Alignment and focusing unit for dual-laser excitation in the fluorescence-activated cell sorter

The two laser beams in a dual-laser fluorescence-activated cell sorter FACS-II can be aligned and focused independently on the sample stream with an additional unit, which can be fitted easily on the optical bench of the FACS. The unit consists of two spherical lenses, which have been mounted in separate holders and can be moved in three directions by way of micrometer gauges. The lenses, which have different focal lengths, have been cut off on one side so each laser beam only passes one lens. The setup has been tested using the flow analysis of a suspension of double-stained chicken red blood cells. The histograms of both fluorescence signals showed normal distributions with a coefficient of variation of approximately 6%. After willful interference with the adjustments, the laser beams could be readily readjusted within five minutes.     

Focusing of heavy ion beams by a high-current plasma lens

Results are presented from studies of the focusing of wide-aperture low-energy (100–400 eV) and moderate-energy (5–25 keV) beams of heavy-metal ions by a high-current electrostatic plasma lens. It is found experimentally that, because of the significant electron losses, the efficient focusing of such beams can be achieved only if the external potentials at the plasma-lens electrodes are maintained constant. Static and dynamic characteristics of the lens are studied under these conditions. It is shown that, as the beam current and the electrode voltage increase, the maximum electrostatic field in the lens tends to a certain limiting value because of the increase in the spatial potential near the lens axis. The role of spherical and moment aberrations in the focusing of wide-aperture low-divergence ion beams is revealed. It is shown that, even when spherical aberrations are minimized, unremovable moment aberrations decrease the maximum compression ratio of a low-energy heavy-ion beam because of the charge separation of multiply charged ions in the focal region. At the same time, as the ion energy increases, the role of the moment aberrations decreases and the focusing of high-current heavy-ion beams by a plasma lens becomes more efficient than the focusing of light-ion (hydrogen) beams. This opens up the possibility of using electrostatic plasma lenses to control ion beams in high-dose ion implanters and high-current accelerators of heavy ions.     

Space-charge lens for focusing negative ion beams by means of an additional electron ionizer

A space-charge lens created at the Institute of Physics, National Academy of Sciences of Ukraine, to focus negative ion beams using an additional electron ionizer is investigated. In the previous version of the lens, in which the gas was ionized by the ion beam itself, the focal power was quite high (the focal length was ? ≤ 20 cm) but the gas pressure was too great (P ～ 10^?3 torr), which resulted in significant charge-exchange losses of the beam ions. The experimental and theoretical study reported here shows that the use of a 100-eV electron beam as an auxiliary ionizer allows the working pressure in the lens to be significantly reduced. As a result, a simple, inexpensive, and efficient lens has been developed that can be used in systems for transportation of negative ion beams.     

Spherical aberration of the fish lens: interspecies variation and age

Summary Spherical aberration of the eyes of a spectrum of freshwater fishes was determined by photographing the refractive effects of excised crystalline lenses on multiple parallel split laser beams. In general, spherical aberration is minimized by the developmentally related variation in lens refractive index. However, spherical aberration is marked and non-monotonic in a non-visual species such as the bullhead. Furthermore, the size and variability of the aberration appears to be related to visual need as indicated by diet and feeding habits. For example, the lenses of predatory sight feeders such as the pike (Esox lucius) or rock bass (Ambloplites rupestris) are optically superior to that of an omnivorous feeder as the carp (Cyprinus carpio).The effect of age was tested by examining rock bass lenses from fish two to seven years of age. Lens quality, as indicated by the amount of change in posterior focal length for beams of varying eccentricity from the optic axis, is optimum in lenses from five year old fish. The significance of this variation in lens quality is uncertain and requires further study with greater attention to specimens of advanced age.     

Wave optical simulation of retinal images in laser safety evaluations

Lasers with wavelengths in the visible and near infrared region, pose a potential hazard to vision as the radiation can be focused on the retina. The laser safety standard IEC 60825–1:2014 provides limits and evaluation methods to perform a classification for such systems. An important parameter is the retinal spot size which is described by the angular subtense of the apparent source. In laser safety evaluations, the radiation is often described as a Gaussian beam and the image on the retina is calculated using the wave optical propagation through a thin lens. For coherent radiation, this method can be insufficient as the diffraction effects of the pupil aperture influence the retinal image. In this publication, we analyze these effects and propose a general analytical calculation method for the angular subtense. The proposed formula is validated for collimated and divergent Gaussian beams.     

Space charge lens for focusing negative-ion beams

A space charge lens is proposed to focus intense beams of negative hydrogen ions. The focal length of the lens is determined as a function of the parameters of the beam and the gas medium. It is demonstrated experimentally that the lens efficiently focuses H-ion beams with currents of up to ～30 mA and energies of ～10 keV. The measured focal lengths are in good agreement with the calculated ones.     

Long-Distance Axial Trapping with Focused Annular Laser Beams

Focusing an annular laser beam can improve the axial trapping efficiency due to the reduction of the scattering force, which enables the use of a lower numerical aperture (NA) objective lens with a long working distance to trap particles in deeper aqueous medium. In this paper, we present an axicon-to-axicon scheme for producing parallel annular beams with the advantages of higher efficiency compared with the obstructed beam approach. The validity of the scheme is verified by the observation of a stable trapping of silica microspheres with relatively low NA microscope objective lenses (NA = 0.6 and 0.45), and the axial trapping depth of 5 mm is demonstrated in experiment.     

A novel method of focus sensing for the laser scanner microscope

An unconventional approach to the focus-sensing problem in a high-speed cell scanner has resulted in a method of directly detecting the position of the cell layer. This is done by using two coaxial but axially shifted beams, one focused on either side of the cell layer. The modulation induced in each beam by the cell's fine structure is compared electronically to yield the focus-error signal. The beams are orthogonally polarized, therefore separable, and are independently detected. The two detector-output signals are used to form both the focus-error signal and the scanner's primary data signal. This focus-sensing technique has been analytically modeled and experimentally demonstrated for a high-numerical-aperture system. In that system, the depth of focus is less than the thickness of the cell layer. It is shown that a lens fabricated from a birefringent crystal is an attractive means of forming the required orthogonally polarized axially shifted beams.     

Axially‐confined in vivo single‐cell labeling by primed conversion using blue and red lasers with conventional confocal microscopes

Green‐to‐red photoconvertible fluorescent proteins have been found to undergo efficient photoconversion by a new method termed primed conversion that uses dual wave‐length illumination with blue and red/near‐infrared light. By modifying a confocal laser‐scanning microscope (CLSM) such that two laser beams only meet at the focal plane, confined photoconversion at the axial dimension has been achieved. The necessity of this custom modification to the CLSM, however, has precluded the wide‐spread use of this method. Here, we investigated whether spatially‐restricted primed conversion could be achieved with CLSM without any hardware modification. We found that the primed conversion of Dendra2 using a conventional CLSM with two visible lasers (473 nm and 635 nm) and a high NA objective lens (NA, 1.30) resulted in dramatic restriction of photoconversion volume: half‐width half‐maximum for the axial dimension was below 5 μm, which is comparable to the outcome of the original method that used the microscope modification. As a proof of this method's effectiveness, we used this technique in living zebrafish embryos and succeeded in revealing the complex anatomy of individual neurons packed between neighboring cells. Because unmodified CLSMs are widely available, this method can be widely applicable for labeling cells with single‐cell resolution.     

Multiple-Wavelength Focusing and Demultiplexing Plasmonic Lens Based on Asymmetric Nanoslit Arrays

A multiple-wavelength focusing and demultiplexing plasmonic lens based on asymmetric nanoslit arrays is designed. The nanoslit arrays are perforated in a gold film and act as metal–insulator–metal plasmonic waveguides. By manipulating the widths of the slit arrays, the plasmonic lens can concentrate two incident plane wave beams to two separated focal points corresponding to their wavelengths. The full wave simulation is performed to verify the designed lens. This work provides a way to design more compact and integrated wavelength-division multiplexing plasmonic devices for nanophotonic communication and spectral imaging.     

The effects of toxicological agents on the optics and mitochondria of the lens and the mitochondria of the corneal epithelium

This review describes how the morphology and distribution of the mitochondria of the epithelium and the superficial fibre layers of the lens were studied using confocal scanning laser microscopy. This research was correlated with an effort to use the optical properties of the intact lens in culture as a proxy for the cornea in measuring ocular toxicity. In turn, this work led to the confocal study of the in vitro and then the in vivo cornea and their possible use in using confocal microscopy to evaluate the effect of various treatments on the integrity of the surface of the eye. Finally, confocal examination of the mitochondria of the lens has provided an avenue to the study of mitochondrial dynamics.     

Dual‐color STED super‐resolution microscope using a single laser source

STED (stimulated emission depletion) microscopy is one of the most promising super‐resolution fluorescence microscopies，due to its fast imaging and ultra‐high resolution. In this paper, we present a dual‐color STED microscope with a single laser source. Polarization beam splitters are used to separate the output from a supercontinuum laser source into four laser beams, including two excitation beams (488, 635 nm) and two depletion beams (592, 775 nm). These four laser beams are then used to build a low cost dual‐color STED system to achieve a spatial resolution of 75 nm in cell samples.     

Action Spectrum for Photobleaching of Human Lenses by Short Wavelength Visible Irradiation

Purpose

Cataract is the world-leading cause of blindness. In search for a new treatment of cataract we have found that the yellow discolouration of aged human lenses can be photobleached using a non-invasive, infra-red, femtosecond laser treatment. These results were presented in an earlier PlosOne publication. The objective of the study was to characterize the single-photon photobleaching action spectrum of the aged human lens in vitro.

Methods

Ninety-one human donor lenses were irradiated with continuous wave laser light at 375, 405, 420, 445, 457 or 473 nm. Photobleaching was monitored by photography and transmission measurements.

Results

The action spectrum peaked at 420 nm followed by, in order of decreasing effect, 445, 457, 473, 405 and 375 nm. Younger and less absorbent lenses showed smaller changes than older and more absorbent lenses. There was a dose-dependent increase in lens transmission with increasing laser irradiation.

Conclusions

For a 75 year old lens an effect corresponding to elimination of 15 years or more of optical ageing was obtained. This study of the spectral characteristics and intensity needed to bleach the human lens with single-photon laser effects found an action-spectrum peak at 420 nm tailing gradually off toward longer wavelengths and more steeply toward shorter wavelengths. The results may be used to guide experiments with two-photon bleaching.     

The Infrared (Far Terahertz) Generation by Nonlinear Interactions of Two Visible Laser Beams in a Metallic Background: Infrared Surface Plasmon Effect

This paper presents an investigation of infrared (IR) radiation generation by nonlinear interaction of two visible laser beams in a metallic background. Two laser beams of Gaussian and Laguerre Gaussian (LG) profiles and background metals such as silver, copper, gold, and aluminum are utilized for IR generation. Effects of laser beam characteristics and structural properties of metals on the evolution of IR electric field amplitude are examined. Considering laser frequencies in the non-transparent region give rises to generation of IR surface plasmon (IRSP). An optimized relation is proposed for achieving efficient surface plasmon waves on a metal surface.

     

Runaway electron beams in the gas discharge for UV nitrogen laser excitation

The review of the methods for obtaining the runaway electron beams in the gas discharge is performed. The new method is offered, using which the beam is first formed in a narrow gap (∼1 mm) between the cathode and the grid and then it is accelerated by the field of the plasma column of the anomalous self-sustained discharge in the main gap (10–20 mm long). The electron beams with an energy of about 10 keV and current density of 10³ A/cm² at a molecular nitrogen pressure of up to 100 Torr have been obtained experimentally. The results of research of the UV nitrogen laser with an excitation via runaway electron beam and radiation of energy of ∼1 mJ are given. The UV nitrogen laser generation with the energy of ∼1 mJ has been obtained by the runaway electron beams.     

Effect of laser light treatment on some biochemical and physiological processes in seeds and seedlings of white lupine and faba bean

The aim of present study was to determine the changes of some biochemical and physiological processes, which occurred in seeds and seedlings of white lupine and faba bean after pre-sowing treatment with laser beams. It was found this treatment of seeds considerably increased the activity of amylolytic enzymes in seeds of both plants. The greatest differentiation of the enzymatic activity was noticed after 120?h from the time of sowing but the activity of these enzymes in the seeds of both tested plants was similar and it had the same course in time. The irradiated seeds of white lupine and faba bean had higher fresh weight at the time of imbibition than the seeds which were not treated with laser beams. It resulted in earlier and more uniform germination. The concentration of free radicals increased considerably in the seeds pre-treated with laser beams and the largest increase in seeds of both plant species was noticed after five exposures to laser beams. Treating seeds with laser beams considerably increased the amount of indole-3-acetic acid (IAA) in the germinating seeds. Three exposures of seeds caused the largest increase of this plant hormone content in the seeds. The activity of IAA in faba bean was slightly higher than in white lupine seeds. Pre-sowing stimulation with laser had a positive influence on the growth and development of seedlings, which had longer hypocotyl and roots in comparison to seedlings which grew from non irradiated seeds.     

Fabrication and Operation of a Nano-Optical Conveyor Belt

The technique of using focused laser beams to trap and exert forces on small particles has enabled many pivotal discoveries in the nanoscale biological and physical sciences over the past few decades. The progress made in this field invites further study of even smaller systems and at a larger scale, with tools that could be distributed more easily and made more widely available. Unfortunately, the fundamental laws of diffraction limit the minimum size of the focal spot of a laser beam, which makes particles smaller than a half-wavelength in diameter hard to trap and generally prevents an operator from discriminating between particles which are closer together than one half-wavelength. This precludes the optical manipulation of many closely-spaced nanoparticles and limits the resolution of optical-mechanical systems. Furthermore, manipulation using focused beams requires beam-forming or steering optics, which can be very bulky and expensive. To address these limitations in the system scalability of conventional optical trapping our lab has devised an alternative technique which utilizes near-field optics to move particles across a chip. Instead of focusing laser beams in the far-field, the optical near field of plasmonic resonators produces the necessary local optical intensity enhancement to overcome the restrictions of diffraction and manipulate particles at higher resolution. Closely-spaced resonators produce strong optical traps which can be addressed to mediate the hand-off of particles from one to the next in a conveyor-belt-like fashion. Here, we describe how to design and produce a conveyor belt using a gold surface patterned with plasmonic C-shaped resonators and how to operate it with polarized laser light to achieve super-resolution nanoparticle manipulation and transport. The nano-optical conveyor belt chip can be produced using lithography techniques and easily packaged and distributed.     

Vortex electron dynamics in a high-current plasma lens

An analytic study is made of the following problems: the instability of a plasma against the excitation of vortex turbulence, the turbulence saturation amplitude, the types and spatial structures of the nascent vortices, and their nonlinear growth rates in an electrostatic plasma lens for focusing high-current ion beams.     

Twin-beam laser velocimeter for the investigation of spermatozoon motility

Previous laser light-scattering studies of spermatozoon motility have been hampered by the large, asymmetric shape of spermatozoa, which causes difficulties in the interpretation of intensity fluctuations in the light scattered from a single laser beam. This paper describes an experimental arrangement for measuring the distribution of transit times for swimming spermatozoa using two slightly separated, focused laser beams. The theory of operation of the instrument is developed to enable the analysis of the experimentally obtained cross-correlation functions. The effects of the pronounced spermatozoon asymmetry and associated intensity modulation in the scattered light are also investigated and shown to be negligible for the twin beam experimental arrangement, provided that the swimming speed distribution has a coefficient of variation (sigma/upsilon greater than 0.1. Results obtained using this apparatus are presented for the velocity distribution of spermatozoa from a variety of bulls.     

Detection and measurement of a single blood cell surface antigen by thermal lens microscopy

A highly sensitive method for detection of antigens on the surface of a single blood cell using thermal lens microscopy is described. Colloidal gold, coated with antibody, was used to stain membrane antigens of leukocytes. Human leukocyte antigens on the lymphocytes and mononuclear leukocytes were observed by new thermal lens microscopy, which involves spectrometry using a laser-induced thermal-lens effect. Antigens of HLA-A, -B, and -C loci on the lymphocytes were identified and quantitated using a single cell. The image of HLA-A, -B, and -C antigen distribution on a mononuclear leukocyte was obtained. Our laser microscope, newly devised for measuring convex surface cells, is a powerful analytical tool for detecting and quantitating localized antigens in a single cell and/or cell-surface-associated molecules.