Application Of Two-photon Microscopy Research Articles

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.

[Bone Cell Biology Assessed by Microscopic Approach. An application of two-photon microscopy for histopathological analysis of bone and cartilage].

Histopathological analysis has long been a gold standard in bio-medical research and clinical medicine. However, it is hard to quantitatively demonstrate histopathological difference. In this review, I will briefly describe principals of two-photon microscopy, and then introduce an application of second harmonic generation-based imaging for quantitative histopathological analysis of cartilage and bone.

Two-photon microscopy in pre-clinical and clinical cancer research

The applications of two-photon microscopy (TPM) on pre-clinical and clinical study of human cancer and diseases are reviewed in this paper. First, the principle of two-photon excitation (TPE) is introduced. The resulting advantages of TPM for imaging studies of animal models and human samples are then elaborated. Subse- quently, the applications of TPM on various aspects of tumor studies, including tumor angiogenesis, invasion and metastasis, tumor microenvironment and metabolism are introduced. Furthermore, studies of TPM on clinical human skin biopsy and the development of two-photon microendoscopy are reviewed. Finally, potential future directions are discussed.

Nonlinear optical microscopy and spectroscopy of ferroelectric and multiferroic materials

The specific features of the application of two-photon microscopy and spectroscopy to the investigation of multiferroic and piezoelectric nanostructures have been considered. It has been shown that the nonlinear optical response of planar multilayer barium strontium titanate/bismuth ferrite nanostructures is characterized by local electrically induced and magnetically induced contributions in specific spectral ranges. For peptide piezoelectric bio-microtubes, it has been found that the maximum value of the second harmonic generation intensity does not change along the tube, whereas the direction of polarization changes significantly.

Two-Photon and Second Harmonic Microscopy in Clinical and Translational Cancer Research

Application of two-photon microscopy (TPM) to translational and clinical cancer research has burgeoned over the last several years, as several avenues of pre-clinical research have come to fruition. In this review, we focus on two forms of TPM-two-photon excitation fluorescence microscopy, and second harmonic generation microscopy-as they have been used for investigating cancer pathology in ex vivo and in vivo human tissue. We begin with discussion of two-photon theory and instrumentation particularly as applicable to cancer research, followed by an overview of some of the relevant cancer research literature in areas that include two-photon imaging of human tissue biopsies, human skin in vivo, and the rapidly developing technology of two-photon microendoscopy. We believe these and other evolving two-photon methodologies will continue to help translate cancer research from the bench to the bedside, and ultimately bring minimally invasive methods for cancer diagnosis and treatment to therapeutic reality.

Knocking at the brain’s door: intravital two-photon imaging of autoreactive T cell interactions with CNS structures

Since the first applications of two-photon microscopy in immunology 10 years ago, the number of studies using this advanced technology has increased dramatically. The two-photon microscope allows long-term visualization of cell motility in the living tissue with minimal phototoxicity. Using this technique, we examined brain autoantigen-specific T cell behavior in experimental autoimmune encephalitomyelitis, the animal model of human multiple sclerosis. Even before disease symptoms appear, the autoreactive T cells arrive at their target organ. There they crawl along the intraluminal surface of central nervous system (CNS) blood vessels before they extravasate. In the perivascular environment, the T cells meet phagocytes that present autoantigens. This contact activates the T cells to penetrate deep into the CNS parenchyma, where the infiltrated T cells again can find antigen, be further activated, and produce cytokines, resulting in massive immune cell recruitment and clinical disease.

Analysis of the dispersion compensation of acousto-optic deflectors used for multiphoton imaging

The acousto-optic deflector (AOD) is highly preferred in laser scanning microscopy for its fast scanning ability and random-addressing capability. However, its application in two-photon microscopy is frustrated by the dispersion of the AOD, which results in beam distortion and pulse lengthening. We report the analysis of simultaneous compensation for the angular dispersion and temporal dispersion of the AOD by merely introducing a single dispersive element such as a prism or a grating. Besides serving as a scanner, the AOD is also a part of the compressor pair by integrating the dispersive nature of the AO interaction. This compensation principle is effective for both one-dimensional (1-D) AOD and two-dimensional (2-D) AOD scanning. Switching from a 1-D to a 2-D system requires proper optical alignment with the compensation element, but does not involve any new components. Analytical expressions are given to illustrate the working principle and to help with understanding the design of the system. Fluorescence images of beads and cells are shown to demonstrate the performance of two-photon microscopy when applying this compensated 2-D AOD as scanner.

Applications of two-photon microscopy in the neurosciences

Thanks to deep tissue penetration of infrared light (IR), two-photon laser scanning microscopy (2PLSM) has become an important tool in modern neuroscience. Examples range from the high-resolution visualization of dendritic spines in single neurons to the monitoring of intracellular Ca++ transients in small neural networks. In the present review we give an overview of the current achievements and discuss emerging applications of the technique.

Emission ratiometric imaging of intracellular zinc: design of a benzoxazole fluorescent sensor and its application in two-photon microscopy.

Zinc and calcium are ubiquitous intracellular metals, and while a variety of quantitative probes have been developed for measuring intracellular changes in calcium concentration, the same is not true of zinc. We describe here the design, synthesis, and properties of the benzoxazole-based, ratiometric zinc probe, Zinbo-5. This bright fluorescent reporter has a quantum yield of 0.1 in the zinc-form, exhibits a Kd for Zn2+ in the nanomolar range, and shows significant changes in both excitation and emission maxima upon zinc binding. The utility of this cell permeable probe is demonstrated in fluorescence microscopy emission ratio imaging experiments on mammalian cells. We further show that Zinbo-5 is well suited for two-photon excitation microscopy ratio imaging and can readily reveal changes in intracellular zinc concentration within optical planes of single cells. To the best of our knowledge, this is the first example of two-photon excitation microscopy applied to ratio imaging of zinc. These methods can be applied to real-time emission or excitation ratio imaging studies of zinc physiology in living cells.

4 Symmetry Breaking in the Zygotes of the Fucoid Algae: Controversies and Recent Progress

Despite its many advantages as an experimental system for the study of the epigenesis of polarity, it is obvious that the fucoid zygote also presents many problems. The development of polarity proceeds largely independently of direct gene action and thus may be considered a problem in cellular physiology. Ca2+ appears to play an important role in the process, but the optical properties of the zygotes (opacity and autofluorescence) hamper the use of modern methods of visualizing the distribution of Ca2+ and other ions. Likewise, other approaches, such as injection of fluorescent-labeled G-actin, in order to study the dynamics of actin filaments, are subject to the same limitations. It may be that the application of two-photon microscopy will enable experimenters to avoid some of these problems. This technique uses excitation wavelengths that are twice the wavelength of maximum absorption by fluorophores, and sufficient photon density for absorption is achieved only in a thin section. The fucoid zygotes are considerably more transparent to longer wavelengths, so attenuation of the exciting light and autofluorescence should be significantly reduced. Perhaps we will then be able to see further into these opaque cells. Another problem concerns the use of different species and genera. This may be unavoidable; for example, those of us who are land-locked tend to rely on Pelvetia, as it travels and stores better than the various species of Fucus and is less seasonal. Our colleagues fortunate enough to work near the ocean prefer to use the species that are locally available. Nevertheless, it is important to be careful about cross-genus and cross-species generalizations. While it is unlikely, based on what we know, that there are fundamental differences in physiological mechanisms among species, there may be small but still important differences in details. Obviously, investigators should directly compare results in more than one species whenever possible. The area of greatest disagreement, perhaps, concerns the mechanism of polarity formation, as opposed to its overt manifestation, germination. Are Ca2+ and actin involved or not? Assuming Ca2+ is involved, is the source internal or external? One basis for the different findings may be the differences in the strength of the polarizing signal provided to the zygotes. Clearly, the cells have powerful mechanisms for amplifying a faint asymmetry and developing an axis in response to an external signal. Furthermore, the fucoids generally develop in the intertidal zone and thus must be adapted to meeting the challenge of a widely varying external environment. They may have alternate mechanisms for responding to unilateral light. We have adopted the approach of presenting the cells with a fairly weak light signal--the minimum required to induce a considerable degree of organization of a population of zygotes. We then determine the effects of various inhibitors on photopolarization. One advantage of this approach is that it has allowed us to find treatments that increase the sensitivity of the zygotes to light, something that would not be possible if the untreated controls were fully polarized. Some of the differences between our results and those of others may be related to their use of a stronger light stimulus. It may be that if given a strong stimulus, a sufficient trace is left in the cells so that they can organize an axis when an inhibitor is removed. Careful consideration of this point may help to reconcile apparently contradictory findings. Despite these difficulties, the fucoid zygotes are likely to continue to be an important experimental system. Technology, including the development of more specific inhibitory reagents, may allow some of the shortcomings of the system to be overcome, and careful consideration of experimental conditions may resolve some of the points of disagreement.