Improvement of confocal microscope performance by shaped annular beam and heterodyne confocal techniques

In order to further improve the performance of a confocal microscope (CM) used for measurement of surface profiles and 3D microstructures, a shaped annular beam heterodyne confocal measurement method based on annular pupil filter technique and reflection confocal microscopy, is proposed to expand the measurement range and to improve the defocused property of CM. The approach proposed uses a confocal dual-receiving light path arrangement and a heterodyne subtraction of two signals received from detectors with axial offset to enable CM to be used for bipolar absolute measurement and to improve the defocused property of CM, and it uses the annular pupil filter technique to produce a binary optical shaped annular beam, which expands the measurement range by expanding the full-width at half-maximum of intensity curve received from two detectors in a heterodyne confocal microscopy system. Theoretical analyses and experimental results indicate that a shaped annular beam heterodyne microscope has a measurement range expanded from 4 to 14 μm, achieved an axial resolution of 2 nm and improved the defocused property, when ε=0.5 and NA=0.65. It can be therefore concluded that the shaped annular beam heterodyne confocal measuring method proposed is a new approach to ultraprecision measurement of surface profiles and 3D microstructures.     

Full-field heterodyne interference microscope with spatially incoherent illumination

A heterodyne interference microscope arrangement for full-field imaging is described. The reference and object beams are formed with highly correlated, time-varying laser speckle patterns. The speckle illumination confers a confocal transfer function to the system, and by temporal averaging, the coherence noise that often degrades coherent full-field microscope images is suppressed. The microscope described is similar to a Linnik-type microscope and allows the use of high-numerical-aperture objective lenses, but the temporal coherence of the illumination permits the use of a low-power achromatic doublet in the reference arm. The use of a doublet simplifies alignment of the microscope and can reduce the cost. Preliminary results are presented that demonstrate full-field surface height precision of 1 nm rms.     

Tomographic imaging by two-wave mixing with a wavelength-scanning laser diode

A method of tomographic imaging is proposed in which two-wave mixing in a photorefractive crystal is used with wavelength scanning of a laser diode and phase modulation of the pump beam. This method provides full optical processing and is effective for weak light from objects because of the use of two-wave mixing. The depth resolution of the method was ~1 cm when the wavelength-scanning width was ~0.02 nm .     

Dual-axis confocal microscope for high-resolution in vivo imaging

We describe a novel confocal microscope that uses separate low-numerical-aperture objectives with the illumination and collection axes crossed at angle theta from the midline. This architecture collects images in scattering media with high transverse and axial resolution, long working distance, large field of view, and reduced noise from scattered light. We measured transverse and axial (FWHM) resolution of 1.3 and 2.1 microm, respectively, in free space, and confirm subcellular resolution in excised esophageal mucosa. The optics may be scaled to millimeter dimensions and fiber coupled for collection of high-resolution images in vivo.     

Guiding a confocal microscope by single fluorescent nanoparticles

Confocal optical microscopes offer unparalleled high sensitivity and three-dimensional (3D) imaging capability but require slow point-by-point scanning; they are inefficient for imaging moving objects. We propose a more efficient solution. Instead of indiscriminate scanning, we let the focus of the microscope pursue the object of interest such that no time is wasted on uninformative background, allowing us to visualize 3D trajectories of fluorescent nanoparticles in solution with millisecond temporal and ~200 nm spatial resolution.     

A shaped annular beam tri-heterodyne confocal microscope with good anti-environmental interference capability

A shaped annular beam tri-heterodyne confocal microscope has been proposed to improve the anti-environmental interference capability and the resolution of a confocal microscope. It simultaneously detects far-, on-, and near-focus signals with given phase differences by dividing the measured light path of the confocal microscope into three sub-paths (signals). Pair-wise real-time heterodyne subtraction of the three signals is used to improve the anti-environmental interference capability, axial resolution, and linearity; and a shaped annular beam super-resolution technique is used to improve lateral resolution. Theoretical analyses and preliminary experiments indicate that an axial resolution of about 1 nm can be achieved with a shaped annular beam tri-heterodyne confocal microscope and its lateral resolution can be better than 0.2 $\mu $m for $\lambda =632.8$~nm, the numerical aperture of the lens of the microscope is NA $=0.85$, and the normalized radius $\varepsilon =0.5$.     

Optimum resolution of laser microscope by using optical heterodyne detection

The ultimate resolution of the optical heterodyne laser microscope is analyzed and experimentally verified. It is shown that the resolution of this system is essentially the same as that of the conventional optical microscope, but has the advantage that the aberration due to the spherical surfaces and the diffusion from neighboring particles do not exist.     

Large field of view MEMS-based confocal laser scanning microscope for fluorescence imaging

Although confocal fluorescence microscopes are widely used in biology and have been proven to be promising diagnostic tools in dermatologic diagnostics, they are at present uncommon in medical practice. This is mainly due to high costs of acquisition and their large and complex outline. With the integration of a MEMS scanner we present a demonstration system of a confocal fluorescence laser scanning microscope which is affordable and portable. It has a field of view of 500 μm × 500 μm and is mainly composed of off-the-shelf components.     

Tracking a single fluorescent molecule with a confocal microscope

We consider the problem of tracking a single fluorescent molecule in both two and three dimensions using a confocal laser scanning microscope. An estimate of the position of the molecule is generated from the measured fluorescence signal through the use of parameter estimation theory. This estimate is used in a nonlinear controller designed both to track the position of the molecule and to provide good measurements for use in the estimation algorithm. The performance of the approach is investigated through numerical simulation for molecules undergoing diffusion and directed transport and the capabilities of the controller relative to experimental limitations are discussed.     

Improved sectioning in a slit scanning confocal microscope

We describe a simple implementation of a slit scanning confocal microscope to obtain an axial resolution better than that of a point-scanning confocal microscope. Under slit illumination, images of a fluorescent object are captured using an array detector instead of a line detector so that out-of-focus light is recorded and then subtracted from the adjacent images. Axial resolution after background subtraction is 2.2 times better than the slit confocal resolution, and out-of-focus image suppression is calculated to attenuate with defocus faster by 1 order of magnitude than in the point confocal case.     

Quantitative amplitude and phase measurement by use of a heterodyne scanning near-field optical microscope

A coherent photon scanning tunneling microscope is presented. The setup employs heterodyne interferometry, allowing both the phase and the amplitude of the optical near field to be measured. Experimental results of measurements on a standing evanescent wave reveal the high resolution that is obtainable with such an approach. In fact we have measured the amplitude and the phase of the near field, with a resolution of 1.6 nm between sample points.     

In vivo imaging of mice auricle vessels using adaptive optical confocal fluorescence microscope

Facilitated with stochastic parallel gradient descent(SPGD) algorithm for wavefront sensorless correcting aberrations, an adaptive optics(AO) confocal fluorescence microscopy is developed and used to record fluorescent signals in vivo. Vessels of mice auricle at 80, 100 and 120 μm depth are obtained, and image contrast and fluorescence intensity are significantly improved with AO correction. The typical 10%–90% rise-time of the metric value measured is 5.0 s for a measured close-loop bandwidth of 0.2 Hz. Therefore, the AO confocal microscopy implemented with SPGD algorithm for robust AO corrections will be a powerful tool for study of vascular dynamics in future.     

Determining the complex point-spread function of a scanning differential heterodyne microscope

For a scanning differential heterodyne microscope, the two-dimensional point spread function and the pupil function are determined from experimental measurements of the intensity distribution of a probing beam. The phase components of these functions were restored from the measured distributions using the Gerchberg-Saxton algorithm. Based on the two-dimensional pupil function, the three-dimensional point-spread function is determined in the scalar approximation using the Debye integral. The aberration function of the microscope is analyzed from the viewpoint of the composition of aberrations and method of their balancing. The focusing adjustment criterion of the microscope for an object under study by the phase response from the test structure is considered.     

Homodyne and heterodyne imaging through a scattering medium

We introduce a novel two-dimensional (2D) homodyne and heterodyne technique for imaging objects through or embedded in a scattering medium. Our imaging approach is based on heterodyning of light with different Doppler broadenings that is scattered from objects of two different textures or from an opaque object and a textured scattering medium. We report on the initial demonstration of pulling signals out of noise for an object hidden behind a scattering medium. Enhancements of signal-to-noise ratio of the order of 50 have been achieved by use of a 2D holographic phase-sensitive detector. We also discuss the experimental feasibility of this approach for objects embedded in a scattering medium.     

Investigation of diffraction gratings with the use of a differential heterodyne microscope

A theory of image formation in a scanning differential heterodyne microscope is developed using the results of observations of a metallized periodic structure with a rectangular profile. The phase and amplitude response functions are calculated for TE and TM polarizations. It is demonstrated that the width of grating elements can be measured with a superresolution. The experimental phase and amplitude responses from a metallized grating with a trapezoidal profile are obtained and compared with the results of theoretical calculations.     

Multispectral imaging with a confocal microendoscope

The concept of a multispectral confocal microscope for in vivo imaging is introduced. To demonstrate the concept we modified a slit-scan fluorescence confocal microendoscope incorporating a fiber-optic catheter for in vivo imaging to record multispectral images. The system was designed to examine cellular structures during optical biopsy and to exploit the diagnostic information contained within the spectral domain. Preliminary experiments were carried out in phantoms and cell cultures to demonstrate the potential of the technique.     

Characterization of a confocal microscope for time-resolved photon counting fluorescence

A confocal microscope setup is developed for time-resolved fluorescence measurements. It is added to a traditional cuvette time-resolved setup, with a pumped Ti-Sa light source. The temporal resolution of 37 ps (FWHM) is not degraded, in comparison with the cuvette setup also described. These setups allow both decay lifetime and anisotropy relaxation time determination. Fluorescence correlation spectroscopy (FCS) is used to determine the observation point size. When associated with the calcium probe calcium green, calcium concentration in single cells can be determined in 10 ms by simultaneous acquisition of early and late fluorescence photons.     

Investigation of the reflection-type double-pass confocal microscope with a phase-conjugate mirror

New experimental results about a reflection-type double-pass confocal microscope with a phase-conjugate mirror are presented. Exploiting the change of polarization of the light, when it is reflected at the object, gives the possibility to enhance the axial resolution of the microscope compared to that of the conventional confocal microscope by a factor of 65%. Furthermore, investigations on the time behavior of the PCM are presented. Received: 11 January 1999 / Revised version: 25 January 1999 / Published online: 7 April 1999     

Miniature near-infrared dual-axes confocal microscope utilizing a two-dimensional microelectromechanical systems scanner

The first, to our knowledge, miniature dual-axes confocal microscope has been developed, with an outer diameter of 10 mm, for subsurface imaging of biological tissues with 5-7 microm resolution. Depth-resolved en face images are obtained at 30 frames per second, with a field of view of 800 x 100 microm, by employing a two-dimensional scanning microelectromechanical systems mirror. Reflectance and fluorescence images are obtained with a laser source at 785 nm, demonstrating the ability to perform real-time optical biopsy.