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.     

Evaluation of a porcine lens and fluorescence assay approach for in vitro ocular toxicological investigations

Cell biology, as monitored with the fluorescent indicator dyes Alamar Blue and 5-carboxyfluorescein diacetate acetoxymethyl ester (CFDA-AM), and lens optical quality, as measured with an in vitro scanning laser system, have been used to evaluate in vitro the condition of porcine lenses after being placed in a culture medium. The measurements, beginning from week one of culture, were compared statistically. Optical quality and cellular viability, as measured with either dye, were unchanged in lenses that had been maintained for 6 weeks in modified M199 medium. Some lenses were treated with 0.152J/cm(2) UVB radiation, and a decline was observed after 48 hours in both optical and metabolic capabilities, as indicated by a decreased capacity of the lenses to reduce Alamar Blue. The measurements with CFDA-AM did not show complete concordance with the other indicators of lens health after UV treatment, making this dye less reliable as applied currently to lens cultures. Overall, the findings suggest that porcine lenses can be maintained for weeks in culture, and that their condition can be evaluated quantitatively by assays that probe cellular functions and optical properties. Such a system should prove valuable for in vitro ocular pharmacotoxicological research.     

High resolution dual laser flow cytometry.

Flow cytometers based on optical sensing utilize external light sources and fluorescent dyes to measure one or more specific components or properties of individual cells or subcellular particles in liquid suspension. To provide for independent excitation of two dyes used in double staining experiments we have constructed a high resolution flow cytometer that uses two laser beams to provide two wavelengths of excitation. These beams are separated spatially so that cells flow through them sequentially, with a time separation of about 20 musec. Since the dyes are excited sequentially their emission occurs at different times and their emission spectra may overlap without causing any difficulty in analysis. We have developed new light collection optics that permit up to four measurements to be made on each cell. This approach greatly increases the number of dye combinations that can be used in flow cytometry, thus removing a significant limitation of single illumination instruments.     

Extended application of flow microfluorometry by means of dual laser excitation

Summary A dual laser beam excitation device for flow analysis of biological particles has been developed. The aid of this arrangement is to increase the range of fluorescent agents employed so far in quantitative and qualitative cytochemistry. Combining an argon ion and a helium-cadmium laser two color fluorescence measurements were performed employing propidium iodide as a DNA stain and fluorescamine which stains total protein in fixed cells. Energy transfer processes between the antibiotic and DNA specific dye mithramycin and propidium iodide both being bound to nuclear chromatin were analyzed. Utilization of energy transfer processes is generally discussed as a mean to extract information about the structure and conformation of nuclear chromatin in situ. The application of a crypton ion laser with three lines near 400 nm and a single line at 350 nm having a light output in each range of nearly one Watt gives the opportunity of utilizing DNA fluorochromes which have an excitation maximum in the deep blue region. DNA spectra are shown employing mithramycin, the benzimidazol derivative 33258 (Hoechst) and the indol compound DAPI which has a high DNA specifity combined with a great stability under UV illumination. By separating two focussed laser beams at their intereecting points with the liquid sample stream the trajectory of each flowing cell crosses the beams sequentially, which causes a solitary dual excitation of each cell. The advantages of a solitary excitation device compared with a simultaneous one is discussed.This work has been supported by the ministry of research and technology (FRG), contract No. 01VH015-B13MT 225a     

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.     

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.     

Cell cytometry with a light touch: sorting microscopic matter with an optical lattice

Lab-on-a-chip design is a key technology for increasing both the reliability and the functionality of many different preparation and diagnostic techniques in biomedicine. The drive towards ever more integrated lab-on-a-chip designs requires increasingly complex microfluidic systems. In order to build these systems, non-invasive actuators such as pumps, filters and mixers are required. We have demonstrated microfluidic sorting based on a 3D interference pattern, formed from multiple coherent laser beams, which has the potential to fulfil all the above criteria. By interfering five laser beams from a fibre laser at 1070 nm, we have formed a 3D optical lattice. When particles flow through the optical lattice their trajectories depend upon the force exerted on the particle by the optical lattice, in combination with the drag force exerted by the fluid flow. Hence, with the strength of a particle's interaction with the lattice determining the total force exerted upon it, its trajectory is determined by its physical properties. These properties include refractive index, size and shape, giving a range of criteria with which to sort an analyte. We have shown separation at 45 degrees of polymer from silica microspheres (by refractive index), the separation of protein microcapsules and the sorting of erythrocytes from lymphocytes. The interference pattern can be tailored to the particles and if a blockage occurs, the laser can simply be switched off, unlike solid-state micro-sorters, so that no jamming occurs. Efficiencies in excess of 95% have been achieved.     

I5S: wide-field light microscopy with 100-nm-scale resolution in three dimensions

A new type of wide-field fluorescence microscopy is described, which produces 100-nm-scale spatial resolution in all three dimensions, by using structured illumination in a microscope that has two opposing objective lenses. Illumination light is split by a grating and a beam splitter into six mutually coherent beams, three of which enter the specimen through each objective lens. The resulting illumination intensity pattern contains high spatial frequency components both axially and laterally. In addition, the emission is collected by both objective lenses coherently, and combined interferometrically on a single camera, resulting in a detection transfer function with axially extended support. These two effects combine to produce near-isotropic resolution. Experimental images of test samples and biological specimens confirm the theoretical predictions.     

MAGNESIUM-RICH INTRALENSAR STRUCTURES IN SCHIZOCHROAL TRILOBITE EYES

Abstract:  The interpretation of the lenses of schizochroal trilobite eyes as aplanatic doublets by Clarkson and Levi-Setti over 30 years ago has been widely accepted. However, the means of achieving a difference in refractive index across the interface between the two parts of each lens to overcome spherical aberration has remained a matter of speculation and lately it has been argued that the doublet structure itself is no more than a diagenetic artefact. Recent advances in technologies for imaging, chemical analysis and crystallographic characterization of minerals at high spatial resolutions have enabled a re-examination of the structure of calcite lenses at an unprecedented level of detail. The lenses in the eyes of the specimen of Dalmanites sp. used in the original formulation of the aplanatic doublet hypothesis are shown to have undergone diagenetic alteration, but its products reflect original differences in mineral chemistry between the upper lens unit and lower intralensar bowl. The turbidity of the bowl and of the core within the upper part of the lens are the result of their greater microporosity and abundance of microdolomite inclusions, both of which were products of diagenetic replacement of original magnesian calcite in these areas. Such a difference in magnesium concentration in the original calcite has long been postulated as one of the ways by which the interface between these lens units could have produced an aberration-free image and the present study provides the first direct evidence of such a chemical contrast, thus confirming the doublet hypothesis.     

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.     

Coherent convergent-beam time-resolved X-ray diffraction

The use of coherent X-ray lasers for structural biology allows the use of nanometre diameter X-ray beams with large beam divergence. Their application to the structure analysis of protein nanocrystals and single particles raises new challenges and opportunities. We discuss the form of these coherent convergent-beam (CCB) hard X-ray diffraction patterns and their potential use for time-resolved crystallography, normally achieved by Laue (polychromatic) diffraction, for which the monochromatic laser radiation of a free-electron X-ray laser is unsuitable. We discuss the possibility of obtaining single-shot, angle-integrated rocking curves from CCB patterns, and the dependence of the resulting patterns on the focused beam coordinate when the beam diameter is larger or smaller than a nanocrystal, or smaller than one unit cell. We show how structure factor phase information is provided at overlapping interfering orders and how a common phase origin between different shots may be obtained. Their use in refinement of the phase-sensitive intensity between overlapping orders is suggested.     

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.     

Design and application of a versatile triple-laser cell and chromosome sorter

A high-resolution triple-laser sorter was designed and constructed to provide flexible switchover and high-resolution sorting of cells or chromosomes with any combination of one, two, or three lasers. These features provide a central facility instrument that currently serves multiple users and analyzes different stain combinations with minimal switchover effort between experiments. Improved optics and mounts that focus the three laser beams independently are able to resolve beads and chromosomes better than our previously reported dual-laser sorter. An improved signal collection unit with electronically controlled reference positions can be focused more quickly and precisely for any signal combination. A removable dye laser extends the range of usable fluorochrome labels. A rapid sheath switchover permits sorting of sterile cells and sterile chromosomes sequentially without additional sterilization or reservoir sheath change. Improved dual-laser chromosome resolution is at least as good, analyzing 8,000 chromosomes/s, compared to the previous dual-laser bench at 2,000/s. Stimulated and unstimulated peripheral blood lymphocytes were analyzed according to simultaneous measurements of cell surface receptors labeled with a fluoresceinated neuropeptide and a Texas red-labeled antibody as well as DNA content during the cell cycle. These results demonstrate the broad range of potential applications of this triple-laser system.     

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.     

A Simple Computer-aided Device for Monitoring Activity of Small Mammals and Insects

A simple computer-aided device was developed to record the activity of small animals. The device comprises of aktographs, opto-couplers, opto-amplifier units, a data acquisition unit (‘Activity Monitor’) and a Pentium processor based personal computer (PC) attached to an addition parallel printer port. The aktographs are devices which facilitate organisms to exhibit its natural activity. The aktographs for fruit flies, ants and mice are tubes with food supply and moisture, petri dishes adequately ventilated with arrangements for constant supply of water, sugar or honey solution, and cages attached with running wheels, respectively. Two mutually perpendicular beams of infra-red (IR) emission form a cross-hair detection system across the aktographs and an interruption to these beams creates an electrical signal (‘transition’) which is amplified using the opto-amplifier unit and sent to the activity monitor which consists of shift registers. The number of moments the IR beams are interrupted is taken as a measure of the amount of activity, based on the assumption that a more active animal would interrupt the IR beams more often compared to a less active one. Programs were written in C++, which facilitated data acquisition and graphical representation in terms of a daily plot of amount of activity as a function of time of the day, and actograms of daily activity were arranged chronologically one below the other. The activity patterns of individual animals (fruit fly, ants, and mice) were monitored using this device. Based on the performance and features of this device, we feel that with some improvement in the software this can substitute some of the existing activity recording devices, which are costly and not flexible.     

A Simple Computer-aided Device for Monitoring Activity of Small Mammals and Insects

A simple computer-aided device was developed to record the activity of small animals. The device comprises of aktographs, opto-couplers, opto-amplifier units, a data acquisition unit ('Activity Monitor') and a Pentium processor based personal computer (PC) attached to an addition parallel printer port. The aktographs are devices which facilitate organisms to exhibit its natural activity. The aktographs for fruit flies, ants and mice are tubes with food supply and moisture, petri dishes adequately ventilated with arrangements for constant supply of water, sugar or honey solution, and cages attached with running wheels, respectively. Two mutually perpendicular beams of infra-red (IR) emission form a cross-hair detection system across the aktographs and an interruption to these beams creates an electrical signal ('transition') which is amplified using the opto-amplifier unit and sent to the activity monitor which consists of shift registers. The number of moments the IR beams are interrupted is taken as a measure of the amount of activity, based on the assumption that a more active animal would interrupt the IR beams more often compared to a less active one. Programs were written in C++, which facilitated data acquisition and graphical representation in terms of a daily plot of amount of activity as a function of time of the day, and actograms of daily activity were arranged chronologically one below the other. The activity patterns of individual animals (fruit fly, ants, and mice) were monitored using this device. Based on the performance and features of this device, we feel that with some improvement in the software this can substitute some of the existing activity recording devices, which are costly and not flexible.     

NONSURGICAL TREATMENT OF CONVERGENT STRABISMUS

It is generally agreed that surgical treatment of convergent strabismus should be withheld until all other less traumatic approaches have proved ineffectual. There are four categories of nonsurgical treatment.One is psychiatric. Too often psychiatric problems in the causation of convergent strabismus are either overlooked or unrecognized.Another is the proper employment of optical devices. For example, spectacle lenses to eliminate the need for excessive accommodation with its associated convergence excess, and the employment of prisms in the lenses to permit the two eyes to see as a unit even though they may not be properly anatomically oriented.Another kind of treatment is orthoptics, the use of exercises and rather complex optical equipment in a laboratory to train the patient in coordination between the two eyes.Treatment with drugs is based on the fact that certain drugs reduce the effort necessary for accommodation (much as eye-glasses do) and therefore lessen the stimulus toward convergence which may possibly tend toward the development of convergent strabismus.     

Hybrid fluorescence and electron cryo-microscopy for simultaneous electron and photon imaging

Integration of fluorescence light and transmission electron microscopy into the same device would represent an important advance in correlative microscopy, which traditionally involves two separate microscopes for imaging. To achieve such integration, the primary technical challenge that must be solved regards how to arrange two objective lenses used for light and electron microscopy in such a manner that they can properly focus on a single specimen. To address this issue, both lateral displacement of the specimen between two lenses and specimen rotation have been proposed. Such movement of the specimen allows sequential collection of two kinds of microscopic images of a single target, but prevents simultaneous imaging. This shortcoming has been made up by using a simple optical device, a reflection mirror. Here, we present an approach toward the versatile integration of fluorescence and electron microscopy for simultaneous imaging. The potential of simultaneous hybrid microscopy was demonstrated by fluorescence and electron sequential imaging of a fluorescent protein expressed in cells and cathodoluminescence imaging of fluorescent beads.     

An improved no-moving-parts video-rate confocal microscope

Several years ago our research program developed a video-rate confocal microscope with no moving parts, based on synchronizing and aligning the scan of an image dissector tube (IDT) with the light returning from a microscope stage that has been acousto-optically scanned by a laser beam. Improvements on the original system have recently been completed. The laser power has been substantially increased and the laser scan is now brought into the Nikon Diaphot inverted microscope through the epi-illumination port. Aberrations in the scanned beam have been reduced by performing the beam shaping required by the acousto-optic deflectors using prisms instead of cylindrical lenses. The IDT is located at the side camera output port at the end of a simple, efficient light path. The new system is described in detail and results obtained using the microscope in reflection and fluorescence mode are presented.     

Assessing Confocal Microscopy Systems for Purchase

Confocal laser scanning microscopy now has so many functions and applications that choosing a new multiuser confocal laser microscopy system can be a daunting task, particularly for a first-time buyer and new users of confocal microscopy. How does one determine which features are most appropriate for any particular laboratory, application, or imaging environment? Each confocal microscopy system has its own individual advantages and limitations, which ultimately defines its market niche. Here, we describe the features that differentiate the four confocal microscopy systems we assessed. The decisive factors in choosing a confocal microscopy system for our anatomical laboratory were user-friendly software for on-line acquisition and off-line processing; the working distances of objective lenses; applicability for a multiuser environment; interactions with company representatives; and turnaround times for questions, service, and accessories.