Confocal theta fluorescence microscopy with annular apertures

In a confocal theta fluorescence microscope, two objective lenses with circular apertures are used, one to illuminate the sample and the other to detect the emitted light at an angle to the illumination axis. We show that annular illumination and detection apertures lead to a reduction in the extent of the point-spread function. A spatial resolution improved by more than 50% can be achieved with a central obstruction blocking the inner 80% of the diameter. For the limit of a very narrow annular aperture and a numerical aperture of 0.75, the volume at half-maximum of the point-spread function is reduced from 15to5 aL. Amixed setup with anannular illumination aperture and a circular detection aperture is also considered.     

Confocal fluorescence polarization microscopy in turbid media: effects of scattering-induced depolarization

We present an experimental and theoretical study of confocal fluorescence polarization microscopy in turbid media. We have performed an experimental study using a fluorophore-embedded polymer rod immersed in aqueous suspensions of 0.1 and 0.5 microm diameter polystyrene microspheres. A Monte Carlo approach to simulate confocal fluorescence polarization imaging in scattering media is also presented. It incorporates a detailed model of polarized fluorescence generation that includes sampling of elliptical polarization, excited-state molecular rotational Brownian motion, and dipole fluorescence emission. Using both approaches, we determine the effects of the number of scattering events, target depth, photon scattering statistics, objective numerical aperture, and pinhole size on confocal anisotropy imaging. From this detailed analysis and comparison of experiment with simulation, we determine that fluorescence polarization is maintained to depths at which meaningful intensity images can be acquired.     

Testing a fast off-axis parabolic mirror by using tilted null screens

We propose the design of tilted null screens for testing off-axis segments of conic surfaces. The tilt allows us to control the size of the screen and the sensitivity of the test. For positive tilt angles the sensitivity is increased while the size of the screen is reduced in the sagittal caustic region and vice versa in the tangential caustic region. Further analysis and preliminary experimental results are presented for a fast off-axis concave parabolic mirror with an elliptical aperture. An offset distance of X(C) = 25.4 mm yields radius of curvature at the vertex R = 20.4 mm; major axis of the mirror D(M) = 49.4 mm; and minor axis D(m) = 29.5 mm.     

Two-photon fluorescence microscopy with a diode-pumped Cr:LiSAF laser

We demonstrate the acquisition of high-quality two-photon fluorescence microscopy images using an all-solid-state self-mode-locked Cr:LiSAF laser. We contrast the performance of the two-photon technique with single-photon confocal fluorescence microscopy images taken with an argon-ion laser. Examples of improved depth penetration and reduced dye bleaching are presented.     

Resolution improvement in two-photon fluorescence microscopy with a single-mode fiber

The dependence of spectral broadening of an ultrashort-pulsed laser beam on the fiber length and the illumination power is experimentally characterized in order to deliver the laser for two-photon fluorescence microscopy. It is found that not only the spectral width but also the spectral blue shift increases with the fiber length and illumination power, owing to the nonlinear response in the fiber. For an illumination power of 400 mW in a 3-m-long single-mode fiber, the spectral blue shift is as large as 15 nm. Such a spectral blue shift enhances the contribution from the short-wavelength components within the pulsed beam and leads to an improvement in resolution under two-photon excitation, whereas the efficiency of two-photon excitation is slightly reduced because of the temporal broadening of the pulsed beam. The experimental measurement of the axial response to a two-photon fluorescence polymer block confirms this feature.     

The deflection of a circular parabolic telescope mirror on an elastic foundation

In this article we describe the deflection due to gravity of a parabolic mirror, supported by an annular shaped elastic foundation. We calculate the deflection by solving the thin plate equation for plates with a non-constant thickness numerically. It will be shown that a central hole in the elastic foundation of a certain diameter will minimize the deflection of the mirror and will result in minimal RMS values. We compare the mirror deflection caused by 18 and 27 axial point support to the deflection of a mirror on an annular shaped elastic foundation. It will be shown that an annular shaped elastic foundation results is much less deflection and smaller RMS values compared to the multiple point axial support for mirrors smaller than 1 m diameter and F# ratios larger than 2. Using this foundation method, mirrors may become thinner which results in lightweight constructions.     

Controlling the contribution of the electric field components to the focus of a high-aperture lens using binary phase structures

We show that the contribution of the electric field components into the focal region can be controlled using binary phase structures. We discuss differently polarized incident waves, for each case suggesting easily implemented binary phase distributions that ensure a maximum contribution of a definite electric field component on the optical axis. A decrease in the size of the central focal spot produced by a high numerical aperture (NA) focusing system comes as the result of the spatial redistribution of the contribution of different electric field components into the focal region. Using a polarization conversion matrix of a high NA lens and the numerical simulation of the focusing system in Debye's approximation, we demonstrate benefits of using asymmetric to polar angle ? binary phase distributions (such as arg[cos ?] or arg[sin 2?]) for generating a subwavelength focal spot in separate electric field components. Additional binary structure variations with respect to the azimuthal angle also make possible controlling the longitudinal distribution of light. In particular, the contribution of the transverse components in the focal plane can be reduced by the use of a simple axicon-like structure that serves to enhance the NA of the lens central part, redirecting the energy from focal plane. As compared with the superimposition of a narrow annular aperture, this approach is more energy efficient, and as compared with the Toraldo filters, it is easier to control when applied to three-dimensional focal shaping.     

Fast analysis method for polarization-dependent performance of a concave diffraction grating with total-internal-reflection facets

A fast simulation method for a waveguide-based concave grating with total-internal-reflection (TIR) facets is presented using the Kirchhoff-Huygens principle. Unlike the conventional scalar method, modifications are made to take into account the influence of the Goos-H?nchen (GH) shift. The simple method is in good agreement with a numerical method based on rigorous coupled-wave analysis for a wide range of practical device parameters and can provide an insightful physical explanation for the numerical results. It is shown that the GH shift is a main contributing factor for the loss and the polarization-dependent loss of an etched diffraction grating demultiplexer with TIR facets.     

Surface-plasmon microscopy with a two-piece solid immersion lens: bright and dark fields

We report bright-field and dark-field surface-plasmon imaging using a modified solid immersion lens and a commercial objective of moderate NA in the epi configuration. The contrast and resolution are extremely good, giving well-resolved images of protein monolayers both in air and in water. We also describe a two-part solid immersion lens that allows the sample to be moved without degrading the image quality in any observable way. The merits of the two-part lens are discussed and compared to commercially available microscope objectives. Finally, we introduce a simple Green's function model that illustrates the key features of both bright-field and dark-field surface-plasmon imaging.     

Analysis and design of a concave diffraction grating with total-internal-reflection facets by a hybrid diffraction method

A novel hybrid diffraction method is introduced to simulate the diffraction and imaging of a planar-integrated concave grating that has total internal reflection (TIR) facets. The Kirchhoff-Huygens diffraction formula is adopted to simulate the propagation of the lightwave field in the free-propagation region, and a rigorous coupled-wave analysis is used to calculate the polarization-dependent diffraction by the grating. The hybrid diffraction method can be used to analyze accurately the imaging properties as well as the polarization-dependent diffraction characteristics of a concave grating. The dependence of several merit parameters of a concave grating with TIR facets on its basic geometric parameters is studied. Compared with one with metallic echelle facets, a concave grating with TIR facets shows a much lower polarization-dependent loss. Since more performance specifications can be considered in the design of a concave grating than with the conventional scalar method, design error can be reduced greatly with the present hybrid diffraction method.     

Two-photon excitation fluorescence microscopy with a high depth of field using an axicon

In conventional two-photon excitation fluorescence microscopy, the numerical aperture of the objective determines the lateral resolution and the depth of field. In some situations, as with functional imaging of dynamic events distributed in live biological tissue, an improved temporal resolution is needed; as a consequence, it is imperative to use optics with a high depth of field to simultaneously image objects at different axial positions. With a conventional microscope objective, increasing the depth of field is achieved at the expense of lateral resolution. To overcome this limitation, we have incorporated an axicon in a two-photon excitation fluorescence microscopy system; measurements have shown that an axicon provides a depth of field in excess of a millimeter, while the lateral resolution is maintained at the micrometer scale. Thus axicon-based two-photon microscopy has been shown to yield a high-resolution projection image of a sample with a single 2D scan of the laser beam while maintaining the improved tissue penetration typical of two-photon microscopy.     

High-resolution image reconstruction in fluorescence microscopy with patterned excitation

We discuss data treatment strategies in structured illumination microscopy, using simulated and experimental data. In the setup, the illumination pattern is generated by projecting a movable pinhole mask into the sample, and a wide-field fluorescence microscope image is acquired for each pattern position. The structured illumination data obtained from a two-dimensional illumination pattern can be treated by projection strategies such as in video confocal microscopy (sum, maximum, maximum minus minimum, and superconfocal), by a scaled subtraction of the out-of-focus estimate, or by a modified version of the Fourier-space treatment, as is known for data from one-dimensional structured illumination. We investigate the influence of some data processing strategies on unwanted effects such as residual patterning and local deviations from linearity in the reconstructed intensity.     

Confined diffusion in tubular structures analyzed by fluorescence correlation spectroscopy on a mirror

In fluorescence correlation spectroscopy (FCS) analysis it is generally assumed that molecular species diffuse freely in volumes much larger than the three-dimensional FCS observation volume. However, this standard assumption is not valid in many measurement conditions, particularly in tubular structures with diameters in the micrometer range, such as those found in living cells (organelles, dendrites) and microfluidic devices (capillaries, reaction chambers). As a result the measured autocorrelation functions (ACFs) deviate from those predicted for free diffusion, and this can shift the measured diffusion coefficient by as much as ~50% when the tube diameter is comparable with the axial extension of the FCS observation volume. We show that the range of validity of the FCS measurements can be drastically improved if the tubular structures are located in the close vicinity of a mirror on which FCS is performed. In this case a new fluctuation time in the ACF, arising from the diffusion of fluorescent probes in optical fringes, permits measurement of the real diffusion coefficient within the tubular structure without assumptions about either the confined geometry or the FCS observation volume geometry. We show that such a measurement can be done when the tubular structure contains at least one pair of dark and bright fringes resulting from interference between the incoming and the reflected excitation beams on the mirror surface. Measurement of the diffusion coefficient of the enhanced green fluorescent protein (EGFP) and IscS-EGFP in the cytoplasm of living Escherichia coli illustrates the capabilities of the technique.     

Time-resolved thermal lens and thermal mirror spectroscopy with sample-fluid heat coupling: a complete model for material characterization

This work presents a theoretical study of a heat transfer effect, taking into account the heat transfer within the heated sample and out to the surrounding medium. The analytical solution is used to model the thermal lens and thermal mirror effects and the results are compared with the finite element analysis (FEA) software solution. The FEA modeling results were found to be in excellent agreement with the analytical solutions. Our results also show that the heat transfer between the sample surface and the air coupling fluid does not introduce an important effect over the induced phase shift in the sample when compared to the solution obtained without considering axial heat flux. On the other hand, the thermal lens created in the air coupling fluid has a significant effect on the predicted time-dependent photothermal signals. When water is used as fluid, the heat coupling leads to a more significant effect in both sample and fluid phase shift. Our results could be used to obtain physical properties of low optical absorption fluids by using a reference solid sample in both thermal lens and thermal mirror experiments.     

Holographic parabolic mirror as a receiver optical front end for wireless infrared communications: experimental study

The inherent multifunctionality of holographic optical elements and their light physical weight make them an attractive solution for the receiver optics of portable terminals in indoor infrared wireless communication systems. A parabolic holographic mirror has been recorded in silver halide at a visible wavelength, and its replay wavelength has been shifted to the near infrared. Employment of proprietary swelling technology resulted in a permanent replay wavelength shift without the need for hologram sealing. Despite the relatively low diffraction efficiency of holograms recorded in silver halide in principle, an improvement in the receiver signal-to-noise ratio of more than 20 dB has been measured. The results of the conducted experiments proved undoubtedly the great potential of curved holographic mirrors as a key element of the receiver optical front end in IR wireless communication systems.     

All-sky camera with a concave mirror

The characteristics of an all-sky camera with a concave mirror are analyzed. A differential equation for a concave aspheric mirror with constant angular magnification is derived for the general dependence of the camera image height on the camera field angle. This equation is solved in parametric form for the case of a concave mirror with a constant angular magnification. The explicit equations for the shape of the aspheric mirror are given for some particular values of the angular magnification. Parametric equations of the surface shape for sevenfold angular magnification are developed into a power series that is used to analyze the imaging performance of such a mirror. The performance of the concave aspheric mirror is compared with that of a spherical mirror. The minimal camera-to-mirror distance is determined as a function of the blur allowed and the camera lens aperture. Some characteristics of convex mirrors are also presented for comparison.     

Image contrast enhancement for two-photon fluorescence microscopy in a turbid medium

Image contrast enhancement is investigated for two-photon excitation fluorescence images of a microscopic sample that is buried underneath a turbid medium. The image contrast, which deteriorates rapidly with sample depth because of scattering loss, is enhanced by an increase in the average excitation power of the focused Gaussian (the TEM(00) mode) beam according to a compensation relation that has been derived by use of a Monte Carlo analysis of the scattering problem. A correct increase in the excitation power results in a detected fluorescence signal that remains invariant with sample depth. The scheme is demonstrated on images of DAPI-stained nuclei cells viewed underneath a suspension of 0.105-mum-diameter polystyrene spheres.     

High-density optical data storage with one-photon and two-photon near-field fluorescence microscopy

A spatially localized photochemical reaction induced by near-field femtosecond laser pulses is demonstrated on a nanometer scale and used for high-density optical data storage. Recorded domains down to 120 and 70 nm are obtained with one-photon and two-photon excitation, respectively. It is shown that the local-field confinement that is due to the quadratic dependence of two-photon excitation on light intensity has the potential to increase the near-field optical storage density.