. Bright luminescence from chlorophyll, centered at 673 and 728 nm, respectively, can be easily observed. Taking advantage of the bright two-photon photoluminescence from chlorophyll, we demonstrated the two-photon scanning paradermal and cross-sectional images of palisade mesophyll cells in live leaves of Arabidopsis thaliana.     

Autofluorescence spectroscopy and imaging of Platymonas subcordiformis irradiated by diode laser based on LSCM

Autofluorescence spectra and optical imaging of Platymonas subcordiformis after irradiation of diode laser were observed via laser scanning confocal microscopy (LSCM). With 488 nm Ar(+) laser excitation, the horizontal and vertical dimensions of a cup-shaped chloroplast of the irradiation group increased about 10% compared with the control group. The fluorescence spectra were similar between irradiation group and control group with a maximum fluorescence band around 682 nm, whereas the former has a higher intensity. Image of a small circular substance with stronger two-photon autofluorescence (TPA) was obtained when using two-photon excitation wavelength of 800 nm in single-channel mode. Further analysis by the 800 nm excitation based on two independent-channels mode showed an emission band of the small circular substance around 376-505 nm, which corresponded to the eyespot of P. subcordiformis. In lambda scanning mode, with two-photon wavelength of 800 nm excitation, six fluorescence peaks that are located at 465, 520, 560, 617, 660 and 680 nm were observed; the fluorescence intensity of the irradiation group was higher than that of the control group, especially at 520, 560 and 617 nm. As a conclusion, diode laser irradiation can promote chloroplast growth of P. subcordiformis cells in the form of expanding area and the increasing content of protein, phospholipids and chlorophyll. LSCM, especially TPA imaging based on femtosecond laser excitation, provides a nondestructive, real-time and accurate method to study changes of living algal cells under laser irradiation and other environmental factors.     

A simple introduction to multiphoton microscopy

Multiphoton microscopy is a powerful technique based on complex quantum mechanical effects. Thanks to the development of turnkey mode-locked laser systems, multiphoton microscopy is now available for everyone to use without extreme complexity. In this short introduction, we describe qualitatively the important concepts underlying the most commonly used type of multiphoton microscopy (two-photon excitation). We elucidate how those properties lead to the powerful results that have been achieved using this technique. As with any technique, two-photon excitation microscopy has limitations that we describe, and we provide examples of particular classes of experiments where two-photon excitation microscopy is advantageous over other approaches. Finally, we briefly describe other useful multiphoton microscopy approaches, such as three-photon excitation and second harmonic generation imaging.     

Three-dimensional laser scanning two-photon fluorescence confocal microscopy of polymer materials using a new,efficient upconverting fluorophore

Three-dimensional confocal imaging of polymer samples was achieved by the use of two-photon excited fluorescence in both positive and negative contrast modes. The fluorophore was a new and highly efficient two-photon induced upconverter, resulting in improved signal strength at low pumping power. Because of the relatively long wavelength of the excitation source (798 nm from a mode-locked Ti:Sap-phire laser), this technique shows a larger penetration depth into the samples than provided by conventional single-photon fluorescence confocal microscopy. Single-photon and two-photon images of the same area of each sample show significant differences. The results suggest the possibility of using two-photon confocal microscopy, in conjunction with highly efficient fluorophores, as a tool to study the surface, interface, and fracture in material science applications.     

Two-photon fluorescence real-time imaging on the development of early mouse embryo by stages

Mouse zygotes are widely used in developmental biology and transgenic animal research. Two-photon laser scanning microscopy is particularly useful in four-dimensional observation of big and thick biological samples, such as mouse embryo. The early mouse embryo development from zygote to 8-cell stage compaction was observed in real-time by stages using two-photon laser scanning microscopy in this paper. During our experiment, several scanning parameters were optimized in different development stages. The initial cleavages of mouse embryo, from the zygote to 2-cell, 2-cell to 4-cell, 4-cell to 8-cell, and the compaction at 8-cell stage were observed. During the first stage, localized intracellular calcium elevation along with the cleavage furrow and the asymmetric zygote cytokinesis was detected. The relation between the asymmetry and the location of the second polar body was investigated. The rotational cleavage of mouse embryo was also observed in the experiment. These results would be helpful to further research on mammalian embryonic development.     

Adapting a compact confocal microscope system to a two-photon excitation fluorescence imaging architecture.

Within the framework of a national National Institute of Physics of Matter (INFM) project, we have realised a two-photon excitation (TPE) fluorescence microscope based on a new generation commercial confocal scanning head. The core of the architecture is a mode-locked Ti:Sapphire laser (Tsunami 3960, Spectra Physics Inc., Mountain View, CA) pumped by a high-power (5 W, 532 nm) laser (Millennia V, Spectra Physics Inc.) and an ultracompact confocal scanning head, Nikon PCM2000 (Nikon Instruments, Florence, Italy) using a single-pinhole design. Three-dimensional point-spread function has been measured to define spatial resolution performances. The TPE microscope has been used with a wide range of excitable fluorescent molecules (DAPI, Fura-2, Indo-1, DiOC(6)(3), fluoresceine, Texas red) covering a single photon spectral range from UV to green. An example is reported on 3D imaging of the helical structure of the sperm head of the Octopus Eledone cirrhosa labelled with an UV excitable dye, i.e., DAPI. The system can be easily switched for operating both in conventional and two-photon mode.     

Compact multiphoton/single photon laser scanning microscope for spectral imaging and fluorescence lifetime imaging

An inverted fluorescence microscope was upgraded into a compact three-dimensional laser scanning microscope (LSM) of 65 x 62 x 48 cm dimensions by means of a fast kHz galvoscanner unit, a piezodriven z-stage, and a picosecond (ps) 50 MHz laser diode at 405 nm. In addition, compact turn-key near infrared femtosecond lasers have been employed to perform multiphoton fluorescence and second harmonic generation (SHG) microscopy. To expand the features of the compact LSM, a time-correlated single photon counting unit as well as a Sagnac interferometer have been added to realize fluorescence lifetime imaging (FLIM) and spectral imaging. Using this unique five-dimensional microscope, TauMap, single-photon excited (SPE), and two-photon excited (TPE) cellular fluorescence as well as intratissue autofluorescence of water plant leaves have been investigated with submicron spatial resolution, <270 ps temporal resolution, and 10 nm spectral resolution.     

A promising new wavelength region for three-photon fluorescence microscopy of live cells

We report three-photon laser scanning microscopy (3PLSM) using a bi-directional pumped optical parametric oscillator (OPO) with signal wavelength output at λ= 1500 nm. This novel laser was used to overcome the high optical loss in the infrared spectral region observed in laser scanning microscopes and objective lenses that renders them otherwise difficult to use for imaging. To test our system, we performed 3PLSM auto-fluorescence imaging of live plant cells at λ= 1500 nm, specifically Spirogyra, and compared performance with two-photon excitation (2PLSM) imaging using a femtosecond pulsed Ti:Sapphire laser at λ= 780 nm. Analysis of cell viability based on cytoplasmic organelle streaming and structural changes of cells revealed that at similar peak powers, 2PLSM caused gross cell damage after 5 min but 3PLSM showed little or no interference with cell function after 15 min. The λ= 1500 nm OPO is thus shown to be a practical laser source for live cell imaging.     

Saturation modified point spread functions in two-photon microscopy

Excitation saturation can dramatically alter the effective imaging point spread function (PSF) in two-photon fluorescence microscopy. The saturation-modified PSF can have important implications for resolution in fluorescence imaging as saturation leads to both an increased fluorescence observation volume and an altered spatial profile for the PSF. We introduce here a computational approach to accurately quantify molecular excitation profiles that represent the modified imaging PSF in two-photon microscopy under the influence of excitation saturation. An analytical model that accounts for pulsed laser excitation is developed to calculate the influence of saturation at any location within the excitation laser profile. The overall saturation modified molecular excitation profiles are then evaluated numerically. Our results demonstrate that saturation can play an important role in two-photon fluorescence microscopy even with relatively modest excitation levels.     

Mapping piezoelectric-field distribution in gallium nitride with scanning second-harmonic generation microscopy

Taking advantage of the electric field-enhanced second-harmonic generation effect in bulk gallium nitride (GaN) and indium gallium nitride (InGaN) quantum wells, we demonstrated the piezoelectric field distribution mapping in bulk GaN and InGaN multiple-quantum-well (MQW) samples using scanning second-harmonic generation (SHG) microscopy. Scanning SHG microscopy and the accompanying third-harmonic generation (THG) microscopy of the bulk GaN sample were demonstrated using a femtosecond Cr:forsterite laser at a wavelength of 1230 nm. Taking advantage of the off-resonant electric field-enhanced SHG effect and the bandtail state-resonance THG effect, the second- and third-harmonic generation microscopic images obtained revealed the piezoelectric field and bandtail state distributions in a GaN sample. Combined with 720 nm wavelength excited two-photon fluorescence microscopy in the same sample, the increased defect density around the defect area was found to suppress bandedge photoluminescence, to increase yellow luminescence, to increase bandtail state density, and to decrease residue piezoelectric field intensity. Scanning SHG microscopy of the InGaN MQW sample was resonant excited with 800 nm femtosecond pulses from a Ti:sapphire laser in order to suppress SHG contribution from the bulk GaN substrate. Taking advantage of the strong piezoelectric field inside the InGaN quantum well, the wavelength resonant effect, and the electric field-enhanced SHG effect of InGaN quantum wells, resonant scanning SHG microscopy revealed the piezoelectric field distribution inside the wells. Combined with accompanying three-photon fluorescence microscopy from the bulk GaN substrate underneath the quantum wells, the direct correspondence between the piezoelectric field strength inside the quantum well and the substrate quality can be obtained. According to our study, the GaN substrate area with bright bandedge luminescence corresponds to the area with strong SHG signals indicating a higher stained-induced piezoelectric field. These scanning harmonic generation microscopies exhibit superior images of the piezoelectric field and defect state distributions in GaN and InGaN MQWs not available before. Combining with scanning multiphoton fluorescence microscopy, these techniques open new ways for the physical property study of this important material system and can provide interesting details that are not readily available by other microscopic techniques.     

激光扫描共聚焦显微镜技术的发展及应用

激光扫描共聚焦显微术是先进的分子和细胞生物学研究技术。它在荧光显微镜成像的基础上加装激光扫描装置,结合数据化图像处理技术,采集组织和细胞内荧光标记图像。在亚细胞水平观察钙等离子水平的变化,并结合电生理等技术观察细胞生理活动与细胞形态及运动变化的相互关系。由于它的应用范围较广泛,已成为形态学、分子细胞生物学、神经科学和药理学等研究领域中很重要的研究技术。     

Advances in laser sources for confocal and multiphoton microscopy

The illumination source for all high-resolution, optical sectioning, scanning microscopes is crucially important to the overall performance of the system. We examine advances that have been made in laser sources for both confocal and multiphoton microscopy where the emphasis has been on the development of potentially low-cost, easy to use sources. Growing interest in temporally and spatially resolved techniques has directed laser research towards addressing these challenges. We present the most recent developments in sources for confocal and multiphoton microscopy along with the considerations that should be made when a new source is being considered.     

Comparison of two-photon excitation laser scanning microscopy with UV-confocal laser scanning microscopy in three-dimensional calcium imaging using the fluorescence indicator Indo-1

Two-photon excitation laser scanning fluorescence microscopy (2p-LSM) was compared with UV-excitation confocal laser scanning fluorescence microscopy (UV-CLSM) in terms of three-dimensional (3-D) calcium imaging of living cells in culture. Indo-1 was used as a calcium indicator. Since the excitation volume is more limited and excitation wavelengths are longer in 2p-LSM than in UV-CLSM, 2p-LSM exhibited several advantages over UV-CLSM: (1) a lower level of background signal by a factor of 6–17, which enhances the contrast by a factor of 6–21; (2) a lower rate of photobleaching by a factor of 2–4; (3) slightly lower phototoxicity. When 3-D images were repeatedly acquired, the calcium concentration determined by UV-CLSM depended strongly on the number of data acquisitions and the nuclear regions falsely exhibited low calcium concentrations, probably due to an interplay of different levels of photobleaching of Indo-1 and autofluorescence, while the calcium concentration evaluated by 2p-LSM was stable and homogeneous throughout the cytoplasm. The spatial resolution of 2p-LSM was worse by 10% in the focal plane and by 30% along the optical axis due to the longer excitation wavelength. This disadvantage can be overcome by the addition of a confocal pinhole (two-photon excitation confocal laser scanning fluorescence microscopy), which made the resolution similar to that in UV-CLSM. These results indicate that 2p-LSM is preferable for repeated 3-D reconstruction of calcium concentration in living cells. In UV-CLSM, 0.18-mW laser power with a 2.φ pinhole (in normalized optical coordinate) gives better signal-to-noise ratio, contrast and resolution than 0.09-mW laser power with a 4.9-φ pinhole. However, since the damage to cells and the rate of photobleaching is substantially greater under the former condition, it is not suitable for repeated acquisition of 3-D images.     

Three-Dimensional imaging of fertilization and early development

The field of biological microscopy has recently enjoyed major technical advances, exemplified by the development of field-emission low-voltage scanning electron microscopes and laser scanning confocal light microscopes. In addition, computer processing of microscopical data is revolutionizing the way morphological information is imaged. In this paper, we illustrate methods by which this new technology can be used to examine events in fertilization and early development in three dimensions. Different types of specimen preparation protocols, using both echinoderm and mammalian gametes and embryos, are evaluated for their ability to preserve accurately the threedimensional organization of these specimens for imaging by both low-voltage scanning electron microscopy and laser scanning confocal light microscopy.     

The use of optical parametric oscillator for harmonic generation and two-photon UV fluorescence microscopy

Ultrafast lasers have found increasing use in scanning optical microscopy due to their very high peak power in generating multiphoton excitations. A mode-locked Ti:sapphire laser is often employed for such purposes. Together with a synchronously pumped optical parametric oscillator (OPO), the spectral range available can be extended to 1,050-1,300 nm. This broader range available greatly facilitates the excitation of second harmonic generation (SHG) and third harmonic generation (THG) due to better satisfaction of phase matching condition that is achieved with a longer excitation wavelength. Dental sections are then investigated with the contrasts from harmonic generation. In addition, through intra-cavity doubling wavelengths from 525-650 nm are made available for effective two-photon (2-p) excitation with the equivalent photon energy in the UVB range (290-320 nm) and beyond. This new capacity allows UV (auto-) fluorescence excitation and imaging, for example, from some amino acids, such as tyrosine, tryptophan, and glycine.