Two-photon Fluorescence Signal Research Articles

Two-photon imaging of rabbit mammary tissue with 3D reconstruction

A broadband two-photon fluorescence (TPF) imaging system, based on a femtosecond oscillator, has been constructed. This system enables TPF imaging of rabbit mammary gland tissue samples by detecting TPF signals generated from the tissue upon excitation by broadband femtosecond pulses. Specifically, TPF imaging utilizes a detection window of 565–615 nm to capture TPF signals from the fluorescent adenine dinucleotide (FAD) present in lactating cells within breast tissue. Moreover, the 3D reconstruction algorithm has been optimized to visualize the 3D structure of the rabbit mammary gland acinar cavity.The optimized three-dimensional reconstruction algorithm can reduce ambiguous signal regions, making the reconstructed three-dimensional structure more reliable and accurate. This is of significant importance for studying the microscopic structure and cell distribution of breast tissue, contributing to a deeper understanding of the tissue's physiological status.

A two-photon fluorescent probe for highly selective detection of Cys over GSH and Hcy based on the Michael addition and transcyclization mechanism and its application in bioimaging and protein straining in SDS-PAGE

BackgroundCysteine (Cys), glutathione (GSH), and homocysteine (Hcy), as three major biothiols are involved in a variety of physiological processes and play a crucial role in plant growth. Abnormal levels of Cys can cause plants to fail to grow properly. To date, although a very large number of fluorescent probes have been reported for the detection of biothiols, very few of them can be used for the selective discrimination of Cys from GSH and Hcy due to their structural similarity, and only a few of them can be used for plant imaging. ResultsHere, three fluorescent probes (o-/m-/p-TMA) based on TMN fluorophore and the ortho-/meta-/para-substituted maleimide recognition groups were constructed to investigate the selective response effect of Cys. Compared to the o-/m-TMA, p-TMA can selectively detect Cys over GSH and Hcy with a rapid response time (10 min) and a low detection limit (0.26 μM). The theoretical calculation confirmed that the intermediate p-TMA-Cys-int has shorter interatomic reaction distances (3.827 Å) compared to o-/m-TMA-Cys (5.533/5.287 Å), making it more suitable for further transcyclization reactions. Additionally, p-TMA has been employed for selective tracking of exogenous and endogenous Cys in Arabidopsis thaliana using both single-/two-photon fluorescence imaging. Furthermore, single cell walls produced obvious two-photon fluorescence signals, indicating that p-TMA can be used for high-concentration Cys analysis in single cells. Surprisingly, p-TMA can be used as a fluorescent dye for protein staining in SDS-PAGE with higher sensitivity (7.49 μg/mL) than classical Coomassie brilliant blue (14.11 μg/mL). SignificanceThe outstanding properties of p-TMA make it a promising multifunctional molecular tool for the highly selective detection of Cys over GSH and Hcy in various complex environments, including water solutions, zebrafish, and plants. Additionally, it has the potential to be developed as a fluorescent dye for a simple and fast SDS-PAGE fluorescence staining method.

Double-clad photonic crystal fibre for two-photon microscopy

Miniaturized and head-mountable scanning nonlinear fibre-optic microscopy offers an exciting opportunity for the visualization of brain activity on freely walking mice. One challenge in the creation of such miniaturized microscopes is the design of optical fibres capable of delivering ultrashort pulses without distortion and collecting fluorescence simultaneously and efficiently. A double-clad photonic crystal fibre (DCPCF) with 37-cell air holes arranged in hexagonal lattice in core is proposed. The full vector finite element method (FEM) is used for numerical analysis. Nearly zero dispersion of −0.34 ps/nm/km at 920 nm wavelength is finally achieved. The single-mode core with an NA of 0.53 at 920 nm wavelength is used to deliver the femtosecond excitation beam and the inner cladding with an NA of 0.54 at 532 nm is used to collect the two-photon fluorescence signal. The nearly zero dispersion, high NA and low nonlinearity will be beneficial to promote its application in two-photon microscopy.

Rapid digital pathology of H&E-stained fresh human brain specimens as an alternative to frozen biopsy

BackgroundHematoxylin and Eosin (H&E)-based frozen section (FS) pathology is presently the global standard for intraoperative tumor assessment (ITA). Preparation of frozen section is labor intensive, which might consume up-to 30 minutes, and is susceptible to freezing artifacts. An FS-alternative technique is thus necessary, which is sectioning-free, artifact-free, fast, accurate, and reliably deployable without machine learning and/or additional interpretation training.MethodsWe develop a training-free true-H&E Rapid Fresh digital-Pathology (the-RFP) technique which is 4 times faster than the conventional preparation of frozen sections. The-RFP is assisted by a mesoscale Nonlinear Optical Gigascope (mNLOG) platform with a streamlined rapid artifact-compensated 2D large-field mosaic-stitching (rac2D-LMS) approach. A sub-6-minute True-H&E Rapid whole-mount-Soft-Tissue Staining (the-RSTS) protocol is introduced for soft/frangible fresh brain specimens. The mNLOG platform utilizes third harmonic generation (THG) and two-photon excitation fluorescence (TPEF) signals from H and E dyes, respectively, to yield the-RFP images.ResultsWe demonstrate the-RFP technique on fresh excised human brain specimens. The-RFP enables optically-sectioned high-resolution 2D scanning and digital display of a 1 cm2 area in <120 seconds with 3.6 Gigapixels at a sustained effective throughput of >700 M bits/sec, with zero post-acquisition data/image processing. Training-free blind tests considering 50 normal and tumor-specific brain specimens obtained from 8 participants reveal 100% match to the respective formalin-fixed paraffin-embedded (FFPE)-biopsy outcomes.ConclusionsWe provide a digital ITA solution: the-RFP, which is potentially a fast and reliable alternative to FS-pathology. With H&E-compatibility, the-RFP eliminates color- and morphology-specific additional interpretation training for a pathologist, and the-RFP-assessed specimen can reliably undergo FFPE-biopsy confirmation.

Controlling Raman gain with atomic coherence

Raman gain is the optical gain generated by stimulated Raman scattering, which makes weak light amplify possible and can play an important role in weak light communication as well as related field. In this paper, experimentally and theoretically, we have observed the controllable Raman gain under the dressing effects in a coherently prepared N-type four-level atomic configuration. By controlling the detuning of the signal field and the coupling field, the electromagnetically induced transparency (EIT) window can be observed in the signal field. With the incidence of a strong pump laser into the atomic system, the two-photon ultranarrow fluorescence signal can overlap with EIT window by changing the frequency detuning of the pump field, meanwhile, a Raman gain can be induced by placing the signal field at possible large detuning. Such Raman gain can be effectively regulated and optimized by tuning the power of the signal field, pump field and the temperature of the atomic system so as to achieve higher contrast. The multi-parameter regulated higher contrast Raman gain with atomic coherence in atomic thermal system has potential applications in optical communication, superluminal laser and biophotonics.

A two-photon metal-organic framework nanoprobe with catalytic hairpin assembly for amplified MicroRNA imaging in living cells and tissues

Monitoring miRNAs can provide critical information for the generation and progress of various cancers. However, the existing DNA amplification strategies-based fluorescent nanoprobes for intracellular miRNAs imaging are often confronted with problems such as shallow light-penetration depth, enzymatic degradation, and low DNA loading efficiency. Herein, we developed a new two-photon nanoprobe (TP-CHA-MOFs) by using MOF as the nanocarrier and hairpins as a two-photon fluorescence signal amplifier for intracellular miRNA imaging. After cell penetration, intracellular phosphate could coordinate with Zr atoms in the MOFs, leading to the release of hairpins from the surfaces of TP-CHA-MOFs. The intracellular miRNA could trigger the formation of the H1-H2 duplex, which led to a turn-on TP fluorescence signal, realizing amplified detection of target miRNA. TP-CHA-MOFs nanoprobe showed high loading capacity of nucleic acid molecules, high biostability toward enzyme, and good biocompatibility. Because of the employment of TP fluorophore, TP-CHA-MOFs achieved sensitive imaging of intracellular miRNA and differentiated tumor cells with different miRNA levels. Moreover, sensitive imaging of miRNA in deep tumor tissues was realized with a penetration depth of 160 µm. The TP-CHA-MOFs nanoprobe is highly promising for future application in medical diagnostics.

Differential characterization of lumbar spine associated tissue histology with nonlinear optical microscopy.

Percutaneous endoscopic lumbar discectomy (PELD) is the major effective treatment for lumbar disc herniation, and rapid histological identification of dissected tissue is critical to guide the discectomy. In this work, we revealed the histological features of different types of peridural tissues of the lumbar spine by label-free multi-modal nonlinear optical microscopy. Stimulated Raman scattering (SRS) was used to extract lipid and protein distributions, while second harmonic generation (SHG) and two-photon excited fluorescence (TPEF) signals were applied to image the collagen and elastin fibers at the same time. Our results demonstrated that the nonlinear optical features of the dura and adjacent soft tissues were significantly different, showing the potentials of our method for intraoperative differentiation of these critical tissues and improving the surgical outcome of PELD.

Understanding femtosecond optical tweezers: the critical role of nonlinear interactions

Typical single-beam optical tweezers use continuous wave (CW) lasers, which can be explained through force balancing the light pressure from a tightly focused laser beam used for trapping microscopic particles. Recent years have also seen a surge in single-beam optical trapping research with high-repetition-rate femtosecond lasers that has shown certain differences from the CW tweezers, one of which is its sensitive detection capability of the ultrashort pulse induced background free two-photon fluorescence signals. The high peak power of each laser pulse is enough to provide instantaneous trapping potential, while the high repetition rate ensures sustained stable trapping from the successive pulses. Though the capability and usefulness of the optical-tweezers are well established, for both CW and pulsed lasers, simulating real-time scenarios to predict optical trapping behaviour remains a challenging problem. This is especially true for femtosecond laser tweezers since high peak powers are involved when the laser is tightly focused for achieving the tweezing action. The nonlinear optical effect and thermal nonlinearity become much more significant for femtosecond optical trapping. We demonstrate the importance of including these nonlinear interactions for femtosecond pulsed laser mediated optical trapping via their effect in scattering and gradient forces in the Rayleigh regime. Our optical-tweezers model includes thermal and optical nonlinear interactions, making it easier to predict the optical-trap stability in real optical trapping scenarios for both CW and pulsed lasers. Our model provides predictive metrics for choosing solvents, probes, and several optical parameters, which can be validated from our experiments.

Monitoring Changes in Biochemical and Biomechanical Properties of Collagenous Tissues Using Label-Free and Nondestructive Optical Imaging Techniques

We demonstrate the ability of nondestructive optical imaging techniques such as second-harmonic generation (SHG), two-photon fluorescence (TPF), fluorescence lifetime imaging (FLIM), and Raman spectroscopy (RS) to monitor biochemical and mechanical alterations in tissues upon collagen degradation. Decellularized equine pericardium (EP) was treated with 50 μg/mL bacterial collagenase at 37 °C for 8, 16, 24, and 32 h. The SHG ratio (defined as the normalized ratio between SHG and TPF signals) remained unchanged for untreated EP (stored in phosphate-buffered solution (PBS)), whereas treated EP showed a trend of a decreasing SHG ratio with increasing collagen degradation. In the fluorescence domain, treated EP experienced a red-shifted emission and the fluorescence lifetime had a trend of decreasing lifetime with increasing collagen digestion. RS monitors collagen degradation, the spectra had less intense Raman bands at 814, 852, 938, 1242, and 1270 cm-1. Non-negative least-squares (NNLS) modeling quantifies collagen loss and relative increase of elastin. The Young's modulus, derived from atomic force microscope-based nanoindentation experiments, showed a rapid decrease within the first 8 h of collagen degradation, whereas more gradual changes were observed for optical modalities. We conclude that optical imaging techniques like SHG, RS, and FLIM can monitor collagen degradation in a label-free manner and coarsely access mechanical properties in a nondestructive manner.

Model-based wavefront shaping microscopy.

Wavefront shaping is increasingly being used in modern microscopy to obtain high-resolution images deep inside inhomogeneous media. Wavefront shaping methods typically rely on the presence of a "guide star" to find the optimal wavefront to mitigate the scattering of light. However, the use of guide stars poses severe limitations. Notably, only objects in the close vicinity of the guide star can be imaged. Here, we introduce a guide-star-free wavefront shaping method in which the optimal wavefront is computed using a digital model of the sample. The refractive index model of the sample, that serves as the input for the computation, is constructed in situ by the microscope itself. In a proof of principle imaging experiment, we demonstrate a large improvement in the two-photon fluorescence signal through a diffuse medium, outperforming state-of-the-art wavefront shaping by a factor of two in imaging depth.

Encrypted wide-field two-photon microscopy with single-pixel detection and compressed sensing

We demonstrate a single-pixel two-photon microscopy using a compact femtosecond fiber laser by spatially tailoring the beam into orthogonal basis for patterned illumination. Such wide-field illumination excites a weak two-photon fluorescence signal that can be detected by a photomultiplier tube. An encrypted hybrid basis with random element sequence of Hadamard basis is adopted to illuminate the sample. The hybrid basis shares the same differential detection with Hadamard basis, and greatly reduces the number of measurements compared with random basis. The reduced number of measurements was demonstrated by using compressed sensing, which allows the minimum image collection and transfer bandwidth.

Intensity distortion in dual-axis galvo mirror scanning TPF imaging system

Two-photon fluorescence (TPF) imaging system with dual-axis galvo mirror as scanning unit is built. Based on the optical path of imaging system built, mechanism of how intensity distortion generating in dual-axis galvo mirror scanning TPF imaging system is theoretically analyzed. In experiment, TPF imaging is realized with Rhodamine B sample through detecting TPF signal generated from Rhodamine B molecule at excitation of broadband femtosecond pulses. The imaging result verifies the existence of intensity distortion with distribution agreeing well with the theoretical analyzation. To eliminate the intensity distortion in TPF imaging result, a mathematical method is proposed in which a calibration matrix is calculated, As the calibration matrix is applied to the distorted imaging result of Rhodamine B sample, intensity distortion is relieved effectively and precise imaging result is achieved.

Characterizing the Two-photon Absorption Properties of Fluorescent Molecules in the 680-1300 nm Spectral Range.

Two-photon laser scanning microscopy (2PLSM) is a state-of-the-art technique used for non-invasive imaging deep inside the tissue, with high 3D resolution, minimal out-of-focus photodamage, and minimal autofluorescence background. For optimal application of fluorescent probes in 2PLSM, their two-photon absorption (2PA) spectra, expressed in absolute cross sections must be characterized. Excitation at optimum wavelength will make it possible to reduce the laser power and therefore minimize photodamage. Obtaining 2PA spectra and cross sections requires correcting the two-photon excited fluorescence signals for a combination of laser properties, including the beam spatial profile, pulse duration, and absolute power, at each wavelength of the tuning range. To avoid such tedious day-to-day laser characterization required in the absolute measurement method, a relative method based on independently characterized 2PA reference standards is often used. By carefully analyzing the available literature data, we selected the most reliable standards for both the 2PA spectral shape and cross section measurements. Here we describe a protocol for measuring the 2PA spectral shapes and cross sections of fluorescent proteins and other fluorophores with the relative fluorescence method using these reference standards. Our protocol first describes how to build an optical system and then how to perform the measurements. In our protocol, we use Coumarin 540A in dimethyl sulfoxide and LDS 798 in chloroform for the spectral shape measurements to cover the range from 680 to 1300 nm, and Rhodamine 590 in methanol and Fluorescein in alkaline water (pH 11) for the absolute two-photon cross section measurements.

Automated label-free detection of injured neuron with deep learning by two-photon microscopy.

Stroke is a significant cause of morbidity and long-term disability globally. Detection of injured neuron is a prerequisite for defining the degree of focal ischemic brain injury, which can be used to guide further therapy. Here, we demonstrate the capability of two-photon microscopy (TPM) to label-freely identify injured neurons on unstained thin section and fresh tissue of rat cerebral ischemia-reperfusion model, revealing definite diagnostic features compared with conventional staining images. Moreover, a deep learning model based on convolutional neural network is developed to automatically detect the location of injured neurons on TPM images. We then apply deep learning-assisted TPM to evaluate the ischemic regions based on tissue edema, two-photon excited fluorescence signal intensity, as well as neuronal injury, presenting a novel manner for identifying the infarct core, peri-infarct area, and remote area. These results propose an automated and label-free method that could provide supplementary information to augment the diagnostic accuracy, as well as hold the potential to be used as an intravital diagnostic tool for evaluating the effectiveness of drug interventions and predicting potential therapeutics.

Characterization of a multicore fiber image guide for nonlinear endoscopic imaging using two-photon fluorescence and second-harmonic generation.

.Multiphoton microscopy (MPM) has the capacity to record second-harmonic generation (SHG) and endogenous two-photon excitation fluorescence (2PEF) signals emitted from biological tissues. The development of fiber-based miniaturized endomicroscopes delivering pulses in the femtosecond range will allow the transfer of MPM to clinical endoscopy. We present real-time SHG and 2PEF ex vivo images using an endomicroscope, which totally complies with clinical endoscopy regulations. This system is based on the proximal scanning of a commercial multicore image guide (IG). For understanding the inhomogeneities of the recorded images, we quantitatively characterize the IG at the single-core level during nonlinear excitation. The obtained results suggest that these inhomogeneities originate from the variable core geometries that, therefore, exhibit variable nonlinear and dispersive properties. Finally, we propose a method based on modulation of dispersion precompensation to address the image inhomogeneity issue and, as a proof of concept, we demonstrate its capability to improve the nonlinear image quality.

TPF imaging of Rhodamine B at different detection windows

Broadband two-photon fluorescence (TPF) imaging system based on femtosecond oscillator is built. Broadband TPF signal generated from Rhodamine B molecule is detected at different detection windows. Both TPF signals detected at short wavelength detection window of 545–615 nm and long wavelength detection window of 642–708 nm are used for TPF imaging. Through switching detection window from short wavelength to long wavelength, deepened imaging depth is achieved. The TPF imaging results, with single [Formula: see text] pixels frame taking time of 4–8 s, demonstrate high performance and the good reliability of the imaging system.

A pilot study of using multiphoton microscopy to diagnose schwannoma

Schwannoma is a kind of slow-growing and rare neoplasm which poses diagnostic challenges owing to its clinical silence at the presentation period. Here, a schwannoma tumor was studied by multiphoton microscopy (MPM). Results indicate that MPM is capable of identifying a schwannoma tumor without the need for labels. Based on the two-photon excited fluorescence signals from cells and blood vessels and second harmonic generation signals from collagens, several significant features for the identification of schwannoma were visualized, including the Antoni A region, Antoni B region, pericellular collagen pattern, hyalinized vessels and hyalinized stroma. These conclusions highlight the potential of MPM taken as a diagnostic method for label-free identification of schwannoma tumors by these histopathologic characteristics.

Simultaneous detection of two-photon fluorescence and backscatter in laser trapping of dielectric nanoparticles

In recent past, high-repetition-rate, ultrafast pulse excitation has been identified to play an important role in stable trapping of dielectric nanoparticles assisted by optical nonlinearity due to its high peak power. We experimentally demonstrate trapping of 100-nm fluorescent polystyrene particles by simultaneous detection of both two-photon fluorescence (TPF) and backscattered signals. Here we show that TPF signal decays over time due to photobleaching, but this signal is useful to know whether a particle is dragged toward the trap while backscattered signal provides detailed information about the particle’s dynamics inside the trap. We also discuss pros and cons of moving-averaging method (used to smooth noisy experimental data). We conclude that both TPF and backscattered detection methods are needed to explore the dynamics of the particle inside the potential well.

Understanding the Dynamic Behavior of an Anticancer Drug, Doxorubicin, on a Lipid Membrane Using Multiple Spectroscopic Techniques.

The interaction, including the adsorption and embedding, of a widely applied anticancer drug, doxorubicin, with a lipid membrane was investigated. Second harmonic generation and two photon fluorescence were used as a powerful combination capable in revealing this dynamic process at the interface. The adsorption, association, deassociation and embedding of doxorubicin on the lipid membrane were clearly identified based on the consistency in the dynamic parameters revealed by the time dependent second harmonic generation and two-photon fluorescence measurements. This work also presents a new approach for in situ measurement of the adsorption density of doxorubicin on lipid membrane, benefiting from the two-photon fluorescence signal of doxorubicin being significantly altered by its chemical environment. The analysis of the location and molecular density based on the fluorescent efficiency of the chromophores makes the fluorescence measurement a "surface sensitive" technique as well. The analytical procedures used in this work are expected to aid in understanding the interaction between fluorescent molecules and lipid membranes in general.

Polarization-insensitive micro-metalens for high-resolution and miniaturized all-fiber two-photon microendoscopic fluorescence imaging

A high transmission efficiency and polarization-insensitive micro-metalens with a 100 μm diameter has been designed for two-photon excited microendoscopic fluorescence imaging. We theoretically calculate the vector field near the focal region of a high NA objective lens to confirm the effectiveness and feasibility of the micro-metalens. The simulation results of the designed micro-metalens are essentially in agreement with that of an objective lens under linearly or radially polarized hollow Gaussian beam (HGB) illumination. A tightly focused spot with an FWHM of 0.56 μm and a beam quality of 72.54% is obtained when the designed micro-metalens is illuminated by radially polarized 5-order HGB at wavelength 915 nm and the corresponding lateral resolution of the two-photon fluorescence signal is 0.39 μm. Moreover, this ultrathin micro-metalens can be fabricated onto a fiber tip and directly penetrate into tissues. This miniaturized imaging system can provide a compact all-fiber two-photon excitation microendoscopic imaging system.