Confocal Aperture Research Articles

Confocal Raman Microspectroscopy through a Planar Interface

We have developed a model to describe the effect of refraction through a planar interface on the collection efficiency and depth of focus when performing confocal Raman microspectroscopy. The planar interface introduces spherical aberration, which can substantially degrade the performance of the microscope, especially for large-numerical-aperture microscope objectives. This spherical aberration will increase the range of focal depths spanned by the paraxial and marginal rays of the illuminating laser beam within the sample. In the collection path, it will also distort the scattering volume defined by the confocal aperture; this results in a dramatic fall in the collected light intensity with increasing depth. We demonstrate that there is an optimum numerical aperture for collected light intensity at a given depth. The prediction of this theoretical model is compared to empirical results obtained by mapping the stress distribution within the diamond anvil of a high-pressure cell. Both the collected Raman intensity and the effective depth of focus are compared to the predictions from the theory.

Trp1, a Candidate Protein for the Store-operated Ca2+Influx Mechanism in Salivary Gland Cells

The trp gene family has been proposed to encode the store-operated Ca(2+) influx (SOC) channel(s). This study examines the role of Trp1 in the SOC mechanism of salivary gland cells. htrp1, htrp3, and Trp1 were detected in the human submandibular gland cell line (HSG). HSG cells stably transfected with htrp1alpha cDNA displayed (i) a higher level of Trp1, (ii) a 3-5-fold increase in SOC (thapsigargin-stimulated Ca(2+) influx), determined by [Ca(2+)](i) and Ca(2+)-activated K(+) channel current measurements, and (iii) similar basal Ca(2+) permeability, and inhibition of SOC by Gd(3+) but not by Zn(2+), as compared with control cells. Importantly, (i) transfection of HSG cells with antisense trp1alpha cDNA decreased endogenous Trp1 level and significantly attenuated SOC, and (ii) transfection of HSG cells with htrp3 cDNA did not increase SOC. These data demonstrate an association between Trp1 and SOC and strongly suggest that Trp1 is involved in this mechanism in HSG cells. Consistent with this suggestion, Trp1 was detected in the plasma membrane region, the proposed site of SOC, of acinar and ductal cells in intact rat submandibular glands. Based on these aggregate data, we propose Trp1 as a candidate protein for the SOC mechanism in salivary gland cells.

Generalized confocal imaging and synthetic aperture imaging

Synthetic aperture imaging is treated as a confocal process. The point-spread function of the synthetic aperture process is narrower than that of conventional imaging by a factor of 0.5, whereas the conventional confocal process offers resolution improvement of a factor of 0.72. The resolution in the regime between conventional confocal and conventional synthetic aperture is explored by computer simulation. Also, experimental results are given.

THE USE OF DYE DILUTION CURVE ANALYSIS IN THE QUANTIFICATION OF INDOCYANINE GREEN ANGIOGRAMS OF THE HUMAN CHOROID

THE USE OF DYE DILUTION CURVE ANALYSIS IN THE QUANTIFICATION OF INDOCYANINE GREEN ANGIOGRAMS OF THE HUMAN CHOROID

An Infinity Corrected All-Reflecting Microscope for Infrared Microspectrometry

A confocal, all-reflecting, infinity corrected microscope was developed to improve infrared microspectrometer (IMS) systems. This system consists of a Fourier transform spectrometer, an infinity corrected all-reflecting microscope, a viewing system for visible light microscopy, a single remote confocal mask for aperturing in either a transmission or reflection mode and an infrared detector. The radiant energy passes through a confocal aperture mask as it travels from the source to the sample and through the same confocal aperture mask as it travels from the sample to the radiant energy detector. A single confocal mask insures that the sample defining mask's size, geometrical shape and orientation are always matched. Mask alignment is assured and simplified since only one adjustment is needed. An aperture beam-splitter is used for reflection measurements. This system simplifies the operation of the microspectrometer, permits the use of standard commercial infinity corrected microscope optical components and reduces the cost of IMS accessory microscopes.

Second Harmonic Generation Imaging (SHG) in the Non-Linear Optical Microscopy of Living Cells

Abstract In recent years there has been considerable interest in two and three-photon excited fluorescence in laser scanning optical microscopy. Because absorption is confined tot he focal plane of the objective, these techniques provide intrinsic optical sectioning without the use of a confocal aperture. In addition, photobleaching and phototoxicity are greatly reduced above and below the focal plane. We have adapted a two-photon microscope to utilize surface second harmonic generation (SHG) as a new contrast mechanism for nonlinear optical biological imaging. Surface SHG was first described by Shen [1] and arises from the second order nonlinear susceptibility, χ(2). Signal will only arise from a non-centrosymmetric environment such as an interfacial region. Thus this technique has the potential to probe cellular membranes at high specificity. Further, since SHG results from an induced polarization and not absorption, photobleaching considerations are greatly reduced over fluorescence based methods.

Application of Multiphoton Imaging to Study of the Vasculature.

Nonlinear or multiphoton fluorescence microscopy utilizes the simultaneous absorption of two (or three) longer wavelength photons to excite fluorophores (Denk et.al., 1990). For example, UV absorbing fluorophores such as DAPI or lndo-1 are excited using 700 nm near infrared light rather than 350 nm UV excitation. This relatively new form of laser scanning fluorescence microscopy has excellent optical sectioning capabilities in thick, highly scattering tissues. The high degree of intrinsic optical sectioning arises from the spatial nature of the excitation dependence, rather than from a confocal aperture or deconvolution algorithm. The fluorescence arising from any point in the specimen depends on the second or third power of the intensity (i.e. two and three photon excitation, respectively). This squaring (or cubing) of the illumination point spread function effectively confines the excitation to a tenth of a femtoliter optical volume when using a high NA lens. 680 to 1100 nm mode-locked lasers producing pulses of 100 femtosecond duration at a repetition rate of 80 Mhz are used to efficiently produce fluorescence excitation at average powers that are usually in the 0.5 to 10 mW range at the sample.

Optical Coherence Microscopy: A New Technique for High-Resolution, Non-Invasive Imaging in Bulk Biological Tissues

Optical coherence microscopy (OCM) is a novel technique complementary to optical coherence tomography (OCT) which combines low-coherence interferometry with confocal microscopy to achieve micron-scale resolution imaging in highly scattering media. OCM may be implemented using a single-mode fiber-optic low-coherence interferometer (See Fig. 1). A high numerical aperture objective is used to focus sample-arm light into the specimen, and the reference arm length of the interferometer is adjusted to match the sample arm focal plane optical depth. The sample arm of the interferometer comprises a scanning confocal microscope, in which either the sample or the probe beam is laterally scanned in a raster pattern, and the optical fiber acts as a single-mode confocal aperture for combined light illumination and collection. The reference arm length of the interferometer establishes the depth position of an interferometric “coherence gate” in the sample, from which backscattered light is preferentially collected. Initial studies of OCM in scattering phantoms have demonstrated that this technique provides increased optical sectioning depth compared to confocal microscopy alone.

Three Dimensional Optical Data Storage Using Two-Photon Induced Photobleaching in a Polymer Matrix

The intensity of two-photon induced fluorescent shows quadratic dependency to the excitation intensity, as a result, one can achieve optical sectioning in fluorescent microscopy without the use of a confocal aperture. When the excitation power is properly adjusted, the fluorescent and photobleached volume is restricted to the vicinity of the focal points; therefore, it is possible to use this mechanism to achieve 3D data storage and microstructure fabrication (stereolithography).The recording medium used in this experiment was poly-HEMA block doped with a high efficient two-photon fluorophore, APSS, (4-[N-(2-hydroxyethyl)-N-methyl) amino phenyl]-4’-(6-hydroxyhexyl sulfonyl) stilbene). The block was made by dissolving APSS in HEMA with an initiator, 2,2’-azobisisobutyronitril (AiBN) (1% mole ratio) and polymerized at 60° C for 48 hours. The polymer was then trimmed into a small rectangular block (3×5×1 mm3) and surfaced by using a ultramicrotome with a glass knife. A modified Biorad MC500 LSM equipped with a home-made computer controlled stage scanner was used for both writing (stage scanning) and read-back (beam scanning).

A high-resolution, confocal laser-scanning microscope and flash photolysis system for physiological studies

We describe the construction of a high-resolution confocal laser-scanning microscope, and illustrate its use for studying elementary Ca 2+ signalling events in cells. An avalanche photodiode module and simple optical path provide a high efficiency system for detection of fluorescence signals, allowing use of a small confocal aperture giving near diffraction-limited spatial resolution (< 300 nm lateral and < 400 nm axial). When operated in line-scan mode, the maximum temporal resolution is 1 ms, and the associated computer software allows complete flexibility to record lines-cans continuously for long (minutes) periods or to obtain any desired pixel resolution in x-y scans. An independent UV irradiation system permits simultaneous photolysis of caged compounds over either a uniform, wide field (arc lamp source) or at a tightly focussed spot (frequency-tripled Nd:YAG laser). The microscope thus provides a versatile tool for optical studies of dynamic cellular processes, as well as excellent resolution for morphological studies. The confocal scanner can be added to virtually any inverted microscope for a component cost that is only a small fraction of that of comparable commercial instruments, yet offers better performance and greater versatility.

3D Microscopy: Confocal, Deconvolution or Both?

Over the past 20 years great improvements have been made in the techniques available for doing 3D fluorescent microscopy on living cells. The first approach, generally referred to as image deconvolution, treats the stack of 2D widefield (WF) image data as merely the sum of a number of discrete point-spread functions (PSF) and uses the computer to find the array of emitters that, when blurred by the PSF, best fits the stored data. If the PSF is known, only presence of statistical and electronic noise in the data, prevents this best-fit set of emitters from being a perfect image of the dye distribution in the specimen. The crucial role played by noise can be appreciated by comparing images from the Hubbell space telescope in its original condition, even after deconvolution, with images taken after the optics had been repaired.The second approach to 3D microscopy requires the introduction of a confocal aperture in front of the photodetector of a scanning laser microscope so that only the fluorescent signal emitted from the plane-offocus is recorded. As a result the image formed represents an “optical section” and a stack of such sections can be recorded at different focus heights to produce a 3D data set.

Real‐time two‐photon confocal microscopy using a femtosecond, amplified Ti:sapphire system

The bilateral imaging approach known from confocal applications operating in the line mode was used to realize real-time two-photon imaging. It is shown that the sectioning inherent to two-photon imaging could be improved by the introduction of a confocal line aperture in the imaging path. Using a high-power, low-repetition-rate amplified Ti:sapphire system, various biological objects were visualized including live boar sperm.

All-solid-state ultrafast lasers facilitate multiphoton excitation fluorescence imaging

Improvements in ultrafast laser technology have enabled a new excitation mode for optical sectioning fluorescence microscopy: multiphoton excitation fluorescence imaging. The primary advantages of this technique over laser scanning confocal imaging derive from the localized excitation volume; additional advantages accrue from the longer wavelength of the excitation source. Recent advances in all-solid-state, ultrafast (subpicosecond) laser technology should allow the technique to gain widespread use as a commercial instrument. In this paper, we review: optical sectioning fluorescence microscopy, multiphoton excitation fluorescence laser scanning microscopy, developments in laser physics which have enabled all-solid-state lasers to be used as excitation sources for multiphoton excitation fluorescence imaging, and provide current data for all-solid-state ultrafast lasers. A direct comparison between confocal (488 nm) imaging and two-photon excitation (1047 nm) imaging of a mouse brain slice stained with the lipophilic dye FM4-64 shows two-photon imaging can provide usable images more than twice as deep as confocal imaging. Multi-mode images (both two- and three-photon excitation) are presented for fixed and living cells as examples of multiphoton excitation fluorescence imaging applied to developmental biology. Also, a comparison of the axial resolution of our system is presented for confocal imaging (488 nm) and two-photon imaging (1047 nm) with and without a confocal pinhole aperture.

Microlaser microscope using self-detection for confocality

The microlaser microscope can use its own source lasers as detectors to provide a matched array of confocal apertures. Detection of the laser light by a single avalanche photodiode makes the electronics simple. We have implemented this in a 64 x 312 pixel format as our first demonstration of the device.

Performance measurements of an infrared digital scanning laser ophthalmoscope

Direct digital acquisition of images using a scanning laser ophthalmoscope (SLO) offers several advantages over the conventional fundus camera; in particular, the ability to produce tomographic images using a confocal aperture. This note describes measurements of the performance of an SLO. Spatial resolution, measured by the modulation transfer function, MTF, was shown to be worse along the direction of scan. As expected, image uniformity was good, with a coefficient of variation of 1.9%. While the effect of using a 100 mu m diameter confocal aperture instead of one with a 400 mu m diameter was to reduce slice thickness from 2600 mu m to 975 mu m, image intensity was reduced by a factor of 30.

Reproducibility of Retinal Nerve Fiber Layer Evaluation by Dynamic Scanning Laser Ophthalmoscopy

Scanning laser ophthalmoscopy is a laser-based image acquisition technique, which greatly improves the quality of the examination of the fundus and the retinal nerve fiber layer. To assess retinal nerve fiber layer imaging by scanning laser ophthalmoscopy and evaluate intra- and interobserver reproducibility in the classification of retinal nerve fiber layer defects, three independent observers evaluated on two separate occasions the videotaped images of 150 eyes of 80 consecutive patients with ocular hypertension or glaucoma. Ophthalmoscopy was performed using argon blue light (488 nm), confocal apertures of 3 to 1 mm, and 40-degree and 20-degree field angles. Of 150 eyes, 20 (13.3%) were excluded from the study because of the poor quality of the images (clinically significant cataract or myopic peripapillary atrophy). The retinal nerve fiber layer was evaluated qualitatively according to a standard classification: normal pattern, slit, wedge, and diffuse defects. Intraobserver reproducibility, evaluated by kappa statistic, was excellent (> or = 0.75): observer A = 0.78 (95% confidence limits, 0.67-0.88); observer B = 0.84 (95% confidence limits, 0.72-0.96); and observer C = 0.79 (95% confidence limits, 0.67-0.91). Interobserver reproducibility was also excellent in all cases: observers A-B = 0.84 (95% confidence limits, 0.71-0.98); observers A-C = 0.76 (95% confidence limits, 0.65-0.87); and observers B-C = 0.80 (95% confidence limits, 0.69-0.92). Kappa values ranged between 0.59 and 0.69 for intraobserver reproducibility and between 0.55 and 0.69 for interobserver reproducibility when using only those eyes in which abnormalities were noted by at least one observer.(ABSTRACT TRUNCATED AT 250 WORDS)

Enhanced three-dimensional reconstruction from confocal scanning microscope images II Depth discrimination versus signal-to-noise ratio in partially confocal images

The enhanced depth discrimination of a confocal scanning optical microscope is produced by a pinhole aperture placed in front of the detector to reject out-of-focus light. Strictly confocal behavior is impractical because an infinitesimally small aperture would collect very little light and would result in images with a poor signal-to-noise ratio (SNR), while a finite-sized partially confocal aperture provides a better SNR but reduced depth discrimination. Reconstruction algorithms, such as the expectationmaximization algorithm for maximum likelihood, can be applied to partially confocal images in order to achieve better resolution, but because they are sensitive to noise in the data, there is a practical trade-off involved. With a small aperture, fewer iterations of the reconstruction algorithm are necessary to achieve the desired resolution, but the low a priori SNR will result in a noisy reconstruction, at least when no regularization is used. With a larger aperture the a priori SNR is larger but the resolution is lower, and more iterations of the algorithm are necessary to reach the desired resolution; at some point the a posteriori SNR is lower than the a priori value. We present a theoretical analysis of the SNR-toresolution trade-off partially confocal imaging, and we present two studies that use the expectationmaximization algorithm as a postprocessor; these studies show that a for a given task there is an optimum aperture size, departures from which result in a lower a posteriori SNR.

A comparison of various readout techniques in magneto-optic recording

The application of confocal, non-confocal, and magnetooptic phase contrast readout techniques to magnetooptic recording is described. Experimental investigations using a scanning laser microscope indicate that the introduction of a confocal aperture into the detector space can sharpen the system step response, reduce intersymbol interference, and provide an in-built focus error signal. It is concluded that the use of magnetooptic phase contrast provides a further alternative to conventional polar Kerr readout and introduces the possibility of particularly simple replay optics.< <ETX xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">&gt;</ETX>

The point‐spread function of a confocal microscope: its measurement and use in deconvolution of 3‐D data

SUMMARYWe have measured the point‐spread function (PSF) for an MRC‐500 confocal scanning laser microscope using subresolution fluorescent beads. PSFs were measured for two lenses of high numerical aperture—the Zeiss plan‐neofluar 63 × water immersion and Leitz plan‐apo 63 × oil immersion—at three different sizes of the confocal detector aperture. The measured PSFs are fairly symmetrical, both radially and axially. In particular there is considerably less axial asymmetry than has been demonstrated in measurements of conventional (non‐confocal) PSFs. Measurements of the peak width at half‐maximum peak height for the minimum detector aperture gave approximately 0·23 and 0·8 μm for the radial and axial resolution respectively (4·6 and 15·9 in dimensionless optical units). This increased to 0·38 and 1·5 μm (7·5 and 29·8 in dimensionless units) for the largest detector aperture examined. The resulting optical transfer functions (OTFs) were used in an iterative, constrained deconvolution procedure to process three‐dimensional confocal data sets from a biological specimen—pea root cells labelled in situ with a fluorescent probe to ribosomal genes. The deconvolution significantly improved the clarity and contrast of the data. Furthermore, the loss in resolution produced by increasing the size of the detector aperture could be restored by the deconvolution procedure. Therefore for many biological specimens which are only weakly fluorescent it may be preferable to open the detector aperture to increase the strength of the detected signal, and thus the signal‐to‐noise ratio, and then to restore the resolution by deconvolution.

Theory of Cavity-Mode-Mixing Effects in Internally Scanned Lasers

An analysis of cavity-mode-mixing effects is given for a variety of cavity geometries and coupling parameters in the presence of a continuous transverse aperture velocity. The paper represents an extension of the previous work by Fox and Li, and Boyd and Gordon to include the effects of time-dependent changes in the transverse aperture location. The average beam profile in the scanning reference frame is calculated through a continuous reexpansion of the optical field in terms of the set of self-reproducing modes characteristic of the same cavity under static transverse boundary conditions. Results are specifically given for square aperture, plane-parallel, concentric, and confocal geometry for light, plane polarized orthogonally to the scan velocity. The effects of a random variation in the mirror-transmission coefficient are calculated, and it is shown that these effects are largely averaged out in the scanning process. Cavities of the type considered earlier by Pole and Myer and by Beiser are specifically considered. A method of evaluating different real systems with the present calculations is discussed; it is shown that a "push-pull"-type scanning laser should have an inherent scan velocity roughly twice that for the single-ended scanning laser geometry previously considered for the same degree of beam degradation and other cavity parameters. A method is developed for the derivation of self-reproducing confocal modes which arise with stationary, continuously varying aperture transmission functions. The method is specifically applied to a confocal Gaussian gain aperture and to a nonsymmetric confocal gain aperture. It is shown that the odd-symmetry mode of the Gaussian gain aperture dominates over the even-symmetry mode at low-gain coefficients, in opposition to the normal diffraction-limited rectangular-transmission-aperture case. An extension of the scanning problem is given to include the transverse displacement of a continuous gain aperture; it is shown formally that the solution of the continuous-aperture problem goes continuously over to the rectangular-aperture results at low values of the gain. The method is specifically applied to a hypothetical scanning laser employing swept Gaussian and nonsymmetric gain apertures.