Two-photon Excitation Efficiency Research Articles

Influence of laser pulse shape and cleanliness on two-photon microscopy

Nonlinear microscopy, including two-photon microscopy, requires pulsed lasers as light source. Typically, when choosing the appropriate pulsed laser for two-photon microscopy, the pulse repetition rate, pulse width, total power output, and output beam diameter are among the critical parameters which are often emphasised. Here, we demonstrate that the pulse shape, often overlooked, can have significant impact on the two-photon microscopy excitation efficiency and the effective signal brightness. We provide metrics to ease practical selection of pulsed laser sources for two-photon microscopy.

Evaluation of compact pulsed lasers for two-photon microscopy using a simple method for measuring two-photon excitation efficiency

Evaluation of compact pulsed lasers for two-photon microscopy using a simple method for measuring two-photon excitation efficiency

Two-photon fluorescence imaging using a tunable spectral window based on fiber supercontinuum.

Two-photon excitation fluorescence (TPEF) microscopy has evolved into a versatile tool in biological research. However, the multiplexing capability of TPEF microscopy is limited by the narrow spectral bandwidth of the light source. In this study, we apply a photonic crystal fiber in TPEF microscopy to broaden the excitation source bandwidth. We tuned the spectral window using a spatial light modulator as a programmable diffraction grating that was placed behind a prism pair. In addition, we combined a grating pair to compensate for dispersion to improve the two-photon excitation efficiency. The combination of a broad spectrum and a programmable grating enabled fast spectral window tuning rate on a time scale of tens of milliseconds. We demonstrate the performance of our method by imaging live B16 cells labeled with four emission spectrum overlapped fluorescent proteins.

Precise 3D computer-generated holography based on non-convex optimization with spherical aberration compensation (SAC-NOVO) for two-photon optogenetics.

Computer-generated holography (CGH) has been adopted in two-photon optogenetics as a promising technique for selective excitation of neural ensembles. However, 3D CGH by nonconvex optimization, the state of art method, is susceptible to imprecise axial positioning, due to the quadratic phase approximation in 3D target generation. Even though the misplacement of targets in conventional CGH can be solved by pre-calibration, it still suffers from low efficiency and poor axial resolution of two-photon excitation. Here, we propose a novel CGH method based on non-convex optimization with spherical aberration compensation (SAC-NOVO). Through numerical simulations and two-photon excitation experiments, we verify that SAC-NOVO could achieve precise axial positioning for single and multiple expanded disk patterns, while ensuring high two-photon excitation efficiency. Besides, we experimentally show that SAC-NOVO enables the suppression of dark target areas. This work shows the superiority of SAC-NOVO for two-photon optogenetics.

In vivo two-photon microscopic observation and ablation in deeper brain regions realized by modifications of excitation beam diameter and immersion liquid.

In vivo two-photon microscopy utilizing a nonlinear optical process enables, in living mouse brains, not only the visualization of morphologies and functions of neural networks in deep regions but also their optical manipulation at targeted sites with high spatial precision. Because the two-photon excitation efficiency is proportional to the square of the photon density of the excitation laser light at the focal position, optical aberrations induced by specimens mainly limit the maximum depth of observations or that of manipulations in the microscopy. To increase the two-photon excitation efficiency, we developed a method for evaluating the focal volume in living mouse brains. With this method, we modified the beam diameter of the excitation laser light and the value of the refractive index in the immersion liquid to maximize the excitation photon density at the focal position. These two modifications allowed the successful visualization of the finer structures of hippocampal CA1 neurons, as well as the intracellular calcium dynamics in cortical layer V astrocytes, even with our conventional two-photon microscopy system. Furthermore, it enabled focal laser ablation dissection of both single apical and single basal dendrites of cortical layer V pyramidal neurons. These simple modifications would enable us to investigate the contributions of single cells or single dendrites to the functions of local cortical networks.

Adaptive optics microspectrometer for cross-correlation measurement of microfluidic flows.

.Mapping flows in vivo is essential for the investigation of cardiovascular pathologies in animal models. The limitation of optical-based methods, such as space-time cross correlation, is the scattering of light by the connective and fat components and the direct wave front distortion by large inhomogeneities in the tissue. Nonlinear excitation of the sample fluorescence helps us by reducing light scattering in excitation. However, there is still a limitation on the signal-background due to the wave front distortion. We develop a diffractive optical microscope based on a single spatial light modulator (SLM) with no movable parts. We combine the correction of wave front distortions to the cross-correlation analysis of the flow dynamics. We use the SLM to shine arbitrary patterns of spots on the sample, to correct their optical aberrations, to shift the aberration corrected spot array on the sample for the collection of fluorescence images, and to measure flow velocities from the cross-correlation functions computed between couples of spots. The setup and the algorithms are tested on various microfluidic devices. By applying the adaptive optics correction algorithm, it is possible to increase up to 5 times the signal-to-background ratio and to reduce approximately of the same ratio the uncertainty of the flow speed measurement. By working on grids of spots, we can correct different aberrations in different portions of the field of view, a feature that allows for anisoplanatic aberrations correction. Finally, being more efficient in the excitation, we increase the accuracy of the speed measurement by employing a larger number of spots in the grid despite the fact that the two-photon excitation efficiency scales as the fourth power of this number: we achieve a twofold decrease of the uncertainty and a threefold increase of the accuracy in the evaluation of the flow speed.

Two-Photon Excitation of Gold Nanorods Interrupted by Extremely Fast Solvent-to-Metal Electron Transfer

Gold nanorods (Au NRs) have been widely exploited for various biomedical applications due to their strong two-photon photoluminescence (2PPL). 2PPL of Au NRs were found significantly quenched in organic solvents, which was originally ascribed to reduced luminescence yields caused by electron transfer quenching. It was recently found that excitation of 2PPL of Au NRs involved two sequential one-photon absorption steps. Here various ultrafast spectroscopic techniques have been employed to demonstrate two different solvent-to-metal electron transfer pathways: electron transfer from organic solvents to sp and d band holes of excited Au NRs, which have different influences on 2PPL of Au NRs. Electron transfer to sp band holes occurs extremely fast (∼25 fs), which blocks absorption of the second photon and reduces overall two-photon excitation efficiency, which is the dominant mechanism for the observed 2PPL quenching. In contrast, electron transfer to d band holes hinders the radiative recombination process an...

Au-Ag core-shell nanoparticles for simultaneous bacterial imaging and synergistic antibacterial activity

Au@Ag nanoparticles (NPs) were recently found to display giant two-photon photoluminescence (2PPL) enhancement, with an enhancement factor up to 815 fold upon aggregate formation. Based on this finding, two-photon imaging of bacteria by Au@Ag NPs under near-infrared (NIR) femtosecond laser pulses was demonstrated in this study, as positively charged Au@Ag NPs can form aggregates on the negatively charged bacterial surface, yielding strong 2PPL emission. The aggregation-enhanced 2PPL of Au@Ag NPs stemmed from higher two-photon excitation efficiency, implying strong two-photon photothermal effects. Au@Ag NPs showed strong antibacterial activity (minimum inhibition concentration as low as 7.5pM against Staphylococcus aureus) and negligible toxicity to human dermal fibroblasts. Their bactericidal activity was further enhanced under NIR irradiation due to strong two-photon photothermal effects. Au@Ag NPs effectively removed 85% of the notorious bacterial biofilm within 4 min under NIR irradiation. These Au@Ag NPs can potentially be used as imaging and antibacterial agents.

Single Particle Studies on Two-Photon Photoluminescence of Gold Nanorod–Nanosphere Heterodimers

The localized surface plasmon resonance (SPR) property of metal nanoparticles has numerous potential applications. Coupling between different SPR modes of adjacent metal nanoparticles can result in significantly enhanced optical responses such as surface-enhanced Raman scattering (SERS) and two-photon photoluminescence (2PPL). In this work, gold (Au) nanorod (NR)–nanosphere (NS) heterodimers with side-linked and end-linked spatial arrangements have been systematically studied on the single-particle level to investigate the plasmon coupling effects on their 2PPL properties. Compared to a single Au NR, both end-linked and side-linked Au NR-NS heterodimers displayed red-shifted scattering spectra with similar cos2θ excitation polarization dependence, which can be understood in terms of plasmon hybridization theory. However, the 2PPL intensities of end-linked and side-linked Au NR-NS heterodimers displayed strikingly different excitation polarization dependence. The 2PPL signals of the end-linked Au NR-NS heterodimer displayed cos4θ excitation polarization dependence, similar to that of Au NR monomer. The 2PPL intensity of the end-linked heterodimer is the largest with polarization along the long axis of Au NR, which is ∼300 times that of the Au NR monomer component and even ∼5 times that of a longer Au NR with a similar overall size to the heterodimer. In a striking contrast, 2PPL intensity of the side-linked heterodimer is largest for excitation along the transverse direction of Au NR, which is ∼25 times that under excitation along the longitudinal direction. Despite these differences, an interesting feature is that the largest 2PPL signals occur along the coupling directions for both end-linked and side-linked heterodimers. Plasmon-coupling-enhanced 2PPL is generally believed to arise from enhanced two-photon excitation efficiency through two effects: improved resonance effects due to the plasmon-coupling-induced red-shifted SPR mode and plasmon-coupling-induced giant local electric field amplification. Stronger 2PPL signals have been observed for coupled nanostructures with excitation along the coupling direction but less favorable overlap between the SPR scattering spectra and excitation wavelength, which unambiguously supports that plasmon-coupling-induced local electric field amplification is the dominant effect responsible for the big difference in 2PPL intensities of different nanostructures. This conclusion has been further confirmed by the excellent agreement between the numerically calculated integrated |E/E0|4 and the experimentally obtained 2PPL signal intensities for various nanostructures. These studies offer some fundamental understanding of plasmon coupling effects on optical responses and excitation mechanisms of 2PPL of metal nanostructures, which provide insight on designing nanostructures with tailored optical properties for various potential photonic and optoelectronic applications.

A simple and general strategy for generating frequency-anticorrelated photon pairs.

Currently, two-photon excitation microscopy is the method of choice for imaging living cells within thick specimen. A remaining problem for this technique is the damage caused by the high photon flux in the excitation region. To reduce the required flux, a promising solution is to use highly frequency-anticorrelated photon pairs, which are known to induce two-photon transitions much more efficiently. It is still an open question what the best scheme is for generating such photon pairs. Here we propose one simple general strategy for this task. As an example, we show explicitly that this general strategy can be realized faithfully within the widely applicable coherently pumped Jaynes-Cummings model. It is shown quantitatively that this strategy can generate highly frequency-anticorrelated photon pairs which can dramatically enhance two-photon excitation efficiency. We believe the proposed strategy can guide new designs for generating frequency-anticorrelated photon pairs.

Exploration of the two-photon excitation spectrum of fluorescent dyes at wavelengths below the range of the Ti:Sapphire laser.

We have studied the wavelength dependence of the two-photon excitation efficiency for a number of common UV excitable fluorescent dyes; the nuclear stains DAPI, Hoechst and SYTOX Green, chitin- and cellulose-staining dye Calcofluor White and Alexa Fluor 350, in the visible and near-infrared wavelength range (540–800 nm). For several of the dyes, we observe a substantial increase in the fluorescence emission intensity for shorter excitation wavelengths than the 680 nm which is the shortest wavelength usually available for two-photon microscopy. We also find that although the rate of photo-bleaching increases at shorter wavelengths, it is still possible to acquire many images with higher fluorescence intensity. This is particularly useful for applications where the aim is to image the structure, rather than monitoring changes in emission intensity over extended periods of time. We measure the excitation spectrum when the dyes are used to stain biological specimens to get a more accurate representation of the spectrum of the dye in a cell environment as compared to solution-based measurements.

Two-Photon Induced Photoluminescence and Singlet Oxygen Generation from Aggregated Gold Nanoparticles

Metal nanoparticles have potential applications as bioimaging and photosensitizing agents. Aggregation effects are generally believed to be adverse to their biomedical applications. Here we have studied the aggregation effects on two-photon induced photoluminescence and singlet oxygen generation of Au nanospheres and Au nanorods of two different aspect ratios. Aggregated Au nanospheres and short Au nanorods were found to display enhanced two-photon induced photoluminescence and singlet oxygen generation capabilities compared to the unaggregated ones. The two-photon photoluminescence of Au nanospheres and short Au nanorods were enhanced by up to 15.0- and 2.0-fold upon aggregation, and the corresponding two-photon induced singlet oxygen generation capabilities were enhanced by 8.3 and 1.8-fold, respectively. The two-photon induced photoluminescence and singlet oxygen generation of the aggregated long Au nanorods were found to be lower than the unaggregated ones. These results support that the change in their two-photon induced photoluminescence and singlet oxygen generation originate from aggregation modulated two-photon excitation efficiency. This finding is expected to foster more biomedical applications of metal nanoparticles as Au nanoparticles normally exist in an aggregated form in the biological environments. Considering their excellent biocompatibility, high inertness, ready conjugation, and easy preparation, Au nanoparticles are expected to find more applications in two-photon imaging and two-photon photodynamic therapy.

Excitation Nature of Two-Photon Photoluminescence of Gold Nanorods and Coupled Gold Nanoparticles Studied by Two-Pulse Emission Modulation Spectroscopy.

Gold nanorods (Au NRs) and coupled gold nanospheres (Au NSs) are known to display strong two-photon photoluminescence (TPPL). Here two-pulse emission modulation (TPEM) and pump-probe measurements were performed on Au NRs and coupled Au NSs to understand their excitation mechanisms. The TPEM cross contributions of Au NRs and coupled Au NSs showed much slower decay compared with a two-photon absorption organic dye. Their decay time constants (4.0 ps for Au NRs and 3.1 ps for coupled Au NSs) match well with the lifetimes of intermediate states measured from pump-probe experiments. These results support the fact that strong TPPL in Au NRs and coupled Au NSs arises from two sequential one-photon absorption steps involving real intermediate states instead of coherent two-photon absorption. These results give direct evidence that previously observed aggregation-enhanced TPPL arises from enhanced two-photon excitation efficiency, which was facilitated by two sequential plasmon-coupling-enhanced one-photon absorption steps via real intermediate states.

Monochromatic visible light-driven photocatalysis realized on 2D ZnO shell arrays

Using sunlight to solve energy shortage and pollution problems is still a hot but challenging issue. Because of this, we have demonstrated a novel and simple approach to fabricating a 2D shell array structure to enhance the interaction between incident light and the material. The enhancement remarkably increased the two-photon excitation efficiency of the material and, utilizing the two-photon induced electron–hole pairs in ZnO, we realized photocatalysis under monochromatic visible light for the first time. The method and theory described in this paper provide the basis for realizing high-efficiency sunlight-driven photocatalysts and also show a new way to improve the performance of photoelectric and photonic devices.

Tunable, low-repetition-rate, cost-efficient femtosecond Ti:sapphire laser for nonlinear microscopy

We report on a broadly tunable, long-cavity Ti:sapphire laser oscillator being mode-locked in the net negative intracavity dispersion regime by Kerr-lens mode-locking, delivering τ FWHM<300 fs pulses at 22 MHz repetition rate. The wavelength of the laser can be tuned over a 170 nm wide range between 712 nm and 882 nm. Having a typical pump power of 2.6 W, the maximum pulse peak power is 60 kW. Comparison of the reported laser with a standard, 76 MHz Ti:sapphire oscillator regarding two-photon excitation efficiency in a laser scanning microscope shows that the 22 MHz laser generates the same fluorescence signal at considerably, 1.82 times lower average power, which is expected to result in a reduced photothermal damage probability of biological samples. This fact along with the broad tunability and a low pump power requirement makes this cost-effective laser an ideal light source for nonlinear microscopy.

Laser-induced fluorescence detection of hydroxyl (OH) radical by femtosecond excitation

The development of a laser-induced fluorescence detection scheme for probing combustion-relevant species using a high-repetition-rate ultrafast laser is described. A femtosecond laser system with a 1 kHz repetition rate is used to induce fluorescence, following two-photon excitation (TPE), from hydroxyl (OH) radicals that are present in premixed laminar flames. The experimental TPE and one-photon fluorescence spectra resulting from broadband excitation into the (0,0) band of the OH A(2)∑(+)-X(2)Π system are compared to simulated spectra. Additionally, the effects of non-transform-limited femtosecond pulses on TPE efficiency is investigated.

Selective two-photon excitation of a vibronic state by correlated photons

We theoretically investigate the two-photon excitation of a molecular vibronic state by correlated photons with energy anticorrelation. A Morse oscillator having three sets of vibronic states is used, as an example, to evaluate the selectivity and efficiency of two-photon excitation. We show that a vibrational mode can be selectively excited with high efficiency by the correlated photons, without phase manipulation or pulse-shaping techniques. This can be achieved by controlling the quantum correlation so that the photon pair concurrently has two pulse widths, namely, a temporally narrow width and a spectrally narrow width. Though this concurrence is seemingly contradictory, we can create such a photon pair by tailoring the quantum correlation between two photons.

Photobleaching and photoenhancement of endogenous fluorescence observed in two-photonmicroscopy with broadband laser sources

We examine the effects of pulse duration tuning on the photodamage inflicted by laser lightillumination on the imaged sample and, thereby, explore the optimization of opticalpulse parameters for multiphoton microscopy imaging under variable conditions.We discuss the dependence of the nonlinear excitation efficiency and associatedphotodamage rates on pulse energy and duration, and use the controlled amount ofsecond-order dispersion (linear chirp), introduced by a pulse shaper, to adjust the pulseduration at the imaging plane of the microscope. The pulse energy is varied tomaintain a constant two-photon excitation efficiency when switching between short (∼14 fs) andlong (∼280 fs) pulses, and the damage is assessed by monitoring the photobleaching rates and samplemorphology. We have found that in addition to the well-known photobleaching effects,significant enhancement of the two-photon excited autofluorescence intensity can beobserved. Photobleaching rates at the onset of the laser light exposure are shown to beindependent of the pulse shape under our experimental conditions, which indicates that theprimary damage (bleaching) mechanism stems from the two-photon excitation process. Thephotoenhancement, however, is found to occur more readily with longer pulses, havinghigher energies per pulse. Experiments are carried out on human melanoma tissue and onrabbit red blood cells.

Efficient selective two-photon excitation by tailored quantum-correlated photons

We theoretically investigate two-photon excitation by correlated photons with energy anticorrelation. A three-level atomic system is used to evaluate selectivity and efficiency of two-photon excitation. It is shown that tailored squeezing of two-photon probability distribution concurrently enhances excitation efficiency and high-contrast selective excitation, without phase manipulation or pulse-shaping techniques. We also discuss candidates to generate such squeezed photons in real material systems.

Real-time analysis of two-photon excitation by correlated photons: Pulse-width dependence of excitation efficiency

We theoretically investigate the dynamics of two-photon excitation by correlated photons with energy anticorrelation in terms of how the excitation efficiency depends on incident pulse width. A three-level atomic system having an intermediate state is used to evaluate the efficiency of two-photon excitation. It is shown that for shorter pulses closer to a monocycle pulse the excitation efficiency by correlated photons is enhanced to become $100$ times as large as that by uncorrelated photons.