Use Of Two-photon Excitation Research Articles

Recent Approaches towards the Development of Ru(II) Polypyridyl Complexes for Anticancer Photodynamic Therapy.

Photodynamic therapy (PDT) is a remarkable alternative or complementary technique to chemotherapy, radiotherapy or immunotherapy to treat certain forms of cancer. The synergistic effect of light, photosensitizer (PS) and oxygen allows for the treatment of tumours with an extremely high spatio-tumoral control, therefore minimizing the severe side effects usually observed in chemotherapy. The currently employed PDT PSs based on porphyrins have, in some cases, some limitations, which include a low absorbance in the therapeutic window, a low body clearance, photobleaching, among others. In this context, Ru(ii) polypyridyl complexes are interesting alternatives. They have low lying excited energy states and the presence of a heavy metal increases the possibility of spin-orbit coupling. Moreover, their photophysical properties are relatively easy to tune and they have very low photobleaching rates. All of these make them attractive candidates for further development as therapeutically suitable PDT PSs. In this review, after having presented this field of research, we discuss the developments made by our group in this field of research since 2017. We notably describe how we tuned the photophysical properties of our complexes from the visible region to the therapeutically suitable red region. This was accompanied by the preparation of PSs with enhanced phototoxicity and high phototoxicity index. We also discuss the use of two-photon excitation to eradicate tumours in nude mice. Furthermore, we describe our approach for the selective delivery of our complexes using targeting agents. Lastly, we report on our very recent synergistic approach to treat cancer using bimetallic Ru(ii)-Pt(iv) prodrug candidates.

Holographic patterning of fluorescent microstructures comprising silver nanoclusters

Metal nanoclusters, which exhibit extraordinary physical and chemical properties that are different from their bulk counterparts, are highly promising nanomaterials for photonics. Recently, the use of two-photon excitation to fabricate silver nanoclusters in polymers was reported but still lacks speed and flexibility which are imperative for applications such as labeling and spectroscopy. Here, we demonstrate the fabrication of fluorescent nanocluster microstructures using spatially phase-shaped laser beams. Using an incident power of 60 mW and exposure time of 8 s, we found that the smallest line-width of the fluorescent microstructures is 478 nm, which is comparable to the line-width achieved with a two-photon laser scanning approach. As a proof-of-principle demonstration, the technique is used to fabricate fluorescent micro-labels that could be used in anti-counterfeiting applications.

Microsecond protein folding events revealed by time-resolved fluorescence resonance energy transfer in a microfluidic mixer.

We demonstrate the combination of the time-resolved fluorescence resonance energy transfer (tr-FRET) measurement and the ultrarapid hydrodynamic focusing microfluidic mixer. The combined technique is capable of probing the intermolecular distance change with temporal resolution at microsecond level and structural resolution at Angstrom level, and the use of two-photon excitation enables a broader exploration of FRET with spectrum from near-ultraviolet to visible wavelength. As a proof of principle, we used the coupled microfluidic laminar flow and time-resolved two-photon excitation microscopy to investigate the early folding states of Cytochrome c (cyt c) by monitoring the distance between the tryptophan (Trp-59)-heme donor-acceptor (D-A) pair. The transformation of folding states of cyt c in the early 500 μs of refolding was revealed on the microsecond time scale. For the first time, we clearly resolved the early transient state of cyt c, which is populated within the dead time of the mixer (<10 μs) and has a characteristic Trp-59-heme distance of ∼31 Å. We believe this tool can find more applications in studying the early stages of biological processes with FRET as the probe.

The back and forth of energy transfer between carotenoids and chlorophylls and its role in the regulation of light harvesting

Many aspects in the regulation of photosynthetic light-harvesting of plants are still quite poorly understood. For example, it is still a matter of debate which physical mechanism(s) results in the regulation and dissipation of excess energy in high light. Many researchers agree that electronic interactions between chlorophylls (Chl) and certain states of carotenoids are involved in these mechanisms. However, in particular, the role of the first excited state of carotenoids (Car S1) is not easily revealed, because of its optical forbidden character. The use of two-photon excitation is an elegant approach to address directly this state and to investigate the energy transfer in the direction Car S1→Chl. Meanwhile, it has been applied to a large variety of systems starting from simple carotenoid-tetrapyrrole model compounds up to entire plants. Here, we present a systematic summary of the observations obtained by two-photon excitation about Car S1→Chl energy transfer in systems with increasing complexity and the correlation to fluorescence quenching. We compare these observations directly with the energy transfer in the opposite direction, Chl→Car S1, for the same systems as obtained in pump-probe studies. We discuss what surprising aspects of this comparison led us to the suggestion that quenching excitonic Car-Chl interactions could contribute to the regulation of light harvesting, and how this suggestion can be connected to other models proposed.

Recent Advances in Two-Photon Photodynamic Therapy

Photodynamic therapy (PDT) is a treatment for tumors and accepted in several countries in the world. Introduction of two-photon absortion (2PA) into PDT allows spatially selective treatment of cancers. Possibilities and limitations of the use of two-photon excitation in PDT are discussed, and many literatures in this area are reviewed. The conclusions are that 2PA-PDT has an advantage for higher selectivity than one-photon absorption PDT, and femtosecond pulsed laser is more suitable for 2PA-PDT than pico- and nanosecond pulses. However, most of photosensitizers used in the past studies had low 2PA cross section values less than 50 GM, and resulted in a low PDT efficiency under two-photon irradiation conditions. To realize 2PA-PDT, much larger 2PA cross sections must be required.

Prospects for precision measurements of atomic helium using direct frequency comb spectroscopy

We analyze several possibilities for precisely measuring electronic transitions in atomic helium by the direct use of phase-stabilized femtosecond frequency combs. Because the comb is self-calibrating and can be shifted into the ultraviolet spectral region via harmonic generation, it offers the prospect of greatly improved accuracy for UV and far-UV transitions. To take advantage of this accuracy an ultracold helium sample is needed. For measurements of the triplet spectrum a magneto-optical trap (MOT) can be used to cool and trap metastable 2^3S state atoms. We analyze schemes for measuring the two-photon $2^3S \to 4^3S$ interval, and for resonant two-photon excitation to high Rydberg states, $2^3S \to 3^3P \to n^3S,D$. We also analyze experiments on the singlet-state spectrum. To accomplish this we propose schemes for producing and trapping ultracold helium in the 1^1S or 2^1S state via intercombination transitions. A particularly intriguing scenario is the possibility of measuring the $1^1S \to 2^1S$ transition with extremely high accuracy by use of two-photon excitation in a magic wavelength trap that operates identically for both states. We predict a ``triple magic wavelength'' at 412 nm that could facilitate numerous experiments on trapped helium atoms, because here the polarizabilities of the 1^1S, 2^1S and 2^3S states are all similar, small, and positive.

Two-Photon Excited Fluorescence Energy Transfer: A Study Based on Oligonucleotide Rulers

The use of two-photon excitation of fluorescence for detection of fluorescence resonance energy transfer (FRET) was studied for a selected fluorescent donor-acceptor pair. A method based on labeled DNA was developed for controlling the distance between the donor and the acceptor molecules. The method consists of hybridization of fluorescent oligonucleotides to a complementary single-stranded target DNA. As the efficiency of FRET is strongly distance dependent, energy transfer does not occur unless the fluorescent oligonucleotides and the target DNA are hybridized. A high degree of DNA hybridization and an excellent FRET efficiency were verified with one-photon excited fluorescence studies. Excitation spectra of fluorophores are usually wider in case of two-photon excitation than in the case of one-photon excitation. This makes the selective excitation of donor difficult and might cause errors in detection of FRET with two-photon excited fluorescence. Different techniques to analyze the FRET efficiency from two-photon excited fluorescence data are discussed. The quenching of the donor fluorescence intensity turned to be the most consistent way to detect the FRET efficiency. The two-photon excited FRET is shown to give a good response to the distance between the donor and the acceptor molecules.

A Two-Photon Antenna for Photochemical Delivery of Nitric Oxide from a Water-Soluble, Dye-Derivatized Iron Nitrosyl Complex Using NIR Light

The experiments described here demonstrate the use of two-photon excitation (TPE) to sensitize nitric oxide (NO) release from a dye-derivatized iron/sulfur/nitrosyl cluster Fe2(mu-RS)2(NO)4 (Fluor-RSE, RS = 2-thioethyl ester of fluorescein) with near-infrared (NIR) light in the form of femtosecond pulses from a Ti:sapphire laser. TPE at 800 nm leads both to weak fluorescence from the organic chromophore at lambda(max) = 532 nm and to NO labilization from the cluster. Since the emission from the reference compound Fluor-Et (the ethyl ester of fluorescein) under identical conditions (50/50 CH3CN/phosphate buffer (1 mM) at pH 7.4) is considerably more intense, the weaker emission from Fluor-RSE and the NO generation indicate that the fluorescein excited states initially formed by TPE are largely quenched by energy transfer to the cluster core. The two-photon absorption (TPA) cross section of Fluor-RSE at 800 nm was determined to be delta = 63 +/- 7 GM via the TPA photoluminescence technique. This can be compared to the TPA cross section of 36 GM reported for fluorescein dye in pH 11 aqueous solution and of 32 +/- 3 GM for Fluor-Et measured under conditions comparable to those used for Fluor-RSE. Pulse intensity dependence studies showed that the quantity of NO released from the latter as the result of NIR photoexcitation follows a quadratic relationship to excitation intensity, consistent with the expectation for a TPE process. These studies demonstrate the potential utility of a two-photon antenna for sensitization of the photochemical release of an active agent (in this case, NO) from a photoactive pro-drug.

Quantitative determination of localized tissue oxygen concentration in vivo by two-photon excitation phosphorescence lifetime measurements.

This study describes the use of two-photon excitation phosphorescence lifetime measurements for quantitative oxygen determination in vivo. Doubling the excitation wavelength of Pd-porphyrin from visible light to the infrared allows for deeper tissue penetration and a more precise and confined selection of the excitation volume due to the nonlinear two-photon effect. By using a focused laser beam from a 1,064-nm Q-switched laser, providing 10-ns pulses of 10 mJ, albumin-bound Pd-porphyrin was effectively excited and oxygen-dependent decay of phosphorescence was observed. In vitro calibration of phosphorescence lifetime vs. oxygen tension was performed. The obtained calibration constants were kq = 356 Torr(-1) x s(-1) (quenching constant) and tau0 = 550 micros (lifetime at zero-oxygen conditions) at 37 degrees C. The phosphorescence intensity showed a squared dependency to the excitation intensity, typical for two-photon excitation. In vivo demonstration of two-photon excitation phosphorescence lifetime measurements is shown by step-wise PO2 measurements through the cortex of rat kidney. It is concluded that quantitative oxygen measurements can be made, both in vitro and in vivo, using two-photon excitation oxygen-dependent quenching of phosphorescence. The use of two-photon excitation has the potential to lead to new applications of the phosphorescence lifetime technique, e.g., noninvasive oxygen scanning in tissue at high spatial resolution. To our knowledge, this is the first report in which two-photon excitation is used in the setting of oxygen-dependent quenching of phosphorescence lifetime measurements.

Seeing the Light: Preassembly and Ligand-Induced Changes of the Interferon γ Receptor Complex in Cells

Our experiments were designed to test the hypothesis that the cell surface interferon gamma receptor chains are preassembled rather than associated by ligand and to assess the molecular changes on ligand binding. To accomplish this, we used fluorescence resonance energy transfer, a powerful spectroscopic technique that has been used to determine molecular interactions and distances between the donor and acceptor. However, current commercial instruments do not provide sufficient sensitivity or the full spectra to provide decisive results of interactions between proteins labeled with blue and green fluorescent proteins in living cells. In our experiments, we used the blue fluorescent protein and green fluorescent protein pair, attached a monochrometer and charge-coupled device camera to a modified confocal microscope, reduced background fluorescence with the use of two-photon excitation, and focused on regions of single cells to provide clear spectra of fluorescence resonance energy transfer. In contrast to the prevailing view, the results demonstrate that the receptor chains are preassociated and that the intracellular domains move apart on binding the ligand interferon gamma. Application of this technology should lead to new rapid methods for high throughput screening and delineation of the interactome of cells.

Two-photon excitation microfluorometer for multiplexed single-step bioaffinity assays

A new type of instrumentation for single-step bioaffinity assays and microvolume fluorometry is presented. The concept is based on the use of two-photon excitation by a low-cost near-infrared laser and individual observation of bioactive fluorescent microparticles. The applicability of the instrument is demonstrated by a microparticle based multiplexed bioaffinity assay where several fluorescent markers are simultaneously excited. This instrument can be applied in the growing fields of drug discovery, in life science research, and in routine laboratory diagnostics.

Three-dimensional observation of internal defects in semiconductor crystals by use of two-photon excitation

We present a method of three-dimensional observation of internal defects in semiconductor crystals for blue lasers by use of two-photon excitation. We excite photoluminescence by using a two-photon process. Since semiconductor materials have intrinsically high absorption in the short-wavelength region, the excitation light of photoluminescence is largely absorbed by the crystals. It is difficult to observe defects in deep regions. Two-photon excitation can overcome this limitation because near-infrared light can be used instead of blue light for excitation of photoluminescence, and the near-infrared light is absorbed at only the focused point. The excitation light can penetrate into the deep regions of the crystal. We succeeded in observing defects in a ZnSe crystal ~200 microm below the crystal surface. This is, as far as we know, the first demonstration of the observation of three-dimensional structures of defects in semiconductor crystals by the use of two-photon excitation.

Fluorescence microscopy of single autofluorescent proteins for cellular biology

In this paper we review the applicability of autofluorescent proteins for single-molecule imaging in biology. The photophysical characteristics of several mutants of the Green Fluorescent Protein (GFP) and those of DsRed are compared and critically discussed for their use in cellular biology. The alternative use of two-photon excitation at the single-molecule level or Fluorescence Correlation Spectroscopy is envisaged for the study of individual autofluorescent proteins. Single-molecule experiments performed in live cells using eGFP and preferably eYFP fusion proteins are reviewed. Finally, the first use at the single-molecule level of citrine, a more photostable variant of the eYFP is reported when fused to a receptor for neurotransmitter in live cells. To cite this article: L. Cognet et al., C. R. Physique 3 (2002) 645–656.

Use of two-photon excitation for erasable–rewritable three-dimensional bit optical data storage in a photorefractive polymer

We report what is believed to be the first use of a photorefractive polymer in erasable-rewritable three-dimensional bit optical data storage under two-photon excitation. We successfully demonstrate writing, erasing, and rewriting of multilayered information in a photorefractive polymer consisting of 2,5-dimethyl-4-(p-nitrophenylazo)anisole, 2,4,7-trinitro-9-fluorenone, 9-ethylcarbazole, and poly(N-vinylcarbazole). A three-dimensional bit density of 5 Gbits/cm(3) is achieved by two-photon absorption under pulsed beam illumination at an infrared wavelength of 800 nm in the recording process. Complete erasing of the recording information is achieved by use of ultraviolet illumination.

Two-photon dissociation of NO near 275 nm investigated by velocity map imaging

The two-photon dissociation of NO at wavelengths near 275 nm has been investigated by velocity map imaging of the O( 3 P 2 ) dissociation product. Two channels are seen, corresponding to formation of the co-product N atoms in their ground ( 4 S ) and excited ( 2 D ) states, and the relative yields and angular distributions measured. In their regions of maximum yield, the channel forming N( 2 D ) produces unaligned products, whereas that producing N( 4 S ) can be described by a β value of ca. 0.4. N( 2 D )/N( 4 S ) ratios vary by a factor of 60 over a narrow range of excitation energies. The use of two-photon excitation of NO as a source of aligned atoms for reaction stereodynamics is discussed.

Image formation and data acquisition in a stage scanning 4Pi confocal fluorescence microscope

We describe the three-dimensional (3-D) image formation and data acquisition in a stage scanning 4Pi confocal fluorescence microscope with the use of two-photon excitation. The 3-D point-spread functions of the 4Pi confocal and regular confocal microscope are measured and compared. Particular emphasis is given to the data acquisition procedure. 4Pi confocal microscopy results in a point-spread function that is 4 times sharper than that of a regular confocal microscope, ultimately leading to superior 3-D imaging of translucent fluorescent specimens. For a two-photon excitation wavelength of approximately 800 nm, we obtain an axial resolution of 140 nm.

Fluorescence of horse liver alcohol dehydrogenase using one- and two-photon excitation

We examined the steady-state and time-resolved emission of liver alcohol dehydrogenase resulting from one-photon and two-photon excitation. Previous studies with one-photon excitation revealed that the two nonidentical tryptophan residues display different emission spectra and decay times. The use of two-photon excitation resulted in similar emission spectra, multiexponential intensity decays, time-resolved emission spectra, and anisotropy decays as was observed for one-photon excitation. These results suggest that both nonidentical tryptophan residues are excited to a similar extent for one- and two-photon excitation. However, the limiting anisotropy (r 0) with two-photon excitation from 585 to 610 nm is below 0.1 and appears distinct from that observed previously forN-acetyl-L-tryptophanamide.

Electronic light microscopy: present capabilities and future prospects

Electronic light microscopy involves the combination of microscopic techniques with electronic imaging and digital image processing, resulting in dramatic improvements in image quality and ease of quantitative analysis. In this review, after a brief definition of digital images and a discussion of the sampling requirements for the accurate digital recording of optical images, I discuss the three most important imaging modalities in electronic light microscopy--video-enhanced contrast microscopy, digital fluorescence microscopy and confocal scanning microscopy--considering their capabilities, their applications, and recent developments that will increase their potential. Video-enhanced contrast microscopy permits the clear visualisation and real-time dynamic recording of minute objects such as microtubules, vesicles and colloidal gold particles, an order of magnitude smaller than the resolution limit of the light microscope. It has revolutionised the study of cellular motility, and permits the quantitative tracking of organelles and gold-labelled membrane bound proteins. In combination with the technique of optical trapping (optical tweezers), it permits exquisitely sensitive force and distance measurements to be made on motor proteins. Digital fluorescence microscopy enables low-light-level imaging of fluorescently labelled specimens. Recent progress has involved improvements in cameras, fluorescent probes and fluorescent filter sets, particularly multiple bandpass dichroic mirrors, and developments in multiparameter imaging, which is becoming particularly important for in situ hybridisation studies and automated image cytometry, fluorescence ratio imaging, and time-resolved fluorescence. As software improves and small computers become more powerful, computational techniques for out-of-focus blur deconvolution and image restoration are becoming increasingly important. Confocal microscopy permits convenient, high-resolution, non-invasive, blur-free optical sectioning and 3D image acquisition, but suffers from a number of limitations. I discuss advances in confocal techniques that address the problems of temporal resolution, spherical and chromatic aberration, wavelength flexibility and cross-talk between fluorescent channels, and describe new optics to enhance axial resolution and the use of two-photon excitation to reduce photobleaching. Finally, I consider the desirability of establishing a digital image database, the BioImage database, which would permit the archival storage of, and public Internet access to, multidimensional image data from all forms of biological microscopy. Submission of images to the BioImage database would be made in coordination with the scientific publication of research results based upon these data.(ABSTRACT TRUNCATED AT 400 WORDS)

Theory for two-photon excitation in pattern photobleaching with evanescent illumination

Although the translational diffusion coefficients of proteins embedded in, or tightly bound to, natural and artificial cell membranes have been extensively studied, very little is known about the diffusion of proteins weakly bound to membranes. One method for measuring the diffusion coefficients of weakly bound species is to combine evanescent interference patterns with fluorescence pattern photobleaching recovery (TIR-FPPR). However, this technique is intrinsically limited in that the maximum post-bleach fluorescence change, normalized to the pre-bleach fluorescence, is 0.22. This limitation severely restricts the precision of the data and the applicability of the method. In the theoretical work described herein, it is shown that the maximum fractional fluorescence change in TIR-FPPR with two-photon bleaching and observation would be 0.36. Thus, the use of two-photon excitation in TIR-FPPR would significantly improve the signal-to-noise ratio and would provide more precise measurements of the translational diffusion of proteins weakly bound to surfaces.

Laser-Induced Fluorescence Detection of NH3 in Flames with the Use of Two-Photon Excitation

Laser-Induced Fluorescence (LIF) has been used for the detection of ammonia molecules, NH3, in flames at atmospheric pressure. Excitation was accomplished by a two-photon step from the ground state, X, to the C′ state, with the detection of the fluorescence radiation from the C′ state to the A state. This excitation scheme was also utilized for the simultaneous flame detection of OH and NH3. NO could also be simultaneously detected with the laser beam resulting from frequency mixing of the IR beam and the doubled dye laser beam from a Nd:YAG-based laser system.