Application Of Multiphoton Microscopy Research Articles

Fluorescent emission and nondestructive 3D textural imaging in geologic materials by multiphoton microscopy.

This work greatly expands the application of multiphoton microscopy to geological investigations by using a tightly focused femtosecond laser beam to excite fluorescent emissions among minimally prepared rock and mineral samples. This novel finding provides a tool for spatially resolving UV-visible fluorescent sources in minerals. Through a combination of harmonic generation and fluorescence, unique opportunities are made available for mineralogical investigations of terrestrial rocks and astromaterials. This report includes the first demonstrations for 3D imaging of fluid inclusions in minerals and radiation-induced luminescence imaging in meteorites. Nonlinear optical mineralogy, enabled by multiphoton microscopy, provides unique insights into mineralogic samples and holds the potential to revolutionize the analysis of geologic and astromaterials samples in the coming years.

Applications of multiphoton microscopy in imaging cerebral and retinal organoids.

Cerebral organoids, self-organizing structures with increased cellular diversity and longevity, have addressed shortcomings in mimicking human brain complexity and architecture. However, imaging intact organoids poses challenges due to size, cellular density, and light-scattering properties. Traditional one-photon microscopy faces limitations in resolution and contrast, especially for deep regions. Here, we first discuss the fundamentals of multiphoton microscopy (MPM) as a promising alternative, leveraging non-linear fluorophore excitation and longer wavelengths for improved imaging of live cerebral organoids. Then, we review recent applications of MPM in studying morphogenesis and differentiation, emphasizing its potential for overcoming limitations associated with other imaging techniques. Furthermore, our paper underscores the crucial role of cerebral organoids in providing insights into human-specific neurodevelopmental processes and neurological disorders, addressing the scarcity of human brain tissue for translational neuroscience. Ultimately, we envision using multimodal multiphoton microscopy for longitudinal imaging of intact cerebral organoids, propelling advancements in our understanding of neurodevelopment and related disorders.

Label-free assessment of pathological changes in pancreatic intraepithelial neoplasia by biomedical multiphoton microscopy.

Pancreatic intraepithelial neoplasia (PanIN) is the most common precursor lesion that has the potential to progress to invasive pancreatic cancer, and early and rapid detection may offer patients a chance for treatment before the development of invasive carcinoma. Therefore, the identification of PanIN holds significant clinical importance. In this study, we first used multiphoton microscopy (MPM) combining two-photon excitation fluorescence and second-harmonic generation imaging to label-free detect PanIN and attempted to differentiate between normal pancreatic ducts and different grades of PanIN. Then, we also developed an automatic image processing strategy to extract eight morphological features of collagen fibers from MPM images to quantify the changes in collagen fibers surrounding the ducts. Experimental results demonstrate that the combination of MPM and quantitative information can accurately identify normal pancreatic ducts and different grades of PanIN. This study may contribute to the rapid diagnosis of pancreatic diseases and may lay the foundation for further clinical application of MPM.

Advances in Ultrafast Fiber Lasers for Multiphoton Microscopy in Neuroscience

Multiphoton microscopy (MPM) has emerged as a vital tool in neuroscience, enabling deeper imaging with a broader field of view, as well as faster and sub-cellular resolution. Recent innovations in ultrafast fiber laser technology have revolutionized MPM applications in living brains, offering advantages like cost-effectiveness and user-friendliness. In this review, we explore the progress in ultrafast fiber laser technology, focusing on its integration into MPM for neuroscience research. We also examine the utility of femtosecond fiber lasers in fluorescence and label-free two- and three-photon microscopy applications within the field. Furthermore, we delve into future possibilities, including next-generation fiber laser designs, novel laser characteristics, and their potential for achieving high spatial and temporal resolution imaging. We also discuss the integration of fiber lasers with implanted microscopes, opening doors for clinical and fundamental neuroscience investigations.

Coherent exciton coupling in fluorescent protein dimers probed by two-photon polarization ratio

Coherent excitonic coupling of two chromophores in a dimer of a GFP-analogue, VenusA206, was discovered previously by using circular dichroism and a combination of photon antibunching and fluorescence correlation spectroscopy. To further elucidate this effect, here we use a new approach based on the measurements of two-photon polarization ratio (Ω) as a function of excitation wavelength. Theoretically, Ω depends on the angle between the vectors of absorption transition dipole moment and permanent dipole moment change upon excitation. Generally, excitonic coupling of two chromophores should provide a change of this angle. We measured Ω for the tandem dimer of Venus (tdV) with two chromophores per protein chain and, as a control - for the tandem dimer with one of the chromophores knocked out by point mutation (tdVx). Compared to tdVx, we observe in tdV a significant increase of Ω in the red side of the pure electronic transition and a significant decrease of Ω in the blue side. At the same time, the two-photon absorption spectra of the two systems virtually coincide. Our observations are consistent with a model where only a minority of dimers are coupled coherently and the two permanent dipoles are oriented in an oblique head-to-head arrangement with an angle ∼400 between them. For multiphoton microscopy applications, it is interesting to investigate if the coherently coupled GFP dimer can provide a cooperative enhancement of the two-photon cross section, i.e., absorb stronger than two uncoupled monomers. This possibility can come from the “motional narrowing” of excitonic transition and will be discussed.

Nonlinear optical microscopy for skinin vivo: Basics, development and applications

Multi-photon microscopy (MPM) and coherent anti-Stokes Raman scattering (CARS) are two advanced nonlinear optical imaging techniques, which provide complementary information and have great potential in combination for noninvasive in vivo biomedical applications. This paper provides a detailed discussion of the basics, development and applications of these technologies for in vivo skin research, covering the following topics: The principle and advantage of MPM and CARS, instrumentation development for in vivo applications, MPM and CARS of normal skin, application of MPM and CARS in skin cancer and disease diagnosis; application of MPM in skin disease intervention, i.e., imaging guided two-photon photothermolysis.

1.7 µm - 1.73 µm tunable ultrafast Raman fiber laser pumped by 1.6 µm dissipative soliton pulses.

Here, we report an all-fiber tunable ultrafast Raman laser synchronously pumped by a home-made 1.6 µm dissipative soliton (DS) picosecond (ps) laser, which produces Stokes light beyond 1.7 µm. The Raman gain medium is a segment of highly germanium-doped (Ge-doped) fiber offering a high Raman gain coefficient at the target wavelength. Once the Raman conversion cavity is synchronized with the pump light, a stable 1.7 µm Raman laser (the first Stokes light) can be obtained at a low pump threshold. The maximum output power of the 1.7 µm Raman laser can reach ∼ 22.62 mW. The wavelength tuning operation is independent of tunable pump source and intra-cavity filter. By adjusting the intra-cavity delay line simply, the different spectral component within the broad Raman gain bandwidth can be selectively synchronized with the pump light so that the Raman laser wavelength can be tuned continuously from 1702.6 nm ∼ 1728.84 nm. This tunable 1.7 µm waveband ultrafast laser will have potential applications in multiphoton microscopy for e.g. deep bio-imaging.

Spectrally resolved point-spread-function engineering using a complex medium.

Propagation of an ultrashort pulse of light through strongly scattering media generates an intricate spatio-spectral speckle that can be described by means of the multi-spectral transmission matrix (MSTM). In conjunction with a spatial light modulator, the MSTM enables the manipulation of the pulse leaving the medium; in particular focusing it at any desired spatial position and/or time. Here, we demonstrate how to engineer the point-spread-function of the focused beam both spatially and spectrally, from the measured MSTM. It consists of numerically filtering the spatial content at each wavelength of the matrix prior to focusing. We experimentally report on the versatility of the technique through several examples, in particular as an alternative to simultaneous spatial and temporal focusing, with potential applications in multiphoton microscopy.

Application of multi-photon microscopy in dermatology

Application of multi-photon microscopy in dermatology

Multiphoton Microscopy of Oral Tissues: Review

Multiphoton microscopy (MPM) is currently acknowledged as a very powerful method for the visualization and analysis of tissues in biomedicine. It allows high resolution, deep optical sectioning and reduced photodamage. MPM does not require labeling and is deployable both in-vivo and ex-vivo, which simplifies the diagnostic procedure compared to traditional histology approaches based on tissue fixation and staining. Among the important applications of MPM in medicine, differentiation of healthy from pathological samples has gained massive interest over the past years, but MPM is also very useful for acquiring new insights on how various pathologies originate and progress. In this work we review the use of MPM in imaging assays focused on investigating unlabeled oral tissues (teeth and oral mucosa) and discuss a series of important results which hold potential for enabling a next generation of oral tissue characterization/diagnostic frameworks. The surveyed literature shows that nonlinear optical imaging tools can significantly contribute to achieve a better understanding of oral cavity tissues, by allowing the accurate analysis of morphological structures and relevant biochemical processes.

Label-Free Identification of Early Stages of Breast Ductal Carcinoma via Multiphoton Microscopy.

Breast cancer can be cured by early diagnosis. Appropriate and effective clinical treatment benefits from accurate pathological diagnosis. However, due to the lack of effective screening and diagnostic imaging methods, early stages of breast cancer often progress to malignant breast cancer. In this study, multiphoton microscopy (MPM) via two-photon excited fluorescence combined with second-harmonic generation was used for identifying the early stages of breast ductal carcinoma. The results showed differences in both cytological features and collagen distribution among normal breast tissue, atypical ductal hyperplasia, low-grade ductal carcinoma in situ, and high-grade ductal carcinoma in situ with microinvasion. Furthermore, three features extracted from the MPM images were used to describe differences in cytological features, collagen density, and basement membrane circumference in the early stages of breast ductal carcinoma. They revealed that MPM has the ability to identify early stages of breast ductal carcinoma label-free, which would contribute to the early diagnosis and treatment of breast cancer. This study may provide the groundwork for the further application of MPM in the clinic.

Comparing the performance of a femto fiber-based laser and a Ti:sapphire used for multiphoton microscopy applications.

Ti:sapphire laser systems are the more extended excitation sources in multiphoton (MP) microscopy. Although tunable, the cost, size, and lack of portability often limit their use in some research fields. Femtosecond fiber-based lasers represent an attractive alternative since they are portable, compact, and affordable. Most MP applications using these devices employ wavelengths beyond 1000 nm. This work evaluates the performance of a mode-locked fiber-based laser emitting at 780 nm (within the spectral region often used with Ti:sapphire devices) for use in MP imaging microscopy. MP images acquired with this laser system have been compared with those obtained with a "regular" solid-state source. Results herein show that the images recorded with both laser sources were similar, independently of the depth location of the imaged plane. The structural information contained in the images hardly differed. Moreover, the images of deeper layers improved by means of adaptive optics were also similar. These ultrafast laser sources are expected to enhance the impact of MP microscopy in basic research, as well as in biomedical environments.

Multimodal multiphoton imaging for label-free monitoring of early gastric cancer

BackgroundEarly gastric cancer is associated with a much better prognosis than advanced disease, and strategies to improve prognosis is strictly dependent on earlier detection and accurate diagnosis. Therefore, a label-free, non-invasive imaging technique that allows the precise identification of morphologic changes in early gastric cancer would be of considerable clinical interest.MethodsIn this study, multiphoton microscopy (MPM) using two-photon excited fluorescence combined with second-harmonic generation was used for the identification of early gastric cancer.ResultsThis microscope was able to directly reveal improved cellular detail and stromal changes during the development of early gastric cancer. Furthermore, two features were quantified from MPM images to assess the cell change in size and stromal collagen change as gastric lesion developed from normal to early cancer.ConclusionsThese results clearly show that multiphoton microscopy can be used to examine early gastric cancer at the cellular level without the need for exogenous contrast agents. This study would be helpful for early diagnosis and treatment of gastric cancer, and may provide the groundwork for further exploration into the application of multiphoton microscopy in clinical practice.

Label-free detection of residual breast cancer after neoadjuvant chemotherapy using biomedical multiphoton microscopy

Neoadjuvant chemotherapy has become a standard treatment for breast cancer as it has been shown to increase the rate of breast preservation and to improve outcome in patients. However, how to accurately detect residual tumors is still a challenge. In this work, we tried to use multiphoton imaging to look for residual breast tumors after preoperative therapy. Imaging results demonstrate that multiphoton microscopy can identify remaining tumor tissues and can even detect rarely residual tumor cells, which would be helpful for surgeons to accurately assess the surgical margin in real time to confirm negative margins during operation. We also performed a quantification analysis of the nuclear area of tumor cells before and after treatment with neoadjuvant chemotherapy. The measurement data show that the tumor cell nuclei after chemotherapy are significantly larger than those without treatment, and there is a statistically significant difference in the nuclear areas between the pre-treatment and post-treatment mammary carcinoma. Our pilot study indicates the potential utility of multiphoton imaging for detecting residual breast carcinoma tissues in fresh, ex vivo specimens without the use of exogenous contrast agents. We foresee real-time intraoperative applications of multiphoton microscopy in evaluating therapy response, and thereby helping clinicians develop individualized treatment plans.

Fluorescent Imaging and Microscopy for Dynamic Processes in Rats.

The rat is a favored model organism to study physiological function in vivo. This is largely due to the fact that it has been used for decades and is often more comparable to corresponding human conditions (both normal and pathologic) than mice. Although the development of genetic manipulations in rats has been slower than in mice, recent advances of new genomic editing tools allow for the generation of targeted global and specific cell type mutations in different rat strains. The rat is an ideal model for advancing imaging techniques like intravital multi-photon microscopy or IVMPM. Multi-photon excitation microscopy can be applied to visualize real-time physiologic events in multiple organs including the kidney. This imaging modality can generate four-dimensional high resolution images that are inherently confocal due to the fact that the photon density needed to excite fluorescence only occurs at the objective focal plane, not above or below. Additionally, longer excitation wavelengths allow for deeper penetration into tissue, improved excitation, and are inherently less phototoxic than shorter excitation wavelengths. Applying imaging tools to study physiology in rats has become a valuable scientific technique due to the relatively simple surgical procedures, improved quality of reagents, and reproducibility of established assays. In this chapter, the authors provide an example of the application of fluorescent techniques to study cardio-renal functions in rat models. Use of experimental procedures described here, together with multiple available genetically modified animal models, provide new prospective for the further application of multi-photon microscopy in basic and translational research.

Wavelength Separation Tunable 2-Color Soliton Generation and Its Application to 2-Color Fluorescence Multiphoton Microscopy

Optical solitons generated through soliton self-frequency shift in optical waveguides, has found the widespread applications in multiphoton microscopy (MPM). The multicolor fluorescence MPM, involving the simultaneous imaging of multiple structures labeled by different fluorophores, typically necessitates multiple-color pulses whose wavelengths match the peak excitation wavelengths of the fluorophores. Here we demonstrate experimentally an efficient method of generating wavelength separation tunable 2-color solitons, based on our recently proposed theoretical scheme of prechirping to manipulate the soliton order of the excitation pulse. Using this method, we reach a maximum soliton separation tuning range of 190 nm, with 1550-nm excitation. As an experimental demonstration of the applicability of this technique, we further show the MPM results using this 2-color soliton source for 2-color, 3-photon fluorescence imaging of fluorescent beads.

Application of a reflective microscope objective for multiphoton microscopy.

Reflective objectives (ROs) mitigate chromatic aberration across a broad wavelength range. Yet, a systematic performance characterisation of ROs has not been done. In this paper, we compare the performance of a 0.5 numerical-aperture (NA) reflective objective (RO) with a 0.55 NA standard glass objective (SO), using two-photon fluorescence (TPF) and second-harmonic generation (SHG). For experiments spanning ∼1 octave in the visible and NIR wavelengths, the SO leads to defocusing errors of 25-40% for TPF images of subdiffraction fluorescent beads and 10-12% for SHG images of collagen fibres. The corresponding error for the RO is ∼4% for both imaging modalities. This work emphasises the potential utility of ROs for multimodal multiphoton microscopy applications.

多光子显微技术在医学诊断中的应用

多光子显微技术在医学诊断中的应用

Multiphoton in vivo imaging with a femtosecond semiconductor disk laser.

We use an ultrafast diode-pumped semiconductor disk laser (SDL) to demonstrate several applications in multiphoton microscopy. The ultrafast SDL is based on an optically pumped Vertical External Cavity Surface Emitting Laser (VECSEL) passively mode-locked with a semiconductor saturable absorber mirror (SESAM) and generates 170-fs pulses at a center wavelength of 1027 nm with a repetition rate of 1.63 GHz. We demonstrate the suitability of this laser for structural and functional multiphoton in vivo imaging in both Drosophila larvae and mice for a variety of fluorophores (including mKate2, tdTomato, Texas Red, OGB-1, and R-CaMP1.07) and for endogenous second-harmonic generation in muscle cell sarcomeres. We can demonstrate equivalent signal levels compared to a standard 80-MHz Ti:Sapphire laser when we increase the average power by a factor of 4.5 as predicted by theory. In addition, we compare the bleaching properties of both laser systems in fixed Drosophila larvae and find similar bleaching kinetics despite the large difference in pulse repetition rates. Our results highlight the great potential of ultrafast diode-pumped SDLs for creating a cost-efficient and compact alternative light source compared to standard Ti:Sapphire lasers for multiphoton imaging.

Energetic ultrafast fiber laser sources tunable in 1030-1215 nm for deep tissue multi-photon microscopy.

We demonstrate an energy scalable approach to implement ultrafast fiber laser sources suitable for deep tissue multi-photon microscopy imaging. Enabled by fiber-optic nonlinearities (dominated by self-phase modulation), these unique ultrafast sources produce nearly transform-limited pulses of 50-90 fs in duration with the center wavelength tunable in the wavelength range of 1030-1215 nm. The resulting pulse energy can be scaled up to 20 nJ by optimizing fiber dispersion, shortening fiber length, and using large-mode-area fibers. We applied such an energetic source to a proof-of-principle study of ex vivo human skin based on harmonics (i.e., second-harmonic generation and third-harmonic generation) imaging. This new type of fiber-format energetic ultrafast source provides a robust solution for multiphoton microscopy applications.