Harmonic Imaging of Stem Cells in Whole Blood at GHz Pixel Rate.

The pre-clinical validation of cell therapies requires monitoring the biodistribution of transplanted cells in tissues of host organisms. Real-time detection of these cells in the circulatory system and identification of their aggregation state is a crucial piece of information, but necessitates deep penetration and fast imaging with high selectivity, subcellular resolution, and high throughput. In this study, multiphoton-based in-flow detection of human stem cells in whole, unfiltered blood is demonstrated in a microfluidic channel. The approach relies on a multiphoton microscope with diffractive scanning in the direction perpendicular to the flow via a rapidly wavelength-swept laser. Stem cells are labeled with metal oxide harmonic nanoparticles. Thanks to their strong and quasi-instantaneous second harmonic generation (SHG), an imaging rate in excess of 10 000 frames per second is achieved with pixel dwell times of 1 ns, a duration shorter than typical fluorescence lifetimes yet compatible with SHG. Through automated cell identification and segmentation, morphological features of each individual detected event are extracted and cell aggregates are distinguished from isolated cells. This combination of high-speed multiphoton microscopy and high-sensitivity SHG nanoparticle labeling in turbid media promises the detection of rare cells in the bloodstream for assessing novel cell-based therapies.

Secondary cavitation bubble dynamics during laser-induced bubble formation in a small container.

We investigated secondary cavitation bubble dynamics during laser-induced bubble formation in a small container with a partially confined free surface and elastic thin walls. We employed high-speed photography to record the dynamics of sub-mm-sized laser-induced bubbles and small secondary bubble clouds. Simultaneous light scattering and acoustic measurements were used to detect the oscillation times of laser-induced bubbles. We observed that the appearance of secondary bubbles coincides with a prolonged collapse phase and with re-oscillations of the laser-induced bubble. We observed an asymmetric distribution of secondary bubbles with a preference for the upstream side of the focus, an absence of secondary bubbles in the immediate vicinity of the laser focus, and a migration of laser-induced bubble toward secondary bubbles at large pulse energies. We found that secondary bubbles are created through heating of impurities to form initial nanobubble nuclei, which are further expanded by rarefaction waves. The rarefaction waves originate from the vibration of the elastic thin walls, which are excited either directly by laser-induced bubble or by bubble-excited liquid-mass oscillations. The oscillation period of thin walls and liquid-mass were Twall = 116 µs and Tlm ≈ 160 µs, respectively. While the amplitude of the wall vibrations increases monotonically with the size of laser-induced bubbles, the amplitude of liquid-mass oscillation undulates with increasing bubble size. This can be attributed to a phase shift between the laser-induced bubble oscillation and the liquid-mass oscillator. Mutual interactions between the laser-induced bubble and secondary bubbles reveal a fast-changing pressure gradient in the liquid. Our study provides a better understanding of laser-induced bubble dynamics in a partially confined environment, which is of practical importance for microfluidics and intraluminal laser surgery.

Laser induced spherical bubble dynamics in partially confined geometry with acoustic feedback from container walls.

We investigated laser-induced cavitation dynamics in a small container with elastic thin walls and free or partially confined surface both experimentally and by numerical investigations. The cuvette was only 8-25 times larger than the bubble in its center. The liquid surface was either free, or two thirds were confined by a piston-shaped pressure transducer. Different degrees of confinement were realized by filling the liquid up to the transducer surface or to the top of the cuvette. For reference, some experiments were performed in free liquid. We recorded the bubble dynamics simultaneously by high-speed photography, acoustic measurements, and detection of probe beam scattering. Simultaneous single-shot recording of radius-time curves and oscillation times enabled to perform detailed investigations of the bubble dynamics as a function of bubble size, acoustic feedback from the elastic walls, and degree of surface confinement. The bubble dynamics was numerically simulated using a Rayleigh-Plesset model extended by terms describing the acoustically mediated feedback from the bubble's environment. Bubble oscillations were approximately spherical as long as no secondary cavitation by tensile stress occurred. Bubble expansion was always similar to the dynamics in free liquid, and the environment influenced mainly the collapse phase and subsequent oscillations. For large bubbles, strong confinement led to a slight reduction of maximum bubble size and to a pronounced reduction of the oscillation time, and both effects increased with bubble size. The joint action of breakdown-induced shock wave and bubble expansion excites cuvette wall vibrations, which produce alternating pressure waves that are focused onto the bubble. This results in a prolongation of the collapse phase and an enlargement of the second oscillation, or in time-delayed re-oscillations. The details of the bubble dynamics depend in a complex manner on the degree of surface confinement and on bubble size. Numerical simulations of the first bubble oscillation agreed well with experimental data. They suggest that the alternating rarefaction/compression waves from breakdown-induced wall vibrations cause a prolongation of the first oscillation. By contrast, liquid mass movement in the cuvette corners result in wall vibrations causing late re-oscillations. The strong and rich interaction between the bubble and its surroundings may be relevant for a variety of applications such as intraluminal laser surgery and laser-induced cavitation in microfluidics.

Dissection of DNA damage and repair pathways in live cells by femtosecond laser microirradiation and free-electron modeling.

Understanding and predicting the outcome of the interaction of light with DNA has a significant impact on the study of DNA repair and radiotherapy. We report on a combination of femtosecond pulsed laser microirradiation at different wavelengths, quantitative imaging, and numerical modeling that yields a comprehensive picture of photon-mediated and free-electron-mediated DNA damage pathways in live cells. Laser irradiation was performed under highly standardized conditions at four wavelengths between 515 nm and 1,030 nm, enabling to study two-photon photochemical and free-electron-mediated DNA damage in situ. We quantitatively assessed cyclobutane pyrimidine dimer (CPD) and γH2AX-specific immunofluorescence signals to calibrate the damage threshold dose at these wavelengths and performed a comparative analysis of the recruitment of DNA repair factors xeroderma pigmentosum complementation group C (XPC) and Nijmegen breakage syndrome 1 (Nbs1). Our results show that two-photon-induced photochemical CPD generation dominates at 515 nm, while electron-mediated damage dominates at wavelengths ≥620 nm. The recruitment analysis revealed a cross talk between nucleotide excision and homologous recombination DNA repair pathways at 515 nm. Numerical simulations predicted electron densities and electron energy spectra, which govern the yield functions of a variety of direct electron-mediated DNA damage pathways and of indirect damage by •OH radicals resulting from laser and electron interactions with water. Combining these data with information on free electron-DNA interactions gained in artificial systems, we provide a conceptual framework for the interpretation of the wavelength dependence of laser-induced DNA damage that may guide the selection of irradiation parameters in studies and applications that require the selective induction of DNA lesions.

Mechanisms of corneal intrastromal laser dissection for refractive surgery: ultra-high-speed photographic investigation at up to 50 million frames per second.

Every year, more than a million refractive eye surgeries using femtosecond lasers are performed but the intrastromal cutting process remains an area of development. We investigated the mechanisms of laser dissection in cornea by ultra-high-speed photography. We found that the intrastromal bubble forms multiple lobes along the elongated laser plasma and the overlying lobes expand along the corneal lamellae. Videography demonstrated that the cutting process relies on crack propagation in the stroma along the bubble lobes with the crack originating from the pre-existing bubble layer. These insights are important for further improvement of the cutting mechanisms in refractive surgery.

Optical Vortex Beam for Gentle and Ultraprecise Intrastromal Corneal Dissection in Refractive Surgery.

Purpose: We introduce a novel focus shaping concept for intrastromal corneal dissection that facilitates cleavage along corneal lamellae, and we analyze laser-tissue interactions governing cutting effectiveness and mechanical side effects. Methods: Focus shaping was achieved by a spiral phase plate that converts an incident Gaussian beam into a Laguerre-Gaussian beam with a helical phase. Such vortex beams have zero intensity at their center, are propagation invariant, and possess a ring focus equal in length to the Gaussian focus but with a larger diameter. Cutting precision and the required absorbed energy for flap dissection were compared for Gaussian and vortex beams on ex vivo porcine corneal specimens at pulse durations between 480 fs and 9 ps. Cutting quality and bubble formation were characterized by scanning electron microscopy and macro photography. Results: With the vortex beam, the cuts were much smoother. Bubble formation was markedly reduced because cutting can be performed close to the bubble threshold, whereas with the Gaussian beam energies well above threshold are needed. Although the incident energy at the flap dissection threshold was slightly larger for the vortex beam, the absorbed energy was much smaller and contributed more effectively to cutting. This reduced plasma-induced pressure more than sevenfold. Conclusions: The vortex beam approach for corneal dissection is a simple, versatile, and cost-effective way of improving the precision of refractive surgery while reducing bubble formation and pressure-related mechanical side effects. Translational Relevance: Phase plates for propagation invariant vortex beams are easily implemented in the beam path of next-generation clinical devices.

Noninvasive two-photon optical biopsy of retinal fluorophores.

High-resolution imaging techniques capable of detecting identifiable endogenous fluorophores in the eye along with genetic testing will dramatically improve diagnostic capabilities in the ophthalmology clinic and accelerate the development of new treatments for blinding diseases. Two-photon excitation (TPE)-based imaging overcomes the filtering of ultraviolet light by the lens of the human eye and thus can be utilized to discover defects in vitamin A metabolism during the regeneration of the visual pigments required for the detection of light. Combining TPE with fluorescence lifetime imaging (FLIM) and spectral analyses offers the potential of detecting diseases of the retina at earlier stages before irreversible structural damage has occurred. The main barriers to realizing the benefits of TPE for imaging the human retina arise from concerns about the high light exposure typically needed for informative TPE imaging and the requirement to correlate the ensuing data with different states of health and disease. To overcome these hurdles, we improved TPE efficiency by controlling temporal properties of the excitation light and employed phasor analyses to FLIM and spectral data in mouse models of retinal diseases. Modeling of retinal photodamage revealed that plasma-mediated effects do not play a role and that melanin-related thermal effects are mitigated by reducing pulse repetition frequency. By using noninvasive TPE imaging we identified molecular components of individual granules in the retinal pigment epithelium and present their analytical characteristics.

Development of a Noninvasive, Laser-Assisted Experimental Model of Corneal Endothelial Cell Loss.

Nd:YAG lasers have been used to perform noninvasive intraocular surgery, such as capsulotomy for several decades now. The incisive effect relies on the optical breakdown at the laser focus. Acoustic shock waves and cavitation bubbles are generated, causing tissue rupture. Bubble sizes and pressure amplitudes vary with pulse energy and position of the focal point. In this study, enucleated porcine eyes were positioned in front of a commercially available Nd:YAG laser. Variable pulse energies as well as different positions of the focal spots posterior to the cornea were tested. Resulting lesions were evaluated by two-photon microscopy and histology to determine the best parameters for an exclusive detachment of corneal endothelial cells (CEC) with minimum collateral damage. The advantages of this method are the precise ablation of CEC, reduced collateral damage, and above all, the non-contact treatment.

Wavelength-Selective Nonlinear Imaging and Photo-Induced Cell Damage by Dielectric Harmonic Nanoparticles.

We introduce a nonlinear all-optical theranostics protocol based on the excitation wavelength decoupling between imaging and photoinduced damage of human cancer cells labeled by bismuth ferrite (BFO) harmonic nanoparticles (HNPs). To characterize the damage process, we rely on a scheme for in situ temperature monitoring based on upconversion nanoparticles: by spectrally resolving the emission of silica coated NaGdF4:Yb3+/Er3+ nanoparticles in close vicinity of a BFO HNP, we show that the photointeraction upon NIR-I excitation at high irradiance is associated with a temperature increase >100 °C. The observed laser-cell interaction implies a permanent change of the BFO nonlinear optical properties, which can be used as a proxy to read out the outcome of a theranostics procedure combining imaging at 980 nm and selective cell damage at 830 nm. The approach has potential applications to monitor and treat lesions within NIR light penetration depth in tissues.

High-resolution imaging of living gut mucosa: lymphocyte clusters beneath intestinal M cells are highly dynamic structures.

In the Peyer's patches of the small intestine, specialized epithelial cells, the membranous (M) cells, sample antigenic matter from the gut lumen and bring it into contact with cells of the immune system, which are then capable of initiating specific immune reactions. Using autofluorescence 2-photon (A2P) microscopy, we imaged living intestinal mucosa at a 0.5-µm resolution. We identified individual M cells without the aid of a marker and in vivo analyzed their sampling function over hours. Time-lapse recordings revealed that lymphocytes associated with M cells display a remarkable degree of motility with average speed rates of 8.2 µm/min, to form new M cell-associated lymphocyte clusters within less than 15 min. The lymphocytes drastically deform the M cells' cytoplasm and laterally move from one lymphocyte cluster to the next. This implies that the micro-compartment beneath M cells is a highly efficient container to bring potentially harmful antigens into contact with large numbers of immunocompetent cells. Our setup opens a new window for high-resolution 3D imaging of functional processes occurring in lymphoid and mucosal tissues.

Correction of hyperopia by intrastromal cutting and liquid filler injection.

Correction of hyperopia requires an increase of the refractive power by steepening of the corneal surface. Present refractive surgical techniques based on corneal ablation (LASIK) or intrastromal lenticule extraction (SMILE) are problematic due to epithelial regrowth. Recently, it was shown that correction of low hyperopia can be achieved by implanting intracorneal inlays or allogeneic lenticules. We demonstrate a steepening of the anterior corneal surface after injection of a transparent, liquid filler material into a laser-dissected intrastromal pocket. We performed the study on ex-vivo porcine eyes. The increase of the refractive power was evaluated by optical coherence tomography (OCT). For a circular pocket, injection of 1 µl filler material increased the refractive power by +4.5 diopters. An astigmatism correction is possible when ellipsoidal intrastromal pockets are created. Injection of 2 µl filler material into an ellipsoidal pocket increased the refractive power by +10.9 dpt on the short and +5.1 dpt on the long axis. OCT will enable to monitor the refractive change during filler injection and is thus a promising technique for real-time dosimetry.

Multi-rate-equation modeling of the energy spectrum of laser-induced conduction band electrons in water.

We study the energy spectrum of laser-induced conduction band (CB) electrons in water by multi-rate equations (MRE) with different impact ionization schemes. Rethfeld's MRE model [Phys. Rev. Lett.92, 187401(2004)Phys. Rev.B 79, 155424(2009)], but the corresponding rate equations are computationally very expensive. We introduce a simplified splitting scheme and corresponding rate equations that still agree with energy conservation but enable the derivation of an asymptotic SRE. This approach is well suited for the calculation of energy spectra at long pulse durations and high irradiance, and for combination with spatiotemporal beam propagation/plasma formation models. Using the energy-conserving MREs, we present the time-evolution of CB electron density and energy spectrum during femtosecond breakdown as well as the irradiance dependence of free-electron density, energy spectrum, volumetric energy density, and plasma temperature. These data are relevant for understanding photodamage pathways in nonlinear microscopy, free-electron-mediated modifications of biomolecules in laser surgery, and laser processing of transparent dielectrics in general.

A light-controlled switch after dual targeting of proliferating tumor cells via the membrane receptor EGFR and the nuclear protein Ki-67.

Using nanotechnology for optical manipulation of molecular processes in cells with high spatial and temporal precision promises new therapeutic options. Especially tumor therapy may profit as it requires a combination of both selectivity and an effective cell killing mechanism. Here we show a dual targeting approach for selective and efficient light-controlled killing of cells which are positive for epidermal growth factor receptor (EGFR) and Ki-67. Liposomes with the covalently linked EGFR antibody Erbitux enabled selective uptake of FITC-labeled Ki-67 antibody TuBB-9 in EGFR-positive cells pre-loaded with the photoactive dye BPD. After irradiation at 690 nm, BPD disrupted the endosomal membranes and delivered the antibodies to the nucleoli of the cells. The second irradiation at 490 nm activated the FITC-labeled TuBB-9, which caused inactivation of the Ki-67 protein and subsequent cell death via apoptosis. Efficient cell killing was possible at nanomolar concentrations of TuBB-9 due to the effective transport by immune liposomes and the high efficacy of the Ki-67 light-inactivation. Delivery of the liposomal constructs and cell destruction correlated well with the EGFR expression pattern of different cell lines (HeLa, OVCAR-5, MCF-7, and human fibroblasts), demonstrating an excellent selectivity.

Intravital autofluorescence 2-photon microscopy of murine intestinal mucosa with ultra-broadband femtosecond laser pulse excitation: image quality, photodamage, and inflammation.

Ultra-broadband excitation with ultrashort pulses may enable simultaneous excitation of multiple endogenous fluorophores in vital tissue. Imaging living gut mucosa by autofluorescence 2-photon microscopy with more than 150 nm broad excitation at an 800-nm central wavelength from a sub-10 fs titanium-sapphire (Ti:sapphire) laser with a dielectric mirror based prechirp was compared to the excitation with 220 fs pulses of a tunable Ti:sapphire laser at 730 and 800 nm wavelengths. Excitation efficiency, image quality, and photochemical damage were evaluated. At similar excitation fluxes, the same image brightness was achieved with both lasers. As expected, with ultra-broadband pulses, fluorescence from NAD(P)H, flavines, and lipoproteins was observed simultaneously. However, nonlinear photodamage apparent as hyperfluorescence with functional and structural alterations of the tissue occurred earlier when the laser power was adjusted to the same image brightness. After only a few minutes, the immigration of polymorphonuclear leucocytes into the epithelium and degranulation of these cells, a sign of inflammation, was observed. Photodamage is promoted by the higher peak irradiances and/or by nonoptimal excitation of autofluorescence at the longer wavelength. We conclude that excitation with a tunable narrow bandwidth laser is preferable to ultra-broadband excitation for autofluorescence-based 2-photon microscopy, unless the spectral phase can be controlled to optimize excitation conditions.

Light-Controlled Delivery of Monoclonal Antibodies for Targeted Photoinactivation of Ki-67.

The selective inhibition of intracellular and nuclear molecules such as Ki-67 holds great promise for the treatment of cancer and other diseases. However, the choice of the target protein and the intracellular delivery of the functional agent remain crucial challenges. Main hurdles are (a) an effective delivery into cells, (b) endosomal escape of the delivered agents, and (c) an effective, externally triggered destruction of cells. Here we show a light-controlled two-step approach for selective cellular delivery and cell elimination of proliferating cells. Three different cell-penetrating nano constructs, including liposomes, conjugates with the nuclear localization sequence (NLS), and conjugates with the cell penetrating peptide Pep-1, delivered the light activatable antibody conjugate TuBB-9-FITC, which targets the proliferation associated protein Ki-67. HeLa cells were treated with the photosensitizer benzoporphyrin monoacid derivative (BPD) and the antibody constructs. In the first optically controlled step, activation of BPD at 690 nm triggered a controlled endosomal escape of the TuBB-9-FITC constructs. In more than 75% of Ki-67 positive, irradiated cells TuBB-9-FITC antibodies relocated within 24 h from cytoplasmic organelles to the cell nucleus and bound to Ki-67. After a second light irradiation at 490 nm, which activated FITC, cell viability decreased to approximately 13%. Our study shows an effective targeting strategy, which uses light-controlled endosomal escape and the light inactivation of Ki-67 for cell elimination. The fact that liposomal or peptide-assisted delivery give similar results leads to the additional conclusion that an effective mechanism for endosomal escape leaves greater variability for the choice of the delivery agent.

Probing the immune and healing response of murine intestinal mucosa by time-lapse 2-photon microscopy of laser-induced lesions with real-time dosimetry.

Gut mucosa is an important interface between body and environment. Immune response and healing processes of murine small intestinal mucosa were investigated by intravital time-lapse two-photon excited autofluorescence microscopy of the response to localized laser-induced damage. Epithelial lesions were created by 355-nm, 500-ps pulses from a microchip laser that produced minute cavitation bubbles. Size and dynamics of these bubbles were monitored using a novel interferometric backscattering technique with 80 nm resolution. Small bubbles (< 2.5 µm maximum radius) merely resulted in autofluorescence loss of the target cell. Larger bubbles (7-25 µm) affected several cells and provoked immigration of immune cells (polymorphonuclear leucocytes). Damaged cells were expelled into the lumen, and the epithelium healed within 2 hours by stretching and migration of adjacent epithelial cells.

A new nanosecond UV laser at 355 nm: early results of corneal flap cutting in a rabbit model.

PURPOSE: A new 355 nm UV laser was used for corneal flap cutting in an animal model and tested for clinical and morphologic alterations. METHODS: Corneal flaps were created (Chinchilla Bastards; n = 25) with an UV nanosecond laser at 355 nm (150 kHz, pulse duration 850 ps, spot-size 1 µm, spot spacing 6 × 6 µm, side cut Δz 1 µm; cutting depth 130 µm) and pulse energies of 2.2 or 2.5 µJ, respectively. Following slit-lamp examination, animals were killed at 6, 12, and 24 hours after treatment. Corneas were prepared for histology (hematoxylin and eosin [HE], TUNEL-assay) and evaluated statistically, followed by ultrastructural investigations. RESULTS: Laser treatment was tolerated well, flap lift was easier at 2.5 µJ compared with 2.2 µJ. Standard HE at 24 hours revealed intact epithelium in the horizontal cut, with similar increase in corneal thickness at both energies. Irrespective of energy levels, TUNEL assay revealed comparable numbers of apoptotic cells in the horizontal and vertical cut at 6, 12, and 24 hours, becoming detectable in the horizontal cut as an acellular stromal band at 24 hours. Ultrastructural analysis revealed regular morphology in the epi- and endothelium, while in the stroma, disorganized collagen lamellae were detectable representing the horizontal cut, again irrespective of energy levels applied. CONCLUSIONS: This new UV laser revealed no epi- nor endothelial damage at energies feasible for corneal flap cutting. Observed corneal swelling was lower compared with existing UV laser studies, albeit total energy applied here was much higher. Observed loss of stromal keratinocytes is comparable with available laser systems. Therefore, this new laser is suitable for refractive surgery, awaiting its test in a chronic environment.

In vivo spectral imaging of different cell types in the small intestine by two-photon excited autofluorescence.

Spectrally resolved two-photon excited autofluorescence imaging is used to distinguish different cell types and functional areas during dynamic processes in the living gut. Excitation and emission spectra of mucosal tissue and tissue components are correlated to spectra of endogenous chromophores. We show that selective excitation with only two different wavelengths within the tuning range of a Ti:sapphire femtosecond laser system yields excellent discrimination between enterocytes, antigen presenting cells and lysosomes based on the excitation and emission properties of their autofluorescence. The method is employed for time-lapse microscopy over up to 8 h. Changes of the spectral signature with the onset of photodamage are demonstrated, and their origin is discussed.

Femtosecond-laser-induced nanocavitation in water: implications for optical breakdown threshold and cell surgery.

We determined the bubble radius R_(max) for femtosecond optical breakdown in water at 347, 520, and 1040 nm with an unprecedented accuracy (+/-10 nm). At threshold, R_(max) was smaller than the diffraction-limited focus radius and ranged from 190 nm to 320 nm. The increase of R_(max) with laser energy E_(L) is slowest at 347 nm, providing optimum control of cell surgery. Experimental results agree with a model of bubble formation in heated and thermoelastically stretched liquids. Theory predicts a threshold temperature T_(th) approximately equal to 168 degrees C. For T>300 degrees C, a phase explosion sets in, and R_(max) increases rapidly with E_(L).

Principles of laser-induced separation and transport of living cells.

Separation and transport of defined populations of living cells grown on a thin membrane can be achieved by laser microdissection (LMD) of the sample of interest, followed by a laser-induced forward transport process [laser pressure "catapulting" (LPC)] of the dissected cell cluster. We investigate the dynamics of LMD and LPC with focused and defocused UV-A laser pulses by means of time-resolved photography. Catapulting is driven by plasma formation when tightly focused pulses are used, and by confined thermal ablation at the bottom of the sample for defocused catapulting. With both modalities, the initial specimen velocity amounts to about 50 to 60 ms. Time-resolved photography of live cell catapulting reveals that in defocused catapulting, strong shear forces arise when the sample is accelerated out of the culture medium covering the cells. By contrast, pulses focused at the periphery of the specimen cause a fast rotational movement that minimizes the flow of culture medium parallel to the sample surface, and thus the resulting shear stresses. Therefore, the recultivation rate of catapulted cells is much higher when focused pulses are used. Compared to collateral damage by mechanical forces, side effects by heat and UV exposure of the cells play only a minor role.