Two-photon Excitation Wavelength Research Articles

Methyl Jasmonate-induced Increase in Intracellular Magnesium Promotes Apoptosis in Breast Cancer Cells.

Methyl jasmonate (MeJa) is a botanical stress hormone that serves as a defense mechanism to inhibit growth in stressed plants. It is well known that MeJa exhibits an anticancer effect by reducing intracellular ATP, activating reactive oxygen species (ROS) production, and promoting mitogen-activated protein kinase (MAPK) activity. Presently, no report has been published on MeJa-induced changes in intracellular Mg2+ concentration ([Mg2+]i), and TRPM7 as an Mg2+ transporter in cancer cells. Therefore, this study aimed to investigate the Mg2+ homeostatic changes and apoptotic effects following MeJa treatment using the MCF-7 human breast cancer cell line. The MTT assay was used to assess the cell viability and half-inhibitory concentration, microscopic two-photon excitation wavelength spectrophotometry was used to measure the [Mg2+]i, a luminescent assay determined intracellular ATP levels, western blot assay measured TRPM7 levels, antioxidant capacities, endoplasmic reticulum (ER) stress, and MAPK signaling pathways, while the fluorescence assay evaluated ROS concentrations and the cell apoptotic index. This study provides evidence that MeJa has an antiapoptotic effect on MCF-7 cells. The increase in [Mg2+]i led to decreased TRPM7 expression, which is related to elevated ROS production, in addition to elevated ER stress and MAPK signaling pathway activity and decreased ATP content. The increase in [Mg2+]i leads to decreased TRPM7 expression and may be the epicenter of MeJa-induced apoptotic cell death in MCF-7 cells.

Graphene quantum dot composite with multiphoton excitation as near infrared-II probe in bioimaging

Non-radiative traps and structures are present on the graphene quantum dots doped with nitrogen and functionalized with amino groups (amino-N-GQDs) and multiple crystalline layers because of cross-link-enhanced emission. Secondary and tertiary amines, which are potential fluorophores, have also been observed on the polyethylenimine (PEI) coating of amino-N-GQDs. Cross-linked PEI coating reduced rotation and vibration, thereby enhancing the photoluminescence quantum yield (PL QY). Introduced nitrogen atoms from N dopants, amino-functionalized groups and PEI, as well as sulfur from polystyrene sulfonate (PSS) enhanced the cooperative effect on the properties of heteroatom-doped materials among electrons captured by new surface states. This enhanced radiative recombination and subsequently enhanced PL QY, therefore, surface conjugation improved the amino-N-GQD surfaces by increasing the quantum confinement of their emissive energy, evidenced by the increased PL QY of amino-N-GQD-PSS-PEI (or amino-N-GQD-polymer composites). In some situations, the maximum available power required for delivery to the two-photon imaging plane without damaging tissues limits imaging depth but the additional brightness provided by amino-N-GQD-polymer composites in this study extended the maximum imaging depth to 240 μm. Amino-N-GQD-polymer composites had favorable two-photon properties under two-photon excitation (self-developed femtosecond Ti–sapphire laser optical system; power: 23.93 nJ pixel−1, 160 scans, approximately 1.09 s of total exposure time; excitation wavelength: 980 nm, near-infrared-II region), indicating that cells treated with amino-N-GQD-polymer composites and the anti-epidermal growth factor receptor antibody can achieve two-photon luminescence with 1/81 of the power required for similar-intensity two-photon autofluorescence (1938.33 nJ pixel−1 with 800 scans, total exposure time of ∼5.44 s). The materials can serve as contrast agents for the non-invasive detection of biological specimens and interior tissues using a two-photon excitation wavelength in the near-infrared region.

In-vivo two-photon visualization and quantitative detection of redox state of cancer.

Glutathione (GSH), the most common and abundant antioxidant in the body, is particularly concentrated in cancer cells (2-10 mM). This concentration is approximately 1000 times that of normal cells, making GSH a specific tumor marker. Overexpression of GSH is critical for mapping the redox state of cancer cells. However, there are few probes and detection methods responsive to GSH that can quantitatively visualize GSH in vivo in two-photon excitation fluorescence (TPEF) imaging mode. The experimental results show that TPEF-GSH could not only target GSH in tumors, but also establish the quantitative relationship between TPEF signal and GSH concentration. We explored the optimal two-photon excitation wavelength of TPEF-GSH, the optimal cell incubation duration with TPEF-GSH, the best imaging time point for GSH in cells, and the quantitative relationship between the TPEF signal and the changes in GSH concentrations. In zebrafish embryo and zebrafish experiments, the ratiometric value of TPEF-GSH increased with the decrease of GSH concentration. Microinjection and co-incubation were used to verify whether the ratiometric value could quantify endogenous GSH in tumor-bearing zebrafish, and the obtained GSH levels were 4.66 mM and 5.16 mM, respectively. The ratio TPEF probe could accurately visualize and quantify GSH in vivo, reflecting the redox status of the tumor. The design of the ratiometric molecular probe provides a reliable strategy for the development of TPEF nanoprobe in vivo. In this article, a new GSH sensitive molecular probe, TPEF-GSH, has been developed with good specificity and sensitivity. TPEF-GSH was successfully used to image cancer cells in vitro and tumor-bearing zebrafish in vivo, and to further detect GSH levels.

Multiplexed Graphene Quantum Dots with Excitation-Wavelength-Independent Photoluminescence, as Two-Photon Probes, and in Ultraviolet-Near Infrared Bioimaging.

In this study, sorted nitrogen-doped graphene quantum dots were prepared and subsequently conjugated with polymers. The synthesized materials exhibited excitation-wavelength-independent photoluminescence emissions ranging from ultraviolet to near-infrared and were 0.9-8.4 nm in size. The materials also exhibited high-photoluminescence quantum yields and excellent two-photon properties. Therefore, in two-photon bioimaging the materials with different emission spectra can be effective two-photon contrast agents. Specific antibodies were used to label organelles in cancer cells and identify nuclear antigens, thereby enabling the simultaneous detection of four targets in cells at a single two-photon excitation wavelength. The sorted nitrogen-doped graphene quantum dot materials were determined to be considerably more advantageous than organic dyes in identifying multiplexed targets, and they can be effective probes in cellular imaging.

Defects induced multicolor down- and up-conversion fluorescence in Se doped ZnO nanorods by single wavelength excitation

Abstract Se doped ZnO nanorods prepared by mechanothermal method with generation of multiple zinc (VZn), oxygen (VO) vacancies and oxygen interstitials (Oi) defects induce multicolor emissions by single and two-photon single wavelength excitations. The photoluminescence spectrum under single-photon excitation exhibits a broad multicolor emission peak from 400 to 800 nm with λmax at ~625 nm and a large Stokes shift of about 250 nm due to overlapping of defects in electronic transitions. The de-convolution peaks within superposition of defects (VZn, VO and Oi) induce multicolor broadband emission to show blue, green and red emissions that are consistent with their corresponding defects. The EPR signals also reveal detail information about these defects and show correlation with optical electronic transition states of blue, green and red emissions in Se doped ZnO NRs. Moreover, the two-photon up-conversion luminescence of Se doped ZnO NRs also shows multicolor (blue, green and red) emissions from channel at 720 nm excitation. Two-photon confocal studies of Se doped ZnO NRs shows multicolor emission at 720, 800 and 860 nm excitations which are consistent with one photon fluorescence at 360, 400 and 430 nm excitations. Therefore, these defects induced multicolor emissions by one and two-photon excitation wavelengths have potential optoelectronic and biomedical applications.

Multiphoton FLIM imaging of NAD(P)H and FAD with one excitation wavelength.

.Two-photon fluorescence lifetime imaging microscopy (FLIM) is widely used to capture autofluorescence signals from cellular components to investigate dynamic physiological changes in live cells and tissues. Among these intrinsic fluorophores, nicotinamide adenine dinucleotide (phosphate) (NAD(P)H) and flavin adenine dinucleotide (FAD)—essential coenzymes in cellular respiration—have been used as intrinsic fluorescent biomarkers for metabolic states in cancer and other pathologies. Traditional FLIM imaging for NAD(P)H, FAD, and in particular fluorescence lifetime redox ratio (FLIRR) requires a sequential multiwavelength excitation to avoid spectral bleed-through (SBT). This sequential imaging complicates image acquisition, may introduce motion artifacts, and reduce temporal resolution. Testing several two-photon excitation wavelengths in combination with optimized emission filters, we have proved a FLIM imaging protocol, allowing simultaneous image acquisition with a single 800-nm wavelength excitation for NADH and FAD with negligible SBT. As a first step, standard NADH and FAD single and mixed solutions were tested that mimic biological sample conditions. After these optimization steps, the assay was applied to two prostate cancer live cell lines: African-American (AA) and Caucasian-American (LNCaP), used in our previous publications. FLIRR result shows that, in cells, the 800-nm two-photon excitation wavelength is suitable for NADH and FAD FLIM imaging with negligible SBT. While NAD(P)H signals are decreased, sufficient photons are present for accurate lifetime fitting and FAD signals are measurably increased at lower laser power, compared with the common 890-nm excitation conditions. This single wavelength excitation allows a simplification of NADH and FAD FLIM imaging data analysis, decreasing the total imaging time. It also avoids motion artifacts and increases temporal resolution. This simplified assay will also make it more suitable to be applied in a clinical setting.

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.

Tuning the Competitive Recombination of Free Carriers and Bound Excitons in Perovskite CH3NH3PbBr3 Single Crystal

Excitation of perovskite CH3NH3PbBr3 crystal generates long-lived carriers, which radiatively recombine via free electrons and holes or localized excitons. Here, we investigate the competitive recombination of free carriers and bound excitons by tuning two-photon excitation wavelength. At excitation far from resonance, the CH3NH3PbBr3 perovskite single crystal shows the free carrier up-conversion emission because the exciton binding energy is comparable with the thermal energy at room temperature. At excitation near resonance (1.97 eV < hνexc < 2.16 eV), the anti-Stokes process with a consistent phonon energy of ∼186 meV is reported for the first time. Furthermore, when the anti-Stokes transition is resonant with localized exciton level below the band, highly efficient up-converted luminescence from the bound exciton recombination is observed. The finding that the excited state recombination kinetics vary versus excitation wavelength, in the present work, is helpful for developing the high-performance opt...

An Efficient Multicolor Two-Photon Imaging of Endogenous Fluorophores in Living Tissues by Wavelength Mixing

Two-photon imaging of endogenous fluorescence can provide important physiological and metabolic information from intact tissues in a label-free and non-invasive way. However, imaging of multiple intrinsic fluorophores, such as NADH, FAD, retinoids and porphyrins in living systems is generally hampered by sequential multi-wavelength excitation resulting in long acquisition times and motion artifacts. We report an efficient and simultaneous multicolor two-photon excitation of endogenous fluorophores with absorption spectra spanning the 700-1040nm range, using wavelength mixing. By using two synchronized pulse trains at two different wavelengths, an additional “virtual” two-photon excitation wavelength is generated, and simultaneous excitation of blue, green and red endogenous fluorophores is achieved. This method permits fast and reliable simultaneous imaging of the metabolic coenzymes NADH and FAD to being implemented, overcoming the difficulties associated with their difference in absorption spectra and disparity in concentration. We achieve efficient ratiometric redox imaging and simultaneous efficient two-photon fluorescence lifetime imaging (FLIM) of NADH and FAD in living tissues. Lifetime gradients of NADH and FAD associated with different cellular metabolic and differentiation states were measured in both reconstructed human skins and live C. elegans worms. Finally, we perform hyperspectral imaging of endogenous fluorophores during early zebrafish development.

RuNH2@AuNPs as two-photon luminescent probes for thiols in living cells and tissues

Two-photon luminescent sensors have emerged as promising molecular tools for imaging biomolecules in living systems. Here, we present hybrid gold nanocomposites RuNH2@AuNPs as luminescence off-on probes in response to thiols, which can replace the Ru(II) complexes on the surfaces of the AuNPs to release the luminophore RuNH2. The liberated Ru(II) complexes exhibit strong two-photon luminescence and a large two-photon absorption cross section by using the two-photon excitation wavelength at 800 nm. Furthermore, the probe responses toward thiols with high selectivity and insensitivity to pH over the biologically relevant pH range. This two-photon probe can visualize biological thiols levels in live cells as well as in living mouse tissues at depths of 80–170 μm by two-photon microscopy.

Multiphoton Excited Fluorescent Materials for Frequency Upconversion Emission and Fluorescent Probes

Recent progress in developing various strategies for exploiting efficient MPA fluorophores for two emerging technological MPA applications including frequency upconversion photoluminescence and lasing as well as 2PA fluorescence bioimaging and biosensing are presented. An intriguing application of MPA frequency-upconverted lasing offers opportunity for the fabrication of high-energy coherent light sources in the blue region which could create new advantages and breakthroughs in various laser-based applications. In addition, multiphoton excitation has led to considerable progress in the development of advanced diagnostic and therapeutic treatments; further advancement is anticipated with the emergence of various versatile 2PA fluorescence probes. It is widely appreciated that the two-photon excitation offers significant advantages for the biological fluorescence imaging and sensing which includes higher spatial resolution, less photobleaching and photodamage as well as deeper tissue penetration as compared to the one-photon excited microscopy. To be practically useful, the 2PA fluorescent probes for biological applications are required to have a site-specificity, a high fluorescence quantum yield, proper two-photon excitation and subsequent emission wavelengths, good photodecomposition stability, water solubility, and biocompatibility besides large 2PA action cross-sections.

Two-Photon Autofluorescence Spectroscopy of Mouse Brains using Serial Two-Photon Tomography

Two-photon microscopy of endogenous fluorescence has proven to be a powerful method for quantification of tissue structure and biochemistry in 3D and with micron resolution. In particular, autofluorescence of mouse brain tissue provides potentially rich signatures for identifying brain anatomical regions and structures without the need for traditional Nissl or other exogenous staining. We quantitatively investigate the identities and distributions of endogneous fluorophores in whole mouse brains using Serial Two-Photon (STP) tomography. We vary the two-photon excitation wavelength over the range of 720 to 980 nm while simultaneously performing spectrally resolved imaging from 400 nm to 650 nm and report on background autofluorescence for use in segmenting, constructing brain atlases, and identifying specific neuronal regions.View Large Image | View Hi-Res Image | Download PowerPoint Slide

Integrated multimodal endomicroscopy platform for simultaneous en face optical coherence and two-photon fluorescence imaging

We report an all-fiber-optic scanning, multimodal endomicroscope capable of simultaneous optical coherence tomography (OCT) and two-photon fluorescence (TPF) imaging. Both imaging modalities share the same miniature fiber-optic scanning endomicroscope, which consists of a double-clad fiber with a core operating in single mode at both the OCT (1310 nm) and two-photon excitation (1550 nm) wavelengths, a piezoelectric two-dimensional fiber-optic beam scanner, and a miniature aspherical compound lens suitable for simultaneous acquisition of en face OCT and TPF images. A fiber-optic wavelength division multiplexer was employed in the integrated platform to combine the low coherence OCT light source and the femtosecond two-photon excitation laser into the same optical path. Preliminary imaging results of cell cultures and mouse tissue ex vivo demonstrate the feasibility of simultaneous real-time OCT and TPF imaging in a scanning endomicroscopy setting for the first time.

Single-Voxel Recording of Voltage Transients in Dendritic Spines

We report sensitive recording of membrane potential in single dendritic spines in cortical neurons within a brain slice using two-photon excitation and a new, fluorinated, intracellularly loaded organic dye, di-2-AN(F)EPPTEA. With a two-photon excitation wavelength of 1060 nm, we achieve voltage sensitivity of >16% change in fluorescence per 100 mV. By targeting single spines in single-voxel recordings, we attain excellent single/noise quality, with back-propagating action potentials (bAPs) visible in single sweeps while recording at 10 kHz. This recording rate allows us to reliably assess fast bAP dynamics on single sweeps including bAP rise times of 0.5 ms. The amplitude and propagation delays of the bAPs are similar among different spines located within the same dendritic region, and this is true despite large differences in spine size. The interregion differences in bAP waveforms in spines vary in relation to their distance from the soma and the caliber of their parent dendrites.

Spectral Characterization and Unmixing of Intrinsic Contrast in Intact Normal and Diseased Gastric Tissues Using Hyperspectral Two-Photon Microscopy

BackgroundLiving tissues contain a range of intrinsic fluorophores and sources of second harmonic generation which provide contrast that can be exploited for fresh tissue imaging. Microscopic imaging of fresh tissue samples can circumvent the cost and time associated with conventional histology. Further, intrinsic contrast can provide rich information about a tissue's composition, structure and function, and opens the potential for in-vivo imaging without the need for contrast agents.Methodology/Principal FindingsIn this study, we used hyperspectral two-photon microscopy to explore the characteristics of both normal and diseased gastrointestinal (GI) tissues, relying only on their endogenous fluorescence and second harmonic generation to provide contrast. We obtained hyperspectral data at subcellular resolution by acquiring images over a range of two-photon excitation wavelengths, and found excitation spectral signatures of specific tissue types based on our ability to clearly visualize morphology. We present the two-photon excitation spectral properties of four major tissue types that are present throughout the GI tract: epithelium, lamina propria, collagen, and lymphatic tissue. Using these four excitation signatures as basis spectra, linear unmixing strategies were applied to hyperspectral data sets of both normal and neoplastic tissue acquired in the colon and small intestine. Our results show that hyperspectral unmixing with excitation spectra allows segmentation, showing promise for blind identification of tissue types within a field of view, analogous to specific staining in conventional histology. The intrinsic spectral signatures of these tissue types provide information relating to their biochemical composition.Conclusions/SignificanceThese results suggest hyperspectral two-photon microscopy could provide an alternative to conventional histology either for in-situ imaging, or intraoperative ‘instant histology’ of fresh tissue biopsies.

Gigantic Two-Photon Absorption Cross Sections and Strong Two-Photon Excited Fluorescence in Pyrene Core Dendrimers with Fluorene/Carbazole as Dendrons and Acetylene as Linkages

We report a series of stiff dendrimers (referred to as T1, T2, T3, and T4) that have both gigantic two-photon absorption (TPA) cross sections up to 25,000 GM and strong two-photon excited fluorescence (TPEF) with fluorescence quantum yield of ∼0.5. The large TPA cross sections and high quantum yields of these dendrimers are directly related to their geometrical structures, where the polycyclic aromatic pyrene is chosen as the chromophoric core because of its planar and highly π-conjugated structure, fluorene moieties as dendrons extend the conjugation length through the planar structure, and carbazole moieties are modified at three-, six-, and nine-positions as electron donor. All of these groups are linked with acetylene linkage for effective π-electron delocalization, leading to large TPA cross section and high fluorescence quantum yield. The spectral properties of all dendrimers are investigated by one- and two-photon excitations. Furthermore, steady-state fluorescence excitation anisotropy and quantum chemical calculation are also employed to determine the structure-related mechanism of these dendrimers with gigantic TPA cross sections and high TPEF efficiency. We then show that the improvement of branched chains in the T-series dendrimers enhances the light-harvesting ability. The core emission spectra, fluorescence quantum yield, and fluorescence lifetime are almost invariable by directly exciting the dendrons. These results will provide a guideline for the design of useful two-photon materials with structural motifs that can enhance the TPA cross-section and fluorescence quantum yield of a molecule without causing a red shift of the one- and two-photon excitation wavelengths for specific applications.

Scanning all-fiber-optic endomicroscopy system for 3D nonlinear optical imaging of biological tissues

An extremely compact all-fiber-optic scanning endomicroscopy system was developed for two-photon fluorescence (TPF) and second harmonic generation (SHG) imaging of biological samples. A conventional double-clad fiber (DCF) was employed in the endomicroscope for single-mode femtosecond pulse delivery, multimode nonlinear optical signals collection and fast two-dimensional scanning. A single photonic bandgap fiber (PBF) with negative group velocity dispersion at two-photon excitation wavelength (i.e. approximately 810 nm) was used for pulse prechirping in replacement of a bulky grating/lens-based pulse stretcher. The combined use of DCF and PBF in the endomicroscopy system made the endomicroscope basically a plug-and-play unit. The excellent imaging ability of the extremely compact all-fiber-optic nonlinear optical endomicroscopy system was demonstrated by SHG imaging of rat tail tendon and depth-resolved TPF imaging of epithelial tissues stained with acridine orange. The preliminary results suggested the promising potential of this extremely compact all-fiber-optic endomicroscopy system for real-time assessment of both epithelial and stromal structures in luminal organs.

Autofluorescence spectroscopy and imaging of Platymonas subcordiformis irradiated by diode laser based on LSCM

Autofluorescence spectra and optical imaging of Platymonas subcordiformis after irradiation of diode laser were observed via laser scanning confocal microscopy (LSCM). With 488 nm Ar(+) laser excitation, the horizontal and vertical dimensions of a cup-shaped chloroplast of the irradiation group increased about 10% compared with the control group. The fluorescence spectra were similar between irradiation group and control group with a maximum fluorescence band around 682 nm, whereas the former has a higher intensity. Image of a small circular substance with stronger two-photon autofluorescence (TPA) was obtained when using two-photon excitation wavelength of 800 nm in single-channel mode. Further analysis by the 800 nm excitation based on two independent-channels mode showed an emission band of the small circular substance around 376-505 nm, which corresponded to the eyespot of P. subcordiformis. In lambda scanning mode, with two-photon wavelength of 800 nm excitation, six fluorescence peaks that are located at 465, 520, 560, 617, 660 and 680 nm were observed; the fluorescence intensity of the irradiation group was higher than that of the control group, especially at 520, 560 and 617 nm. As a conclusion, diode laser irradiation can promote chloroplast growth of P. subcordiformis cells in the form of expanding area and the increasing content of protein, phospholipids and chlorophyll. LSCM, especially TPA imaging based on femtosecond laser excitation, provides a nondestructive, real-time and accurate method to study changes of living algal cells under laser irradiation and other environmental factors.

Multiphoton Microscopy of Endogenous Fluorescence Differentiates Normal, Precancerous, and Cancerous Squamous Epithelial Tissues

This study characterizes the morphologic features and the endogenous fluorescence in the stratified squamous epithelia of the 7,12-dimethylbenz(a)anthracene-treated hamster cheek pouch model of carcinogenesis using multiphoton laser scanning microscopy (MPLSM). MPLSM allows high-resolution, three-dimensional image data to be collected deeper within thick tissue samples with reduced phototoxicity compared with single-photon imaging. Three-dimensional image stacks of normal (n = 13), precancerous (dysplasia, n = 12; carcinoma in situ, n = 9) and cancerous tissue [nonpapillary squamous cell carcinoma (SCC), n = 10, and papillary SCC, n = 7] sites in the hamster cheek pouch were collected in viable, unsectioned tissue biopsies at a two-photon excitation wavelength of 780 nm. Five features were quantified from the MPLSM images. These included nuclear density versus depth, keratin layer thickness, epithelial thickness, and the fluorescence per voxel in the keratin and epithelial layers. Statistically significant differences in all five features were found between normal and both precancerous and cancerous tissues. The only exception to this was a lack of statistically significant differences in the keratin fluorescence between normal tissues and papillary SCCs. Statistically significant differences were also observed in the epithelial thickness of dysplasia and carcinoma in situ, and in the keratin layer thickness of dysplasia and SCCs (both nonpapillary and papillary). This work clearly shows that three-dimensional images from MPLSM of endogenous tissue fluorescence can effectively distinguish between normal, precancerous, and cancerous epithelial tissues. This study provides the groundwork for further exploration into the application of multiphoton fluorescence endoscopy in a clinical setting.

Two-photon excitation of a phytofluor protein

Phytofluors are highly fluorescent proteins in which the chromophore in a phytochrome is replaced with phycoerythrobilin (PEB), the pigment precursor of the cyanobacterial light harvesting protein phycoerythrin. We examined the fluorescence spectra of the N-terminal region of the cyanobacterial phytochrome 1 from cyanobacterium Synechocystis sp. Pcc 6803 bound to PEB. This protein, Cph1(N514)-PEB, displayed a good two-photon cross-section of 20–30GM for excitation at 792nm. This phytofluor also exhibits a high fundamental anisotropy at most practical two-photon excitation (2PE) wavelengths from 700 to 900nm. Identical lifetimes and correlation times with one and 2PE indicates that the phytofluor is not adversely affected by the intensities needed for 2PE. The one-photon absorption extends well beyond the absorption spectrum and even beyond the emission spectrum to 700nm. The phytofluor thus appears to be a suitable probe for 2PE and/or cellular imaging.