Optical wavefront shaping is a powerful technique to control the distribution of light in the focus of a microscope. Combined with optogenetics, it holds great promise for a precise manipulation of neuronal activity with light.
A better understanding of complex brain circuits however, requires advanced and flexible optical methods capable of simultaneously photo-exciting multiple neurons, possibly using dedicated excitation shapes, arbitrarily distributed in the three dimensions, with single-cell resolution. At the same time, the study of deep brain structures with all optical techniques, even in the multi-photon regime, is limited by scattering to a depth of few hundreds µm.
Here we first present a new optical scheme, based on the spatio-temporal shaping of a pulsed laser beam, to project several tens of spatially confined two photon excitation patterns in a large volume. Using two spatial light modulators and the temporal focusing technique we are able to produce at least 4 different extended excitation patterns, with single cell axial confinement, that we independently multiplex at the sample volume an arbitrary number of times. We fully characterise the optical response of the system, discuss the possibility of simplifying it at the expenses of flexibility, and subsequently exploit it to perform multi-cell volumetric excitation in both Drosophila and zebrafish larvae. Finally, we summarise our recent efforts towards the extension of such method to a micro endoscope, which could be used for the study of complex neural circuits in deep brain structures, thus overcoming the limitations imposed by scattering.
Genetically encoded light-sensitive channels and reporters enable both neuronal activity optical control and read-out. Full explotation of these optogenetic tools requires single-cell scale methods to pattern light into neural tissue.
Computer Generated Holography (CGH) can powerfully enhance optogenetic stimulation by efficiently shaping light onto multiple cellular targets. However, a linear proportionality between lateral shape area and axial extent degrades axial precision for cases demanding extended lateral patterning i.e., to cover entire soma of multiple cells. To address this limitation, we previously combined CGH with temporal focusing (TF) to stretch laser pulses outside of the focal plane, which combined with two-photon’s nonlinear fluorescence dependence, axially confines fluorescence regardless of lateral extent. However, this configuration restricts nonlinear excitation to a single spatiotemporal focal plane: which is the objective focal plane.
Here we demonstrate a novel scheme enabling generation of spatiotemporally focused pattern generation in three dimensions. We demonstrate that this approach enables simultaneous photoconversion of tens of zebrafish larvae spinal cord neurons occupying separate axial planes.
Voltage-sensitive fluorescence indicators enable tracking neuronal electrical signals simultaneously in multiple neurons or neuronal subcompartments difficult to access with patch electrodes. However, efficient widefield epifluorescence detection of rapid voltage fluorescence transients necessitates that imaged cells and structures lie sufficiently far from other labeled structures to avoid contamination from out of focal plane and scattered light. We overcame this limitation by exciting dye fluorescence with one-photon computer-generated holography shapes contoured to axons or dendrites of interest, enabling widefield detection of voltage fluorescence with high spatial specificity. By shaping light onto neighboring axons and dendrites, we observed that dendritic back-propagating action potentials were broader and slowly rising compared with axonal action potentials, differences not measured in the same structures illuminated with a large “pseudowidefield” (pWF) spot of the same excitation density. Shaped illumination trials showed reduced baseline fluorescence, higher baseline noise, and fractional fluorescence transient amplitudes two times greater than trials acquired with pWF illumination of the same regions.
Multiphoton excitation has recently found application in the fields of bioimaging, uncaging and lithography. In
order to fully exploit the advantages of nonlinear excitation, in particular the axial resolution due to nonlinearity,
most systems to date operate with point or multipoint excitation, while scanning either the laser beam or the
sample to generate the illumination pattern. Here we combine the recently introduced technique of scanningless
multiphoton excitation by temporal focusing with recent advances in digital holography to generate arbitrarily
shaped, depth resolved, two-dimensional excitation patterns completely without scanning. This is of particular
importance in applications requiring uniform excitation of large areas over short time scales, such as neuronal
activation by multiphoton uncaging of neurotransmitters. We present an experimental and theoretical analysis of
the effect of spatial patterning on the depth resolution achieved in temporal focusing microscopy. It is shown that
the depth resolution for holographic excitation is somewhat worse than that achieved for uniform illumination.
This is also accompanied by the appearance of a speckle pattern at the temporal focal plane. The origin of the
two effects, as well as means to overcome them, are discussed.
A passive, optical cell sorter is created using the light pattern of a 'nondiffracting' beam—the Bessel beam. As a precursor to cell sorting studies, microspheres are used to test the resolution of the sorter on the basis of particle size and refractive index. Variations in size and, more noticeably, refractive index, lead to a marked difference in the migration time of spheres in the Bessel beam. Intrinsic differences (size, refractive index) between native (unlabeled) cell populations are utilized for cell sorting. The large difference in size between erythrocytes and lymphocytes results in their successful separation in this beam pattern. The intrinsic differences in size and refractive index of other cells in the study (HL60 human promyelocytic leukaemic cells, murine bone marrow, and murine stem/progenitor cells) are not large enough to induce passive optical separation. Silica microsphere tags are attached to cells of interest to modify their size and refractive index, resulting in the separation of labeled cells. Cells collected after separation are viable, as evidenced by trypan blue dye exclusion, their ability to clone in vitro, continued growth in culture, and lack of expression of Caspase 3, a marker of apoptosis.
In this work we present a laser trapping system based on an Ar+ laser and the calibration method we used for the optical
trapping force measurements on polystyrene spheres. The method uses the dielectrophoretic force exerted on a dielectric
particle from an inhomogeneous electric field for the calculation of the optical force. We used polystyrene microspheres
on a dielectrophoretic chamber with planar electrodes in the optical tweezers setup. For the determination of the
dielectrophoretic force the method is based on Green's theorem. A sphere is trapped in the optical tweezers at various
laser powers. An electrical field is applied between the electrodes and thus a dielectrophoretic force is exerted on the
particle. The electric field is increased very slowly until the cell is pulled out from the trap. At this moment the optical
force equals the dielectrophoretic force, which can be theoretically calculated. This algorithm is repeated several times
and the optical force is graphically represented as a function of laser power. Forces of some pN were calculated.
A relatively new method for measuring optically induced forces on microparticles and cells, different from the conventional Brownian motion and viscous drag force calibration methods widely used, is introduced. It makes use of the phenomenon of dielectrophoresis for the calibration of optical tweezers through the dielectrophoretic force calculations. A pair of microelectrodes is fabricated by photolithography on a microscope slide and it is connected to a high-frequency generator. The calibration of the optical tweezers setup is performed by the manipulation of polystyrene beads and yeast cells. Calibration diagrams of the transverse forces versus power are deduced for different cell radii and numerical apertures of the objective lenses. The optical system and the related technique provide a fast and easy method for optical tweezers calibration.
Microscopic particles with varying optical properties may be induced to move in different ways when placed on a sculpted optical potential due to differences in shape, size or polarisability. The separation of red blood cells (erythrocytes) and white blood cells (lymphocytes) is achieved in a non-invasive manner and in the absence of any microfluidic systems using a 'non-diffracting' circularly symmetric Bessel beam. The Bessel beam, which consists of a series of concentric rings, each of equal power and of 3.2μm thickness with a spacing of 2μm around a central maximum of 5μm diameter (and is akin to a rod of light as its propagation distance is 3mm), is directed upward into a sample chamber containing blood. Fluctuations in Brownian motion cause cells to escape from individual rings of the Bessel beam and travel towards the beam centre, where the intensity of the rings increases. However, these cells must be able to overcome the potential barrier of each ring which gets larger toward the central maximum. Lymphocytes - spherical in shape and 7μm in diameter (therefore overlapping two rings) - are transported, due to the gradient force of the optical field, to the beam centre where they are guided upwards and form a vertical stack, whereas erythrocytes re-align on their sides in the outer rings and are then guided upwards, because once aligned they cannot escape the potential barrier and 'lock-in' to that ring. The optical power required for optimal sorting in this static sorter which requires no fluid flow is investigated.
The increasing use of lasers in biomedical research and clinical praxis leads to the development and application of new, non-invasive, therapeutic, surgical and diagnostic techniques. In laser surgery, the theory of ablation dictates that pulsed mid-infrared laser beams exhibit strong absorption by soft and hard tissues, restricting residual thermal damage to a minimum zone. Therefore, the development of high quality 3 μm lasers is considered to be an alternative for precise laser ablation of tissue. Among them are the high quality oscillator-two stages amplifier lasers developed, which will be described in this article. The beam quality delivered by these lasers to the biological tissue is of great importance in cutting and ablating operations.
As the precision of the ablation is increased, the cutting laser interventions could well move to the microsurgery field. Recently, the combination of a laser scalpel with an optical trapping device, under microscopy control, is becoming increasingly important. Optical manipulation of microscopic particles by focused laser beams, is now widely used as a powerful tool for 'non-contact' micromanipulation of cells and organelles. Several laser sources are employed for trapping and varying laser powers are used in a broad range of applications of optical tweezers. For most of the lasers used, the focal spot of the trapping beam is of the order of a micron. As the trapped objects can vary in size from hundreds of nanometres to hundreds of microns, the technique has recently invaded in to the nanocosomos of genes and molecules. However, the use of optical trapping for quantitative research into biophysical processes requires accurate calculation of the optical forces and torques acting within the trap.
The research and development efforts towards a mid-IR microbeam laser system, the design and realization efforts towards a visible laser trapping system and the first results obtained using a relatively new calibration method to calculate the forces experienced in the optical trap are discussed in detail in the following.
The interaction of a TEA (Transversally Excited Atmospheric pressure) corona preionized oscillator double amplifier HF (hydrogen fluoride) laser beam with dentin tissue is reported. Pulses of 39 ns in the wavelength range of 2.65-3.35 μm and output energies in the range of 10-45 mJ, in a predominantly TEM00 beam were used to interact with dentin tissue. Ablation experiments were conducted with the laser beam directly focused on the tissue. Several samples of freshly extracted human teeth were used, cut longitudinally in facets of about 1mm thick and stored in phosphate buffered saline after being cleaned from the soft tissue remains. The experimental data (ablation thresholds, ablation rates) are discussed with respect to the ablation mechanism(s). Adequate tissue removal was observed and the ablation behavior was, in the greates part of the available fluences, almost linear. From the microscopic examination of teh samples, in a scanning electron microscope (SEM), the irradiated surfaces displayed oval craters (reflecting the laser beam shape) with absence of any melting or carbonization zone. It is suggested that the specific laser removes hard tissue by a combined photothermal and plasma mediated ablation mechanism, leaving a surface free from thermal damage and with a well-shaped crater.
In this study a frustrated total internal reflection (FTIR) Q-switched and free-running Er:YAG laser, as well as a novel design transversally excited atmospheric pressure (TEA) oscillator-double amplifier corona preionised high beam quality Hydrogen-Fluoride (HF) laser system, all developed in our lab, were used in dentin ablation experiments. In the case of the Er:YAG laser, pulses of 190 ns in Q-switched operation and of 80 μs pulse width in free-running operation at 2.94 μm were used, while HF laser pulses of 39 ns in the wavelength range of 2.6-3.1 μm in a predominantly TEM00 beam were also used to interact in vitro with dentin tissue. Several samples of freshly extracted human teeth were used, cut longitudinally in facets of 0.4-1.5 mm thick. Ablation experiments were conducted with the laser beam directly focused on the tissue or after being waveguided through suitable mid-IR fiber/waveguide alternatively ended with quartz end-sealing caps. The correlation between the various laser beam parameters, as wavelength, pulse duration, repetition rate, energy and spatial distribution of the beam profile and the ablative characteristics (ablation rates, tissue surface morphology) of dentin surface were investigated.
The hard tissue laser ablation and the relevant laser applications on dental tissues have attracted several investigators, since the ablation efficiency, the lateral thermal and/or mechanical damage and the final surface characteristics must be optimised, in order to fulfil the efficient and safe removal of pathological dental areas in a clinical environment. For example, it was theoretically predicted that the lateral tissue thermal damage might be limited when the laser intensity is high and the interaction time is short. In this case the Q-switched Er:YAG laser operating with a pulse length below the thermal relaxation time of the irradiated tissue, is considered to be very effective. Therefore, the efficacy of the Q-switched Er:YAG laser, versus the free-running operation mode, on dentine ablation was studied, as well as the morphological changes at the irradiated dentine surface. Additionally, the interaction of nanosecond UV laser pulses and IR/visible picosecond laser pulses with hard tissue samples was investigated for comparative evaluation of the different laser ablation mechanisms.
An intensive development effort is going on throughout the world, in order to develop reliable lasers emitting in the 3 μm wavelength range, as this wavelength is strongly absorbed by the water and the other components of soft and hard tissue and thus its use is important in various medical applications. In parallel, good flexible delivery systems, in the mid-IR wavelength region, are needed in order to deliver the laser beam to the tissue. In this work High Power (HP) Oxide Glass fibers are tested for determining their maximum capabilities in delivering free-running and Q-switched Er:YAG laser radiation at 2.94 μm. Oxide glass is a new material in solid core fiber fabrication for medical applications, and its performance at the wavelength of 2.94 μm, for various laser characteristics is of great importance. Also a comparison is made between results obtained with the two different Er:YAG lasers, afree-running and a Q-switched one, and the results obtained at 2.78 μm, with a chemical HF laser.
Transmission measurements of Q-switched Er:YAG laser radiation, through cyclic olefin polymer-coated silver hollow glass waveguides, were performed under straight and bent conditions and the beam quality at the output of the waveguide was studied.
The effect of the 2.94 μm Er:YAG laser radiation propagation through sapphire fibers with diameters varying from 250 μm to 550 μm, on the quality of the laser beam is investigated. A comparison was made between the fibers performance in free-running and Q-switched Er:YAG laser radiation.
In the last few years, there has been an increasing interest for the 3 m laser radiation in various medical applications, as this wavelength is strongly absorbed by the water and the other components of soft and hard tissue. An intensive development effort is going on throughout the world, in order to develop reliable lasers emitting in the 3 m wavelength range. Our laser development effort with the Q-switched Er:YAG laser is briefly described in this article. Additionally for medical applications there is a great demand for good flexible delivery systems, in the mid-IR wavelength region. In this work the radiation transmission of a Q-switched Er:YAG laser, emitting at 2.94 m, through high power (HP) oxide glass fibers of 450 ?m core diameter was studied. Attenuation measurements were obtained as a function of the laser energy input and as a function of curvature, at 90o, 180o and 360o bending angle. The output beam quality was studied using a beam profiler. Experiments with the same delivery system transmitting free-running Er:YAG laser radiation, were performed for comparison. The results are promising for the delivery of Q-switched Er:YAG laser radiation, as the fibers exhibited attenuation of 0.7 dB/m, and no damage of them was observed.
The determination of the tissue optical parameters and the study of light propagation in multicomponent tissues is an important tool for real time laser treatment dosimetry and non-invasive laser diagnostics. The purpose of this work was the evaluation of a small portable spectrophotometry system, for non-invasive laser dosimetry, as compared to a more sophisticated, tabletop, spectroscopic system. The small portable system consists of an optical spectrum analyzer, an oscilloscope and appropriate fibers, while the complete laser spectroscopy system consists of a spectrograph, an integrating sphere and a photodiode array as a detector element. The determination of the optical properties of soft and hard biological tissues -- in vitro -- was performed with both systems. Spectral information obtained, the appropriate mathematical modeling and the comparative results are discussed.
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