Highly efficient and photostable laser parameters were observed for the diiodinated BODYPY derivatives 1 – 3 both in liquid phase and incorporated into polymeric matrices. The laser samples were transversely pumped at wavelengths near their absorption maxima (515 and 532 nm) at 5 mJ/pulse and 10 Hz repetition rate; these are conditions that would induce photodegrdation of many laser-active fluors. Under these extreme conditions, the new dyes exhibit laser action from 530 nm to 625 nm with remarkable efficiencies, up to 55% in liquid solutions and 45% in poly(methylmethacrylate), and with high photostability since the laser output remains at the initial level, with no sign of degradation, after 100000 pump pulses in the same position of the sample. The efficiencies and photo stabilities of the new dyes outperform those of one presently commercialized and considered benchmarks over this spectral region (i.e. coumarines, xanthenes, perilendiimides). The enhanced optical properties recorded under drastic laser pumping conditions suggest that these new photonic systems could be outstanding in biophotonic applications like optical microscopy and nanoscopy, since they would allow very long observation times and improved spatial resolution.
© 2011 OSA
Many new applications for lasers are emerging; for instance there is considerable interest in developing them to give more useful as illumination sources for microscopy . Laser-based microscopy is already well established in biomedicine and in life sciences, yielding three-dimensional images with high spatial resolutions, high sensitivities and discrete chemical or biomolecular selectivities [2–7]. This trend is particularly evident in single-molecule based detection technologies, where fluorescence correlation spectroscopy, fluorescence intensity distribution analysis, and photon-counting histogram analysis may create images showing sample properties that are not evident in ordinary microscopy [8–12].
Ongoing research on microscopy and imaging requires that organic fluorophores satisfy certain properties to be useful as fluorescence markers . In the first place, high absorption cross-sections, fluorescence quantum yields, solubilities in water, and labeling efficiencies are required. Secondly, low rates of triplet, or dark-state formation, are desirable to assure high contrasted-images. Finally, high photostabilities are required so that the fluorescent markers can withstand large excitation intensities of laser light. This last requirement is demanding for most commonly used dyes, which exhibit good photostabilities under low laser intensities but high photobleaching rates when the excitation intensity increases, shortening the observation time and leading to a loss in brightness and, consequently, in the image resolution.
4,4-Difluoro-4-bora-3a,-4a-diaza-s-indacene (BODIPY or BDP) derivatives have favorable photophysical properties such as high absorption coefficients at visible wavelengths, high fluorescence quantum yields, low triplet-triplet absorptions, low tendencies for aggregation, and high photostabilities relative to other dyes (i.e. xanthenes, fluorescein, hemicyanines) [14–16]. Moreover, they are amenable to structural modifications involving introduction of substituents to tune their optical properties [14–16]. Unfortunately, some of BDP derivatives lack of photostability under intense laser irradiation, and display sensitivity to the polarity and/or pH of the solvent [17–22]. Conversely, systems with overall enhanced optical properties are likely to be facilitated new applications of BDP dyes as labels for biomolecules, chromogenic probes, fluorescent sensors, and as tunable laser dyes for liquid phase and in solid-state applications.
Featured here are studies to highlight the lasing potential of a series of diiodinated BDP dyes 1 – 3 shown in Fig. 1 . These dyes have been previously reported as doping agents in polyfluorenes, tuning the emission wavelengths of these polymers ; they have fluorescence emissions dispersed between 530 and 625 nm. We find BDPs 1 – 3 exhibit highly efficient and photostable wavelength tunable laser actions under drastic laser-pumping conditions, in both liquid phase and solid state. Their properties, in this regard, are superior to those exhibited by some commercially available laser dyes like some coumarines, xanthenes, perilendiimides.
Details of the synthesis of the new BDP dyes have been reported elsewhere . Commercial laser dye Coumarin 522 (laser grade, Exciton) was used as received with a purity > 99% (checked by spectroscopic and chromatographic methods). Solvents for laser studies were of spectroscopic grade (Merck, Aldrich or Sigma) and were used without purification. Methyl methacrylate (Merck) was distilled under reduced pressure before use. The initiator: 2,2´-azobis(isobutyronitrile) (AIBN) (Acros) was also purified by recrystallization in ethanol before use.
2.2. Preparation of solid polymeric samples
The new BDP derivatives were incorporated into poly(methylmethacrylate) (PMMA) following a previously described procedure . The solid monolith laser samples were cast in cylindrical shapes, forming rods of 10 mm diameter and 10 mm length. A cut was made parallel to the axis of the cylinder to obtain a lateral flat surface of ca. 6 mm × 10 mm. This surface as well as the ends of the laser rods were prepared for lasing experiments by using a grinding and polishing machine (Phoenix Beta 4000, Buehler) until optical-grade finished. The planar grinding stage was carried out with a P4000 (~6 µm) silicon carbide disk (Tecmicro) as an abrasive with mineral oil as a lubricant. The final polishing stage was realized using a cloth disk Mastertex (Buehler) with diamond of 1µm in mineral oil as an abrasive type.
2.3. Laser experiments
Liquid solutions of dyes were contained in 1 cm optical-path quartz cells that were carefully sealed to avoid solvent evaporation during experiments. The solutions of the BDP dyes as well as the solid samples doped with these new dyes were transversely pumped at different wavelengths matching the maximum absorption of the corresponding dyes: at 532 nm, with 5 mJ/pulse, 6 ns full width at half maximum FWHM pulses from a frequency-doubled Q-switched Nd:YAG laser (Monocrom OPL-10) and at 515 nm, with 5 mJ, 12 ns FWHM pulses from a Nd:YAG-pumped dye laser (Spectron SL800 with an ethanolic solution of Coumarin 522). The exciting pulses were line-focused onto the cell (or onto the lateral flat surface of the solid sample) providing pump fluences on the active medium of 180 mJ/cm2. The oscillation cavity (2 cm length) consisted of a 90% reflectivity aluminum mirror, with the lateral face of the cell (or the end face of the solid sample) as output coupler.
The photostability of these dyes in liquid phase was analyzed under experimental conditions identical to those selected to irradiate the fluorophores when embedded in solid polymeric matrices, allowing a comparison, of their stability in both liquid solution and solid phase under laser irradiation. The irradiated volume in solid samples under the selected experimental conditions was estimated to be 10 μL, so capillary Pyrex tube (1 cm height, 1 mm internal diameter) carefully sealed into which solutions were incorporated to provide irradiated volumes similar to those analyzed in the solid samples, thus preventing the refreshing of the pumped molecules by molecular diffusion and maintaining the same laser pump conditions in both cases.
The optical quality of the capillaries mentioned above precludes laser emission from the dyes, but information on photostabilities can be obtained by monitoring the decrease in laser-induced fluorescence intensity, excited transversally to the capillary, as a function of the number of pump pulses. The fluorescence emission was monitored perpendicular to the exciting beam, and its collection and analysis were carried out with the same set-up selected to characterize the laser emission from dyes incorporated into solid samples. In short, the fluorescence was collected by an optical fiber, and imaged onto the input slit of a monochromator (Acton Research corporation) and detected with a charge-coupled device (CCD) (SpectruMM:GS128B). The fluorescence emission was recorded by feeding the signal to the boxcar (Stanford Research, model 250) to be integrated before being digitized and processed by a computer. Each experience was repeated at least three times. The estimated error of the energy measurements was 10% and the experimental error in the photostability measurements was estimated to be on the order of 7%. Details of the experimental setup can be found elsewhere .
3. Results and discussion
3.1. Liquid phase
Initially, the laser derivatives were pumped at 532 nm, a usual wavelength utilized to pump laser dyes with emission in the yellow-red region of the visible spectra. An optical density for the dye solutions of ca. 20 was used so that the incoming pump radiation penetrates the sample (i.e. is absorbed) to a depth similar to the thickness of the pump stripe at the input face of the cell onto which the pumping radiation is focused (≈0.3 mm). This gives rise to an emitted beam with near circular-cross-section, enhancing the laser efficiency (ratio between the energy of the dye laser output and the energy of the pump laser incident on the sample surface).
The absorption intensity of dye 1 at 532 nm was low so relatively concentrated solutions were used to reach the required optical density. Consequently, solubility problems and aggregation issues for this probe prevented the laser action or, in the best cases, decreased the lasing efficiency to a mere 0.6%. For this reason, this particular dye was analyzed by transverse pumping with laser radiation at 515 nm, i.e. a wavelength near to its absorption maximum . Under these experimental conditions, broad-line-width laser emission with pump threshold energy of ≈0.8 mJ, beam divergence of ≈5 mrad and pulse duration of ≈8 ns FWHM was obtained from the new dyes when placed in a simple plane-plane non-tunable resonator. The dependence of the laser action of the new BDP derivatives on the corresponding dye concentration was analyzed in dichloromethane, a solvent in which the dyes show good solubility, varying the optical densities while keeping constant all the other experimental parameters (Figs. 2 and 3 ).
The lasing efficiency of all the analyzed dyes increase significantly with dye concentration, reaching a maximum value for solutions with concentrations in the range 2.5-4.8 × 10−4 M, depending on the dye. From this point on, further increases in dye concentration result in a slight decrease in lasing efficiency that can be related to the increase in re-absorption/re-emission processes with deleterious effect in the laser action (Fig. 2) .
The laser emission spectra of 1 – 3 in solution follow the same dependence on their concentrations, with their laser emission shifted to lower energies as the concentration increases (Fig. 3). This trend must also be related to the effect of re-absorption/re-emission phenomena because the possibility of exciting molecules by absorption of a photon previously emitted by another molecule in the medium depends on the overlapping between absorption and fluorescence spectra, which is affected by the dye concentration .
The lasing behaviour of the new dyes shows good correlations with their photophysical properties  as evidenced by the following observations: 1) the higher the fluorescence quantum yield is, the higher the lasing efficiency becomes; 2) the low fluorescence quantum yield of dye 3 (Φ = 0.48) could, to some extent, be compensated by its high Stokes shift (1565 cm−1), which reduces the losses at the resonator cavity by re-absorption/re-emission effects, giving rise to high lasing efficiencies, similar to those registered from dye 1 with higher fluorescence quantum yield (Φ = 1); 3) The laser emission of dye 2 shifts up to 56 nm with respect to that of dye 1, while the presence of (2-metoxyphenyl) substituent at positions 3 and 5 of the BDP core in dye 3 increases this red-shift up to 86 nm.
Solvent effects on the dye laser action were probed by using apolar, polar protic and polar nonprotic media at optimal dye concentrations for the laser efficiencies of each dye (Table 1 ). We learned from these experiments that the dyes exhibit minimal dependence on solvent polarities and hydrogen-bonding characteristics; this characteristic represents a great advantage with respect to other pyrromethene derivatives which exhibit stronger environment dependence [14–16,26,28–37].
Lasing photostabilities (evolution of the laser-induced emission with the number of pump pulses at 10 Hz) of 1 – 3 in ethyl acetate at concentrations for optimal laser action were recorded (Fig. 4 ).
Dyes 1 - 3 are highly photostable since, after 100000 pump pulses, they maintained their initial emission intensities without any sign of degradation. For instance, the emission intensity of dye 1 drops by less than 10% with respect to its initial output. Thus, the new BDP derivatives are both extremely efficient and photostable at high laser intensities, which should result in an excellent performance when used in fluorescence microscopy. This is particularly so for single-molecule experiments where high photon rates are crucial.
Lasing parameters of three well-known dyes with similar lasing wavelengths were measured in solutions under similar experimental conditions to calibrate the performance of 1 – 3. The efficiency of Coumarin 540A, (1.5 × 10−3 M) at ca. 535 nm was 25%, and this fell to 35% after just 30000 pump pulses. Rhodamine 6G (4 × 10−4 M) lases at 580 nm with an efficiency of 25% but completely losses its emission after 50000 pump pulses. Perylene Red (5 × 10−4 M) lases at 614 nm with an efficiency of 26% maintaining 70% of its initial emission after 100000 pump pulses. Laser performances exhibited by 1 – 3 are significantly more than those recorded here for the common commercially available BDP dyes (PM567, PM597, and PM650) and their 8-alkyl, 8-phenyl, 8-polyphenylene, 2,6-alkyl or 3-styryl analogues [26,28–30,38–40], pumped under identical experimental conditions. For instance, the laser efficiencies recorded for PM567, PM597 and PM650 in ethyl acetate solutions are 48%, 58% and 20%, respectively, but, with low photostabilities compared with those exhibited by our BDP derivatives, since the commercial BDP dyes lost their initial emission after just 50000, 80000 and 20000 pump pulses, respectively.
3.2. Solid state
Solid state experiments were carried out using samples with dye concentrations that had the highest lasing efficiencies in ethyl acetate solutions. MMA (methyl methacrylate) was chosen as the main monomeric component of the formulations because this ester mimics ethyl acetate, a solvent where the studied dyes gave rise to high lasing efficiencies.
Broad-band and efficient laser emission, with beam divergence of ca. 5 mrad and pulse duration of ca. 5 ns FWHM, was registered from the dyes incorporated as true solutions into the solid homopolymer PMMA (Table 2 ). No significant differences were observed in the wavelength of the maximum laser emission of each dye between their liquid and solid solutions (Fig. 5 ). The lasing efficiencies of the solid materials, in the range of 25-45%, are lower than those of the corresponding liquid solutions. Surface finishings of the solid samples in these experiments were not laser-grade, and higher lasing efficiencies are expected with laser-grade surfaces. The lasing stabilities of the dye-doped solid matrices were studied by following the evolution of the laser output as a function of the number of pump pulses in the same position of the sample, at 10 Hz repetition rate. Dyes 1 – 3 exhibited high photostabilities, following with the structure a similar dependence to that described in liquid phase.
The laser action of these BDP derivatives doped PMMA improves the lasing properties of commercial BDP dyes PM567, PM597 and PM650 incorporated to PMMA, which exhibited lasing efficiencies of 28%, 40% and 13%, respectively, with lower photostabilities than those reported for these dyes in liquid phase.
In summary, the BDP dyes studied here lase with high efficiencies, up to 55% in the liquid phase and 45% when incorporated into solid-state matrices. They possess high photostabilities and their laser emissions remain at the initial value without sign of degradation or, in the worst case, less than 10% loss from of their initial value after 100000 pump pulses at 10 Hz repetition rate in the same position of the sample. The position of the emission band was modulated by the type of substituent attached to the BDP core, while maintaining a high laser performance. The new dyes outperform commercial dyes emitting in the same spectral region, being versatile and efficient as active media in dye lasers both in liquid phase and incorporated into solid matrices. Furthermore, the high efficiency and high photostability recorded under drastic laser pumping conditions suggest that these dyes could perform outstandingly well in biophotonic applications, such as optical microscopy and nanoscopy, since serving as fluorescent markers would allow very long observation times and improved spatial resolution.
This work was supported by the Spanish MICINN (Projects MAT2010-20646-C04-01 and TRACE2009-0144), and by the National Institutes of Health (GM0879811) and The Robert Welch Foundation (A-1121). V. Martín and M. E. Pérez-Ojeda thank CSIC for her JAE postdoctoral contract and predoctoral grant, respectively.
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