Simultaneous Two-photon Fluorescence Research Articles

Exploring Idiopathic Pulmonary Fibrosis Biomarker by Simultaneous Two-Photon Fluorescence Imaging of Cysteine and Peroxynitrite.

Idiopathic pulmonary fibrosis (IPF) has been characterized as a chronic inflammatory disease that leads to irreversible damage to pulmonary function. However, there is no specific IPF biomarker that can be used to distinguish IPF and not pneumonia. Endoplasmic reticulum (ER) stress is prominent in IPF. To search for a specific biomarker of IPF, we developed two ER-targeting two-photon (TP) fluorescent probes, TPER-ONOO and TPER-Cys, for peroxynitrite (ONOO-) and cysteine (Cys) imaging, respectively. A significant increase in Cys levels in the lungs was discovered only in mice with IPF, which implied that Cys might be an IPF biomarker candidate. Furthermore, we uncovered the mechanism of glutathione (GSH) deficiency in IPF, which was not due to Cys shortage but instead was attributable to impaired glutamate cysteine ligase and glutathione synthetase activities via ONOO--induced post-transcriptional modification. This work has potential to provide a new method for IPF early diagnosis and drug efficacy evaluation.

Confined photo-release of nitric oxide with simultaneous two-photon fluorescence tracking in a cellular system

Nitric oxide (NO) is a key signaling molecule in biological systems. New tools are required to therapeutically modulate NO levels with confined precision. This study explores the photoactivatable properties of an NO releasing compound (CPA), based on cupferron O-alkylated with an anthracene derivative. Upon light stimulation, CPA uncages two species: cupferron, which liberates NO, and an anthrylmethyl carbocation, which evolves into a fluorescent reporter. Proof-of-principle is demonstrated using one- and two-photon excitation (1PE and 2PE) in a cellular system (A431 cells). It was found that 1PE induces cell toxicity, while 2PE does not. Since 1PE using UV light is more likely to generate cellular photodamage, the cell toxicity observed using 1PE is most likely a combinatory effect of NO release and other UV-induced damage, which should be subject to further investigation. On the other hand, absence of phototoxicity using 2PE suggests that NO alone is not cytotoxic. This leads to the conclusion that the concept of 2PE photorelease of NO from CPA enable opportunities for biological studies of NO signaling with confined precision of NO release with minimal cytotoxicity.

Multicolor two-photon imaging of endogenous fluorophores in living tissues by wavelength mixing

Two-photon imaging of endogenous fluorescence can provide physiological and metabolic information from intact tissues. However, simultaneous imaging of multiple intrinsic fluorophores, such as nicotinamide adenine dinucleotide(phosphate) (NAD(P)H), flavin adenine dinucleotide (FAD) and retinoids in living systems is generally hampered by sequential multi-wavelength excitation resulting in motion artifacts. Here, we report on efficient and simultaneous multicolor two-photon excitation of endogenous fluorophores with absorption spectra spanning the 750–1040 nm range, using wavelength mixing. By using two synchronized pulse trains at 760 and 1041 nm, an additional equivalent two-photon excitation wavelength at 879 nm is generated, and achieves simultaneous excitation of blue, green and red intrinsic fluorophores. This method permits an efficient simultaneous imaging of the metabolic coenzymes NADH and FAD to be implemented with perfect image co-registration, overcoming the difficulties associated with differences in absorption spectra and disparity in concentration. We demonstrate ratiometric redox imaging free of motion artifacts and simultaneous two-photon fluorescence lifetime imaging (FLIM) of NADH and FAD in living tissues. The lifetime gradients of NADH and FAD associated with different cellular metabolic and differentiation states in reconstructed human skin and in the germline of live C. Elegans are thus simultaneously measured. Finally, we present multicolor imaging of endogenous fluorophores and second harmonic generation (SHG) signals during the early stages of Zebrafish embryo development, evidencing fluorescence spectral changes associated with development.

Highly efficient, conjugated-polymer-based nano-photosensitizers for selectively targeted two-photon photodynamic therapy and imaging of cancer cells.

Two-photon photodynamic therapy (2P-PDT) is a promising noninvasive treatment of cancers and other diseases with three-dimensional selectivity and deep penetration. However, clinical applications of 2P-PDT are limited by small two-photon absorption (TPA) cross sections of traditional photosensitizers. The development of folate receptor targeted nano-photosensitizers based on conjugated polymers is described. In these nano-photosensitizers, poly{9,9-bis[6''-(bromohexyl)fluorene-2,7-ylenevinylene]-co-alt-1,4-(2,5-dicyanophenylene)}, which is a conjugated polymer with a large TPA cross section, acts as a two-photon light-harvesting material to significantly enhance the two-photon properties of the doped photosensitizer tetraphenylporphyrin (TPP) through energy transfer. These nanoparticles displayed up to 1020-fold enhancement in two-photon excitation emission and about 870-fold enhancement in the two-photon-induced singlet oxygen generation capability of TPP. Surface-functionalized folic acid groups make these nanoparticles highly selective in targeting and killing KB cancer cells over NIH/3T3 normal cells. The 2P-PDT activity of these nanoparticles was significantly improved, potentially up to about 1000 times, as implied by the enhancement factors of two-photon excitation emission and singlet oxygen generation. These nanoparticles could act as novel two-photon nano-photosensitizers with combined advantages of low dark cytotoxicity, targeted 2P-PDT with high selectivity, and simultaneous two-photon fluorescence imaging capability; these are all required for ideal two-photon photosensitizers.

Trapping and two-photon fluorescence excitation of microscopic objects using ultrafast single-fiber optical tweezers

Analysis of trapped microscopic objects using fluorescence and Raman spectroscopy is gaining considerable interest. We report on the development of single fiber ultrafast optical tweezers and its use in simultaneous two-photon fluorescence (TPF) excitation of trapped fluorescent microscopic objects. Using this method, trapping depth of a few centimeters was achieved inside a colloidal sample with TPF from the trapped particle being visible to the naked eye. Owing to the propagation distance of the Bessel-like beam emerging from the axicon-fiber tip, a relatively longer streak of fluorescence was observed along the microsphere length. The cone angle of the axicon was engineered so as to provide better trapping stability and high axial confinement of TPF. Trapping of the floating objects led to stable fluorescence emission intensity over a long period of time, suitable for spectroscopic measurements. Furthermore, the stability of the fiber optic trapping was confirmed by holding and maneuvering the fiber by hand so as to move the trapped fluorescent particle in three dimensions. Apart from miniaturization capability into lab-on-a-chip microfluidic devices, the proposed noninvasive microaxicon tipped optical fiber can be used in multifunctional mode for in-depth trapping, rotation, sorting, and ablation, as well as for two-photon fluorescence excitation of a motile sample.

Use of tapered amplifier diode laser for biological-friendly high-resolution optical trapping

A 1064 nm laser is commonly used for biological optical trapping. However, it has the problem of generating reactive oxygen species in the presence of a sensitizer, which leads to photo damage in biological samples. Here we constructed optical tweezers using a tapered amplifier diode laser that operates at 830 nm. Compared to a 1064 nm laser, this laser is friendly to live cells, eliminates photo damage associated with reactive oxygen species, and allows simultaneous two-photon fluorescence imaging of green fluorescent proteins in live mammalian cells. All these advantages could significantly benefit future application of this single molecule technique in biological studies.

New Dyes With Fast Voltage-Dependent Changes In Membrane SHG

Laser scanning second harmonic generation (SHG) microscopy has shown significant promise for membrane potential imaging with voltage sensitive dyes (VSDs), possessing significant advantages over fluorescence-based imaging modalities. Through simultaneous patch-clamping and non-linear imaging of cells, SHG has been found to exhibit sensitivities to trans-membrane potential that are up to four times better than those obtained under optimal conditions using one-photon fluorescence imaging (Millard et al., 2004). For styryl dyes, while electrochromism is the dominating photophysical mechanism of fluorescence, some ANEP-based dyes display slow SHG voltage responses, suggesting that chromophore membrane reorientation or redistribution may be involved. The mechanism of the SHG response is not entirely understood and necessitates additional study in order to fully optimize this imaging modality. We report on our further investigation of the time dependence of the voltage sensitivity of SHG and simultaneous two-photon fluorescence imaging, using “fast” voltage-switching experiments. The response kinetics of resonance enhanced SHG from several styryl dyes developed in our laboratory, including di-3-ANEPPDHQ, as well as di-4-ANEPPTEA and a new fluorinated derivative, have been determined. Voltage-clamped neuroblastoma cells stained with these dyes were imaged with 1064 nm excitation from a mode locked fiber-based laser source. For SHG, these VSDs were found to exhibit moderately large voltage sensitivities in addition to fast kinetic responses. Our results suggest that voltage sensitive dyes can be developed which have both large SHG signal changes and the requisite speed for use as a practical tool for measuring electrical activity in neuronal systems.(Supported by NIH grant EB001963).

Innovations in two-photon deep tissue microscopy.

The goal of this article is to provide a review of deep tissue studies based on two-photon microscopy. We will further explore three new developments that have significantly enhanced the power of two-photon imaging: (1) simultaneous two-photon fluorescence and confocal reflected-light imaging, (2) two-photon video-rate imaging, and (3) fluorescent spectroscopic measurements in deep tissue.