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.     

The Ring Dye Laser as a Source for Two-Photon Excited Fluorescence

ABSTRACT

The near-Gaussian beam profile, low pulse-to-pulse amplitude fluctuation and amenability to simple signal processing make synchronously-pumped dye lasers, both continuous and cavity-dumped, excellent sources for two-photon spectroscopy. In recent years attention has shifted from the more common standing wave configuration to a traveling wave ring laser of synchronous length. The continuous variant has proven to be a source with a detectivity for two-photon excited fluorescence equal to the cavity-dumped, standing wave laser but without the added optical and electronic complexity of the Bragg cell. However, cavity gain/loss, bandwidth and length changes influence pulse characteristics to a different extent than in standing wave lasers. These effects are discussed relative to both the two-color, two-photon experiment and overall instrumental design considerations.     

Picosecond pulsed two-photon imaging with repetition rates of 200 and 400 MHz

We show the viability of high-resolution two-photon fluorescence imaging of fixed and live cells by exciting the fluorophores with a train of near-infrared pulses with duration in the picosecond range. This is exemplified with a compact, diode-pumped Nd:YVO₄ laser, emitting trains of 7-ps pulses at a wavelength of 1064 nm, with a repetition rate of 200 MHz at two separate outputs. Incoherent combination of the outputs enabled two-photon excitation with a repetition rate of 400 MHz. For a numerical aperture of 1.4 (oil), we used an average illumination power of up to 20–40 mW at the sample. The pulses were coupled into a beam scanning microscope, either directly or through a single mode glass fibre. Compared with standard femtosecond titanium–sapphire excitation conditions, our experiments were performed with a 2.5 or 5 times higher repetition rate, 30–70 times longer pulses and 10–35 times lower pulse peak intensity. The experiments indicate the possibility of significantly relaxing the temporal pulse width constraints for a series of applications.     

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.     

Acousto-optic modulator system for femtosecond laser pulses

Femtosecond laser pulses have made a revolution in multiphoton excitation microscopy, micromachining, and optical storage for their unprecedented high peak power. However, modulation of their intensity with acousto-optic modulator (AOM) is frustrated by dispersion which results in a significant stretch in pulse width. Here we report a scheme composed of two acousto-optic deflectors (AODs) to modulate the intensity of the femtosecond laser pulses with simultaneous compensation for the temporal dispersion. With commercial AODs, we demonstrated such an AOM system for the femtosecond laser pulses with overall transmission efficiency of around 80%. The pulse width of the exit beam is 115-177 fs for an input pulse of 110 fs, across the wavelength range of 720-920 nm when the temporal dispersion compensation is optimally tuned at 800 nm. The fluorescence intensity in a two-photon microscopy experiment performed using this system increased 5.5-fold over that of the uncompensated AOM.     

Cr:Forsterite-laser-based fiber-optic nonlinear endoscope with higher efficiencies

We demonstrate a beam-scanning nonlinear light endoscope based on a flexible fiber bundle. Excited with a femtosecond Cr:Forsterite laser, the degradation in multiphoton multiharmonic excitation efficiency due to the pulse-broadening effect is significantly reduced without utilizing any external devices. The system resolution has been characterized to be 5.4 microm in the two-photon fluorescence endoscope, limited by the sampling theory. Finally, several image examples have been given.     

Spectral characteristics of autofluorescence and second harmonic generation from ex vivo human skin induced by femtosecond laser and visible lasers

The spectral properties of one-photon, two-photon excited autofluorescence and second harmonic generation (SHG) from ex vivo human skin induced by a femtosecond (fs) laser and three visible lasers in backscattering geometry are systematically investigated. Our experimental results indicate that peak position of autofluorescence spectra from the dermis and epidermis shift toward long wavelengths, and the fluorescent intensity decreases when the excitation wavelength increases due to an effect of the excitation wavelength on autofluorescence signals. However, the intensity of the SHG signal in collagen has the maximal value of 800 nm excitation wavelength. This may be the result that the energy of the SHG signal is in resonance with an electronic absorption band. The two-photon excited autofluorescence and SHG intensity all obey a quadratical dependence on the excitation power. Compared with the two-photon excited fluorescence and SHG, the one-photon excited fluorescence in the dermis and epidermis exhibits different spectral characteristics. The investigation of the spectral characteristics of autofluorescence and SHG from ex vivo human skin can provide new insights into morphologic structures and biochemical components of tissues, which are vital for improving the application of laser-induced autofluorescence and SHG spectroscopy technique for noninvasive in vivo tissue diagnostics.     

基于飞秒激光双光子的微齿轮加工技术研究

针对传统加工方法很难实现微机电系统(Micro electromechanical systems,MEMS)零部件高质量加工的问题,在微细加工技术研究基础上,提出采用飞秒激光双光子聚合加工技术加工标准渐开线微齿轮的方法.采用钛蓝宝石激光器自行搭建的飞秒激光双光子加工系统,能够输出波长为800 nm的飞秒激光用于双光子聚合加工,利用AutoCAD软件设计标准渐开线微齿轮,通过理论和试验两种方法研究激光功率与单个固化点尺寸之间的关系,进而研究扫描步距与加工精度和表面粗糙度之间的关系,结果表明,激光功率越小,加工分辨率越高;扫描步距越小,加工变形越大,但表面质量提高.采用优化后的加工工艺参数加工出高质量的标准渐开线微齿轮,其表面粗糙度Ra27.66 nm.因此,飞秒激光双光子加工技术能够为微齿轮或其他MEMS零部件的加工提供一条有效途径.     

Fluorescence lifetime images and correlation spectra obtained by multidimensional time-correlated single photon counting

Multidimensional time-correlated single photon counting (TCSPC) is based on the excitation of the sample by a high-repetition rate laser and the detection of single photons of the fluorescence signal in several detection channels. Each photon is characterized by its arrival time in the laser period, its detection channel number, and several additional variables such as the coordinates of an image area, or the time from the start of the experiment. Combined with a confocal or two-photon laser scanning microscope and a pulsed laser, multidimensional TCSPC makes a fluorescence lifetime technique with multiwavelength capability, near-ideal counting efficiency, and the capability to resolve multiexponential decay functions. We show that the same technique and the same hardware can be used for precision fluorescence decay analysis and fluorescence correlation spectroscopy (FCS) in selected spots of a sample.     

Two-photon image correlation spectroscopy and image cross-correlation spectroscopy

We introduce two-photon image correlation spectroscopy (ICS) using a video rate capable multiphoton microscope. We demonstrate how video rate two-photon microscopic imaging and image correlation analysis may be combined to measure molecular transport properties over ranges typical of biomolecules in membrane environments. Using two-photon ICS, we measured diffusion coefficients as large as 10⁻⁸ cm² s⁻¹ that matched theoretical predictions for samples of fluorescent microspheres suspended in aqueous sucrose solutions. We also show the sensitivity of the method for measuring microscopic flow using analogous test samples. We demonstrate explicitly the advantages of the image correlation approach for measurement of correlation functions with high signal-to-noise in relatively short time periods and discuss situations when these methods represent improvements over non-imaging fluorescence correlation spectroscopy. We present the first demonstration of two-photon image cross-correlation spectroscopy where we simultaneously excite (via two-photon absorption) non-identical fluorophores with a single pulsed laser. We also demonstrate cellular application of two-photon ICS for measurements of slow diffusion of green fluorescent protein/adhesion receptor constructs within the basal membrane of live CHO fibroblast cells.     

Effect of a confocal pinhole in two-photon microscopy.

We investigated the effect of a finite-sized confocal pinhole on the performance of nonlinear optical microscopes based on two-photon excited fluorescence and second-harmonic generation. These techniques were implemented using a modified inverted commercial confocal microscope coupled to a femtosecond Ti:sapphire laser. Both the transverse and axial resolutions are improved when the confocal pinhole is used, albeit at the expense of the signal level. Therefore, the routine use of a confocal pinhole of optimized size is recommended for two-photon microscopy wherever the fluorescence or harmonic signals are large.     

基于非线性偏振旋转锁模技术的光纤飞秒激光振荡器

基于非线性偏振旋转（nonlinear polarization rotation, NPR）锁模机制的光纤激光器因其结构紧凑、可靠性高而备受关注。基于这一锁模原理设计并搭建了掺镱光纤飞秒激光器。当双向泵浦功率为380 mW,在1 030 nm波段获得了基频重复率为22.8 MHz的锁模脉冲。脉冲宽度为224 fs,平均功率180 mW,单脉冲能量8 nJ,10 dB带宽约为40 nm,信噪比大于50 dB。该激光器采用环形腔结构产生稳定的锁模飞秒脉冲输出,可实现自启动锁模。泵浦功率增加到1.6 W可观察到最高三阶被动谐波锁模,三次谐波对应68.5 MHz重复频率。该激光器由于在线宽、脉宽、脉冲能量上的优势,在光谱测量、拉曼成像等领域具有应用意义。     

掺铒单模光纤飞秒脉冲激光器和放大器

为了获得一种被动锁模掺铒光纤振荡器及功率放大器,数值模拟出超短脉冲在光纤中的传输和演化过程,并基于此搭建了一种被动锁模掺铒光纤飞秒振荡器及功率放大器。实验获得了中心波长1560 nm、重复频率100 MHz、输出功率30 mW、脉冲宽度85 fs超短脉冲。通过采用PPLN晶体进行倍频,进一步获得了输出功率5 mW,中心波长780 nm的飞秒脉冲。该光纤激光器为全保偏光纤结构,具有体积小巧、可靠性高、稳定性好的特点。     

Control of microstructure shape and morphology in femtosecond laser ablation of imprint rollers

With increasing demand for microstructure shape accuracy for MEMS and optoelectronic devices, controllability of shape and morphology in micro-fabrication has become increasingly crucial. In this paper, the effects of processing parameters on the shape and morphology of microstructures in femtosecond laser fabrication of imprint roller are explored. An optimized fabrication process is proposed to acquire high accuracy microstructures, in which a two-step inclination ablation process and optimal laser focus position are adopted. Adjusting and matching the processing parameters is a basic method to acquire well-defined shapes, but the ablation results indicate that the draft angle of microstructures can only be adjusted in a limited range due to the intensity distribution of laser beam. A two-step inclination ablation process is adopted to increase the draft angle. In the two-step inclination ablation process, the laser beam irradiates the target surface with an angle and the microstructure with a much steeper draft angle forms after the two-step fabrication. Laser focus position is explored as an important parameter affecting the morphology, and an optimal laser focus position is obtained to enhance the ablation quality. By matching the laser fluence and laser focus position, this morphology enhancement method can realize the high-quality ablation of microstructures with a wide range of dimensions without changing the focusing objective lens.     

Extension of imaging depth in two-photon fluorescence microscopy using a long-wavelength high-pulse-energy femtosecond laser source

We experimentally demonstrate, for the first time to the best of our knowledge, two-photon fluorescence imaging with a femtosecond optical parametric amplifier. In particular, we systematically compare the imaging depths of two-photon fluorescence microscopes based on three different excitation sources, including a femtosecond oscillator, a femtosecond regenerative amplifier and the optical parametric amplifier. The results show that the optical parametric amplifier can greatly extend the penetration depth by approximately 227% as compared with that obtained with the femtosecond oscillator due to effective suppression of scattering at longer wavelength and enhanced excitation efficiency enabled by higher pulse energy.     

用于去除非共振背景的圆偏振宽带CARS光谱研究

相干反斯托克斯拉曼散射(CARS)是一种受激拉曼现象,在显微成像时,存在非共振背景,会导致光谱产生峰位偏移和谱线变形。本文利用飞秒激光作为光源,通过光栅滤波系统产生窄带泵浦光,飞秒激光激发光子晶体光纤产生超连续谱作为斯托克斯光,两束光被调制为圆偏振光后同时激发样品产生CARS光谱。通过模拟计算说明圆偏振光可以有效去除各向异性材料CARS光谱中的非共振背景,从而使CARS光谱具有和自发拉曼相似的谱线形状。聚苯乙烯微球和液晶样品的CARS光谱实验结果与模拟计算基本相符,说明圆偏振是一种有效去除CARS光谱非共振背景的方法。     

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.     

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.     

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.     

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.