All-optical constant-force laser tweezers

Optical tweezers are a powerful tool for the study of single biomolecules. Many applications require that a molecule be held under constant tension while its extension is measured. We present two schemes based on scanning-line optical tweezers to accomplish this, providing all-optical alternatives to force-clamp traps that rely on electronic feedback to maintain constant-force conditions for the molecule. In these schemes, a laser beam is rapidly scanned along a line in the focal plane of the microscope objective, effectively creating an extended one-dimensional optical potential over distances of up to 8 microm. A position-independent lateral force acting on a trapped particle is created by either modulating the laser beam intensity during the scan or by using an asymmetric beam profile in the back focal plane of the microscope objective. With these techniques, forces of up to 2.69 pN have been applied over distances of up to 3.4 microm with residual spring constants of <26.6 fN/microm. We used these techniques in conjunction with a fast position measurement scheme to study the relaxation of lambda-DNA molecules against a constant external force with submillisecond time resolution. We compare the results to predictions from the wormlike chain model.     

High-sensitivity DNA detection with a laser-excited confocal fluorescence gel scanner

A high-sensitivity, laser-excited confocal fluorescence gel scanner has been developed and applied to the detection of fluorescently labeled DNA. An argon ion laser (1-10 mW at 488 nm) is focused in the gel with a high-numerical aperture microscope objective. The laser-excited fluorescence is gathered by the objective and focused on a confocal spatial filter, followed by a spectral filter and photodetector. The gel is placed on a computer-controlled scan stage, and the scanned image of the gel fluorescence is stored and analyzed in a computer. This scanner has been used to detect DNA separated on sequencing gels, agarose mapping gels and pulsed field gels. Sanger sequencing gels were run on M13mp18 DNA using a fluoresceinated primer. The 400-microns-thick gels, loaded with 30 fmol of DNA fragments in 3-mm lanes, were scanned at 78-microns resolution. The high resolution of our scanner coupled with image processing allows us to read up to approximately 300 bases in four adjacent sequencing lanes. The minimum band size that could be detected and read was approximately 200 microns. This instrument has a limiting detection sensitivity of approximately 10 amol of fluorescein-labeled DNA in a 1 x 3-mm band. In applications to agarose mapping gels, we have exploited the fact that DNA can be prestained with ethidium homodimer, followed by electrophoresis and fluorescence detection to achieve picogram sensitivity. We have also developed methods using both ethidium homodimer and thiazole orange staining which permit two-color detection of DNA in one lane.(ABSTRACT TRUNCATED AT 250 WORDS)     

An evaluation of confocal versus conventional imaging of biological structures by fluorescence light microscopy

Scanning confocal microscopes offer improved rejection of out-of-focus noise and greater resolution than conventional imaging. In such a microscope, the imaging and condenser lenses are identical and confocal. These two lenses are replaced by a single lens when epi-illumination is used, making confocal imaging particularly applicable to incident light microscopy. We describe the results we have obtained with a confocal system in which scanning is performed by moving the light beam, rather than the stage. This system is considerably faster than the scanned stage microscope and is easy to use. We have found that confocal imaging gives greatly enhanced images of biological structures viewed with epifluorescence. The improvements are such that it is possible to optically section thick specimens with little degradation in the image quality of interior sections.     

Acousto-optic laser-scanning cytometer

An instrument has been developed that uses a computer-controlled rapidly scanning laser beam to make cytometric measurements on cells or particles and which can measure low levels of fluorescence when using low-power lasers (Gershman, Hoffman, and O'Connell, "Methods and Apparatus for Analysis of Particles and Cells.") The method used is based upon acousto-optic principles of light diffraction. A vertically polarized 5-mW He-Ne laser is directed into an acousto-optic Bragg cell in which a portion of the incident light undergoes a small angular variation or deflection. Suitable optics focus the beam to a 25 microns diameter spot, at the 1/e2 point, in a sample cuvette while translating the angular variation into a linear scan. The cuvette enclosing the sample is slowly moved (approximately 1 micron/ms) via a stepper drive into the scanning beam while the forward angle light scatter sensor is monitored for the presence of valid signal events. When an event occurs, appropriate software optimizes the position of the focused laser beam onto the cell. Subsequently, scanning is stopped to allow for cell interrogation times that last for milliseconds or longer.     

一种目视激光显微镜

本文介绍一种目视激光显微镜。该装置采用白炽灯和激光做光源。通过调压器衬底亮度可以调整到零。由于激光的高亮度和强相干性,与普通显微镜相比,该显微镜具有景深长,分辨率高,层次丰富的特点。使用该显微镜时能实现镜象的假色彩编码,且镜象具有立体感。文中报导了该显微镜的原理和使用效果。     

Ultrafast laser scanner microscope.

Advances in monolayer deposition of cervical cells have removed one of the last serious obstacles to the design of high-resolution automated diagnostic assessment systems. In this article, we describe the design considerations for a system that is capable of acquiring, within 60 sec, a 0.5 micron digitized image of a 4 cm2 area on a standard glass slide. The most feasible approach is found to be a system using a rotating polygon to sweep the focused spot from a laser across a 2-mm scan line while the slide is uniformly translated perpendicular to the scan direction the use of laser sources (a helium-neon laser at 632 nm and a krypton ion laser at 568 and/or 476 nm) as compared to the incoherent light sources used in conventional microscope systems alleviates many of the optical design problems and provides the proper wavelengths needed for recognition of Papanicolaou stained cells. We also find that focus control of the scanning spot should be achievable using a technique involving a holographic grating. Other relevant considerations such as sample heating problems, multiphoton absorption by the sample, detector signal-to-noise ratios, laser amplitude noise control, and the digitization and buffering of the data stream are also discussed.     

Confocal scanning optical microscopy and three-dimensional imaging

Summary— Confocal scanning optical microscopy has significant advantages over conventional fluorescence microscopy: it rejects the out-of-locus light and provides a greater resolution than the wide-field microscope. In laser scanning optical microscopy, the specimen is scanned by a diffraction-limited spot of laser light and the fluorescence emission (or the reflected light) is focused onto a photodetector. The imaged point is then digitized, stored into the memory of a computer and displayed at the appropriate spatial position on a graphic device as a part of a two-dimensional image. Thus, confocal scanning optical microscopy allows accurate non-invasive optical sectioning and further three-dimensional reconstruction of biological specimens. Here we review the recent technological aspects of the principles and uses of the confocal microscope, and we introduce the different methods of three-dimensional imaging.     

Statistical evaluation of confocal microscopy images

BACKGROUND: The coefficient of variation (CV) is defined as the standard deviation (sigma) of the fluorescent intensity of a population of beads or pixels expressed as a proportion or percentage of the mean (mu) intensity (CV = sigma/mu). The field of flow cytometry has used the CV of a population of bead intensities to determine if the flow cytometer is aligned correctly and performing properly. In a similar manner, the analysis of CV has been applied to the confocal laser scanning microscope (CLSM) to determine machine performance and sensitivity. METHODS: Instead of measuring 10,000 beads using a flow cytometer and determining the CV of this distribution of intensities, thousands of pixels are measured from within one homogeneous Spherotech 10-microm bead. Similar to a typical flow cytometry population that consists of 10,000 beads, a CLSM scanned image consists of a distribution of pixel intensities representing a population of approximately 100,000 pixels. In order to perform this test properly, it is important to have a population of homogeneous particles. A biological particle usually has heterogeneous pixel intensities that correspond to the details in the biological image and thus shows more variability as a test particle. RESULTS: The bead CV consisting of a population of pixel intensities is dependent on a number of machine variables that include frame averaging, photomultiplier tube (PMT) voltage, PMT noise, and laser power. The relationship among these variables suggests that the machine should be operated with lower PMT values in order to generate superior image quality. If this cannot be achieved, frame averaging will be necessary to reduce the CV and improve image quality. There is more image noise at higher PMT settings, making it is necessary to average more frames to reduce the CV values and improve image quality. The sensitivity of a system is related to system noise, laser light efficiency, and proper system alignment. It is possible to compare different systems for system performance and sensitivity if the laser power is maintained at a constant value. Using this bead CV test, 1 mW of 488 nm laser light measured on the scan head yielded a CV value of 4% with a Leica TCS-SP1 (75-mW argon-krypton laser) and a CV value of 1.3% with a Zeiss 510 (25-mW argon laser). A biological particle shows the same relationship between laser power, averaging, PMT voltage, and CV as do the beads. However, because the biological particle has heterogeneous pixel intensities, there is more particle variability, which does not make as useful as a test particle. CONCLUSIONS: This CV analysis of a 10-microm Spherotech fluorescent bead can help determine the sensitivity in a confocal microscope and the system performance. The relationship among the factors that influence image quality is explained from a statistical endpoint. The data obtained from this test provides a systematic method of reducing noise and increasing image clarity. Many components of a CLSM, including laser power, laser stability, PMT functionality, and alignment, influence the CV and determine if the equipment is performing properly. Preliminary results have shown that the bead CV can be used to compare different confocal microscopy systems with regard to performance and sensitivity. The test appears to be analogous to CV tests made on the flow cytometer to assess instrument performance and sensitivity. Published 2001 Wiley-Liss, Inc.     

Fluorescence correlation spectroscopy with high count rate and low background: analysis of translational diffusion

An epi-illuminated microscope configuration for use in fluorescence correlation spectroscopy in bulk solutions has been analyzed. For determining the effective sample dimensions the spatial distribution of the molecule detection efficiency has been computed and conditions for achieving quasi-cylindrical sample shape have been derived. Model experiments on translational diffusion of rhodamine 6G have been carried out using strong focusing of the laser beam, small pinhole size and an avalanche photodiode in single photon counting mode as the detector. A considerable decrease in background light intensity and measurement time has been observed. The background light is 40 times weaker than the fluorescence signal from one molecule of Rh6G, and the correlation function with signal-to-noise ratio of 150 can be collected in 1 second. The effect of the shape of the sample volume on the autocorrelation function has been discussed. Correspondence to: R. Rigler     

Axial resolution enhancement of light‐sheet microscopy by double scanning of Bessel beam and its complementary beam

The side lobes of Bessel beam will create significant out‐of‐focus background when scanned in light‐sheet fluorescence microscopy (LSFM), limiting the axial resolution of the imaging system. Here, we propose to overcome this issue by scanning the sample twice with zeroth‐order Bessel beam and another type of propagation‐invariant beam, complementary to the zeroth‐order Bessel beam, which greatly reduces the out‐of‐focus background created in the first scan. The axial resolution can be improved from 1.68 μm of the Bessel light‐sheet to 1.07 μm by subtraction of the two scanned images across a whole field‐of‐view of up to 300 μm × 200 μm × 200 μm. The optimization procedure to create the complementary beam is described in detail and it is experimentally generated with a spatial light modulator. The imaging performance is validated experimentally with fluorescent beads as well as eGFP‐labeled mouse brain neurons.      

Laser surgery: using the carbon dioxide laser.

In 1917 Einstein theorized tha through an atomic process a unique kind of electromagnetic radiation could be produced by stimulated emission. When such radiation is in the optical or infrared spectrum it is termed laser (light amplification by stimulated emission of radiation) light. A laser, a high-intensity light source, emits a nearly parallel electromagnetic beam of energy at a given wavelength that can be captured by a lens and concentrated in the focal spot. The wavelength determines how the laser will be used. The carbon dioxide laser is now successfully employed for some surgical procedures in gynecology, otorhinolaryngology, neurosurgery, and plastic and general surgery. The CO2 laser beam is directed through the viewing system of an operating microscope or through a hand-held laser component. Its basic action in tissue is thermal vaporization; it causes minimal damage to adjacent tissues. Surgeons require special training in the basic methods and techniques of laser surgery, as well as in the safety standards that must be observed.     

Confocal reader for biochip screening and fluorescence microscopy

We developed a fluorescence reader for the sensitive detection of surface-generated fluorescence. The system is applicable for high resolution imaging as well as for the readout of large biochips. The surface of a microscope coverslip is scanned with a laser beam focused to a waist diameter of 500 nm (FWHM) by means of a single aspheric lens. Scanning large areas with a focused beam usually evokes the need of automatic control elements to adjust the laser spot to the designated position at the surface. Due to the special design of the reader, the focus keeps at the plane of the surface even when scanning large areas, obviating the requirement of any real time control. Thus the instrument is straightforward and inexpensive. Nevertheless it features a high sensitivity and high optical resolution. The versatility of the instrument is demonstrated by imaging cells and reading out a DNA-chip. The excellent sensitivity is shown by detecting single fluorescently labeled antibodies.     

A system for imaging transverse distribution of scattered light and chlorophyll fluorescence in intact rice leaves

In order to examine the transverse distribution of scattered light and chlorophyll fluorescence in intact rice leaves, a micro-fluorescence imaging system was devised using a microscope, a CCD camera with an image intensifier, an Ar and a He-Ne laser light source, an image processor, and a microcomputer. A laser light was projected vertically on to the surface of a rice leaf segment at a cut-edge, and scattered light and induced fluorescence were observed at the cut-section from a 90° angle to the axis of the laser beam. The intensity of scattered light showed a maximum at several micrometres depth from the leaf surface and a steep gradient afterwards. Fluorescence reached a maximum crossing with the decline curve of the scattered light. The maximum of fluorescence measured at 741 nm was observed at a greater depth from the leaf surface than that at 687 nm, suggesting that part of the fluorescence of the longer wavelength was emitted due to absorption of fluorescence of the shorter wavelength. Profiles of the scattered light and the chlorophyll fluorescence depended on leaf anatomy.     

High-performance confocal system for microscopic or endoscopic applications

We designed a high-performance confocal system that can be easily adapted to an existing light microscope or coupled with an endoscope for remote imaging. The system employs spatially and temporally patterned illumination produced by one of several mechanisms, including a micromirror array video projection device driven by a computer video source or a microlens array scanned by a piezo actuator in the microscope illumination path. A series of subsampled "component" video images are acquired from a solid-state video camera. Confocal images are digitally reconstructed using "virtual pinhole" synthetic aperture techniques applied to the collection of component images. Unlike conventional confocal techniques that raster scan a single detector and illumination point, our system samples multiple locations in parallel, with particular advantages for monitoring fast dynamic processes. We compared methods of patterned illumination and confocal image reconstruction by characterizing the point spread function, contrast, and intensity of imaged objects. Sample 3D reconstructions include a diatom and a Golgi-stained nerve cell collected in transmission.     

Current state of whole slide imaging use in cytopathology: Pros and pitfalls

Whole slide imaging (WSI) allows generation of large whole slide images and their navigation with zoom in and out like a true virtual microscope. It has become widely used in surgical pathology for many purposes, such as education and training, research activity, teleconsultation, and primary diagnosis. However, in cytopathology, the use of WSI has been lagging behind histology, mainly due to the cytological specimen's characteristics, as groups of cells of different thickness are distributed throughout the slide. To allow the same focusing capability of light microscope, slides have to be scanned at multiple focal planes, at the cost of longer scan times and larger file size. These are the main technical pitfalls of WSI for cytopathology, partly overcome by solutions like liquid‐based preparations. Validation studies for the use in primary diagnosis are less numerous and more heterogeneous than in surgical pathology. WSI has been proved effective for training students and successfully used in proficiency testing, allowing the creation of digital cytology atlases. Longer scan times are also a barrier for use in rapid on‐site evaluation, but WSI retains its advantages of easy sharing of images for consultation, multiple simultaneous viewing in different locations, the possibility of unlimited annotations and easy integration with medical records. Moreover, digital slides set the laboratory free from reliance on a physical glass slide, with no more concern of fading of stain or slide breakage. Costs are still a problem for small institutions, but WSI can also represent the beginning of a more efficient way of working.     

Optical tweezers based force measurement system for quantitating binding interactions: system design and application for the study of bacterial adhesion

An optical force measurement system for quantitating forces in the pN range between micrometer-sized objects has been developed. The system was based upon optical tweezers in combination with a sensitive position detection system and constructed around an inverted microscope. A trapped particle in the focus of the high numerical aperture microscope-objective behaves like an omnidirectional mechanical spring in response to an external force. The particle's displacement from the equilibrium position is therefore a direct measure of the exerted force. A weak probe laser beam, focused directly below the trapping focus, was used for position detection of the trapped particle (a polystyrene bead). The bead and the condenser focus the light to a distinct spot in the far field, monitored by a position sensitive detector. Various calibration procedures were implemented in order to provide absolute force measurements. The system has been used to measure the binding forces between Escherichia coli bacterial adhesins and galabiose-functionalized beads.     

Directed positioning of nuclei in living Paramecium tetraurelia: Use of the laser optical force trap for developmental biology

We have constructed a laser optical force trap (“laser tweezers”) by coupling an Nd:YAG laser to an optical microscope with a high numerical aperture objective. The laser beam (approximately 0.1 W power) is focused to a diffraction-limited spot at the specimen plane of the objective: the wavelength chosen (1,064 nm) is not strongly absorbed by most biological materials and is thus not ablative. Because the intensity of the laser beam increases towards the center of the focal spot, small particles brought near the spot will be attracted to the center and held there. Movement of the laser beam will tend to move any trapped particles with it. The laser tweezers can permit precise, nondestructive repositioning of small structures inside a living cell, without recourse to micromanipulators. Initial work has involved the use of laser tweezers on cells of Paramecium tet-raurelia held by a rotocompressor. We have been able to trap and reposition small organelles, especially the highly refractile structures known as crystals. Using a trapped crystal as a “tool”, we have been able to push micronuclei and other structures for many micrometers to virtually any desired location in a cell. In spite of extended exposure of specific structures and of individual cells to the laser beam, no damage has been detectible. Exposed cells, which were removed from the rotocompres-sor and cultured, showed complete viabilty. The laser tweezers technique shows tremendous potential for applications to the study of many fundamental cellular and developmental phenomena in paramecia and other ciliates. For example, we intend to use this technique to investigate temporal and spatial characteristics of nuclear determining regions during sexual reorganization in Paramecium. © 1992 Wiley-Liss, Inc.     

Double-beam flying spot scanner for two-dimensional polyacrylamide gel electrophoresis

The construction of a double-beam photometer in which the light source is a cathode ray oscilloscope is described. The light spot from the oscilloscope was focused and reduced in size at the gel plane to give a diameter of less than 0.15 mm and make it possible to scan over a 50 X 59-mm rectangle; using reduced spatial resolution (spot less than 0.2 mm) the area scanned becomes 70 X 90 mm. The light from the CRT was divided into two beams; one was directed through the transparent object to a photomultiplier and the other to a reference photomultiplier. The signals from these two detectors were converted to the logarithm of the ratio by a logging amplifier to give a direct measure of absorbance. Positioning of the spot, control of light intensity, and measurement of absorbance were carried out through an interface to a 16-bit computer. The relationship between measured and actual absorbance was linear over the range of absorbance 0 to 2, which could be raised to 1 to 3 by placing a neutral filter in the reference beam. The system generated an image containing 256 X 256 pixels in about 5 min, the scanning speed was determined by the persistence time of the P4 phosphor on the cathode ray tube, and faster scans can be made using A6 phosphor.     

Microscope-based multiparameter laser scanning cytometer yielding data comparable to flow cytometry data

We describe a computer-controlled 10 microns spot size laser scanning cytometer for making multiple wavelength fluorescence and scatter measurements of unconstrained cells on a surface such as a microscope slide. Designated areas of slides placed on a microscope stage are automatically scanned, and cells which generate above-threshold scatter or fluorescence values are found and individually processed to determine a list of measurement parameters. For each fluorescence or scatter measurement parameter, this list contains the integrated and peak values and bit pattern images of a scan window centered on the cell. The measurement time, the position of the cell on the slide, and two segmentation indices are also included in the list. Measurement time, cell position, and properties derived from the bit patterns are used interchangeably with integrated or peak measurement values as coordinates of multiproperty displays. Cells may be selected for counting, data display in various forms, or visual observation based on their meeting complex criteria among a chain of two property screens. Cells with selected properties may be viewed during an experiment or retrospectively. A designated specimen field may be repeatedly remeasured to perform kinetic cell studies. An argon ion and a HeNe- based laser instrument have been constructed and software has been written and evaluated with the specific goal of increasing the precision of propidium iodide-stained cellular DNA measurements. Some of the capabilities of the instrument and its current performance are described.     

A new method for on-line measurement of diurnal change in potato tuber growth under controlled environments

An on-line laser micrometer system was applied to measurement of diurnal change in potato (Solanum tuberosum L.) tuber growth. Diameters of the potato tuber were scanned by moving a laser micrometer along the longitudinal axis of the tuber at constant speed, and tuber volume was evaluated as an aggregate of thin discs. A single potato tuber, without competitive sink tubers in the plant, was grown in controlled air at 20 degrees C and 80% RH, and tuber volume was measured at 30 min intervals. During the growth experiment, the potato tuber increased in size without any inhibitory effect of periodical laser beam irradiation. Greatest expansion generally occurred during the early night, and transient contraction of the tuber occurred at the beginning of the light period.