The intrinsic confined photo-interacting volume in multi- photon fluorescence microscopy provides the possibility of obtaining fluorescence spectrum from specific cellular structure in a tissue. In this article, we demonstrated that it is feasible to obtain useful two-photon pumped fluorescence spectrum from cell wall and single chloroplast. The difference in fluorescence spectra obtained with single- and two-photon excitation indicates that a significant shift in fluorescence maximum may occur due to the non-linear nature of excitation. Therefore, in order to properly interpret two-photon fluorescence micrographs, it is important to characterize the fluorescence spectrum of the specimen and the commonly used fluorescence probes. The fluorescence spectra will in turn be useful in the selection of filter sets in multi-photon fluorescence microscopy.

3D imaging using a multiphoton scanning confocal microscope is ultimately limited by aberrations of the system. We describe a system to adaptively compensate the aberrations with a deformable mirror. We have increased the transverse scanning range of the microscope by three with compensation of off-axis aberrations.We have also significantly increased the longitudinal scanning depth with compensation of spherical aberrations from the penetration into the sample. Our correction is based on a genetic algorithm that uses second harmonic or two-photon fluorescence signal excited by femtosecond pulses from the sample as the enhancement parameter. This allows us to globally optimize the wavefront without a wavefront measurement. To improve the speed of the optimization we use Zernike polynomials as the basis for correction. Corrections can be stored in a database for look-up with future samples.

The interaction of macromolecules in space and time are known to be important for the regulation of many biochemical reactions. Image correlation spectroscopy (ICS) was recently introduced as an imaging analog of fluorescence correlation spectroscopy optimized for measuring the aggregation state of fluorescently labeled macromolecules on the surface of biological cells. We present two novel developments of dynamic ICS that will greatly enhance our abilities to measure molecular interactions as a function of time and space in living cells. We illustrate the use of a rapid scan two-photon microscope system to collect image series at high time resolution (30 frames/s) for dynamic ICS analysis. Secondly, we demonstrate the implementation of two-color image cross-correlation spectroscopy (ICCS) with a CLSM using multiple wavelength excitation, and with two-photon excitation of samples containing two different fluorescent species. Cross-correlation analysis allows the degree of co- localization of two different fluorophores to be measured directly. By performing two-color ICCS, we can monitor the interactions of non-identical labeled macromolecules as a function of time and space. We describe the experimental setup for both methods and illustrate the application for measurements of the diffusion coefficients of singly and doubly labeled fluorescent microspheres in aqueous solutions.

We describe a Programmable Array Microscope (PAM) system which is implemented using a single Ferroelectric Liquid Crystal Spatial Light Modulator in a double pass configuration. The SLM array is placed such that it is in both the source and detector planes of a confocal microscope. The pixels of the SLM are arranged to form an aperture array similar to the type found in confocal direct- view microscopes (DVMs). Among the advantages of the PAM system over DVM systems are a lack of moving parts, and complete control over the aperture function. We present optical sectioning curves taken using scanned grids of square apertures of varying number and spacing, showing how these parameters affect the confocal behavior. In particular, we demonstrate the effect which the finite contrast ratio of the SLM pixels has on the optical sectioning curve and introduce a simple theory which explains this effect. Finally, we show confocal images captured from test samples using the PAM system.

We describe a simple method to obtain an arbitrary complex optical field. The system employs a ferroelectric liquid crystal spatial light modulator acting as a binary optical element in a 4-f optical system. The ability to produce an arbitrary wavefront permits us to modify the pupil function of an objective lens in any desired manner. Examples of pupil plane filters which enhance certain aspects of the point spread function are described.

There exist a number of fluorescent probes whose lifetimes change in response to ion concentrations (for example H⁺ and Ca²⁺) in the surrounding medium. We describe a technique for utilizing this property in a confocal scanning laser microscope. The technique is based on intensity-modulated laser illumination of the specimen, and phase-sensitive lock-in detection of the fluorescent light. In this way we get a lifetime-dependent output signal which, after calibration, can provide information concerning ion concentrations. In the current study we have used a pH sensitive fluorophore, SNAFL-2, to study the performance of this technique. We find that the sensitivity is such that a pH difference of 0.1 units can easily be detected in an 8- bit digital image. Noise measurements show that under realistic conditions we can expect a pixel-to-pixel standard deviation of approximately one to two pH units.

In this paper we study the spatial distribution and wavefront characteristics of third harmonic generation in relation to some material and interface conditions over the focal region of the fundamental beam. We investigate, mostly from an experimental point of view, the implications the physics of the THG generation process has in situations where THG may be employed for 3D imaging. Due to the non- linear character of the THG generation process it is inherently suitable for this application. For the first time images of the distribution of the THG radiation, as the interface is moved through focus, are shown. Experiments on closely spaced interfaces or bilayers confirm unambiguously the correctness of the vector model for THG generation (Ward et al. 1969) in uniform media. In view of these and other data the image formation, especially for biological objects, with THG radiation will be discussed.

A confocal microscope permits 3D imaging of volume objects by the inclusion of a pinhole in the detector path which eliminates out of focus light. This configuration is however very sensitive to aberrations induced by the specimen or the optical system and would therefore benefit from an adaptive optics approach. We present a wavefront sensor capable of measuring directly the Zernike components of an aberrated wavefront and show that it is particularly applicable to the confocal microscope since only those wavefronts originating in the focal region contribute to the measured aberration.

Fluorescence microscopy is a powerful instrument for the study of cells and other biological objects. One of the actual tasks of the modern microscopy is the study of drugs transportation in cells and it's affect on the different cell organelles. The authors have proposed and tested the new type of the microscope with superimposed interference and fluorescence images of the living cell. The optical setup of the fluorescence interference microscope is based on confocal Linnik's type interference microscope. The fluorescence image gives the information about the distribution of drugs in different parts of the cell. The interference image gives the information about the distribution of dry mass or protein in the different parts of the cell.

The use of a volume holographic filter as collector element in a confocal system imaging through scattering (turbid) material is described. We show that the penetration depth of the volume holographic system is de-coupled from the scatter noise discrimination properties, and is potentially more advantageous than the traditional confocal microscope. Since the volume-holographic filter is a matched-filter, the penetration depth is dependent on the mismatch of the refractive index of the sample being imaged relative to the recording conditions. We present a method to overcome this limitation based on using a pre-compensating index matching film during the recording of the volume hologram. An improvement of the penetration depth is shown experimentally.

Polarization control and analysis have proven to be a powerful tool both in conventional and confocal microscopies, but most of these approaches have used homogeneously polarized pupils. Other groups have had varying levels of success producing beams with spatially inhomogeneous polarization. In this paper, we describe the generation and focusing properties of such beams and their application to scanning laser microscopy. We specifically consider microscopy using the lowest-order azimuthally polarized beam.

Multiple beam interference system is used in conjunction with a conventional scanning confocal microscope to examine the morphology and construction of 3D images of Histolytic Ameba and parasite Candida Albicans. The present combination permits to adjoin advantages of both systems, namely the vertical high contrast and optical sectioning. The interference pattern obtained from a multiple internal reflection of a simple, sandwiched between the glass plate and the cover plate, was focussed on an objective of a scanning confocal microscope. According to optical path differences, morphological details were revealed. The combined features, namely improved resolution in z axis, originated from the interference pattern and the optical sectioning of the confocal scanning system, enhance the resolution and contrast dramatically. These features permitted to obtain unprecedented images of Histolytic Ameba and parasite Candida Albicans. Because of the improved contrast, several details like double wall structure of candida, internal structure of ameba are clearly visible.

The purpose of the investigation was to elaborate a new method of functional imaging of living tumor cells. Human colon carcinoma cells HCT116 were investigated with a conventional light microscope, confocal laser scanning microscope and with a laser phase microscope (LPM). The LPM is a functional imaging technique providing information about cell morphology which is imposed by the physiological inhomogeneity of the refractive index. The phase of the light wave passing through an object contains quantitative information about the object thickness, the shape, and the spatial distribution of the refractive index varying with morphology and chemical composition inhomogeneity inside the object. The new method of investigation of the cells in different stages of the cell cycle is developed. Every phase image of the investigated cells has been compared with conventional light microscopic and confocal microscopic images of the same cell. the relation between the cell state, their morphological peculiarities and the phase characteristics of the measured cell is determined. Data thus acquired, quantitatively characterizing intra- and intercellular processes during the cell cycle, and the method of measurements can be used to investigate with high optic resolution the mechanisms of different physical, chemical and biomolecular interactions with the tumor cells.

Circumventing the optical diffraction limit is important for data storage, photolithography, and imaging. The Solid Immersion Lens (SIL), in which light is focussed through a high refractive-index lens held close to a sample, offers a method for improving resolution. Microfabrication of SILs enables the use of lenses as small as a few wavelengths in diameter. When these are integrated onto cantilevers, the SIL can be scanned over topography with control of lens- sample separation. We have extended Mie theory to investigate aberration in small lenses and the effect of lens size on a converging beam. We also report progress on the fabrication and first results from micromachined silicon SILs.

In time-lapse microscopy image quality is limited by instrumental and specimen characteristics. Moreover, an enormous amount of data may be accumulated. This challenges not only the development of new tools for image processing, but typically forces methods to be executed in combination. To remedy essential problems, an image processing pipeline is employed which features image restoration, image compression, visualization and analysis. To control the flow of information within this pipeline and to optimize the parameter settings like compression rates or number of iterations, the information content is evaluated on the basis of the ratio of signal and noise.

Image restoration algorithms provide efficient tools for recovering part of the information lost in the imaging process of a microscope. We describe recent progress in the application of deconvolution to confocal microscopy. The point spread function of a Biorad-MRC1024 confocal microscope was measured under various imaging conditions, and used to process 3D-confocal images acquired in an intact preparation of the inner ear developed at Karolinska Institutet. Using these experiments we investigate the application of denoising methods based on wavelet analysis as a natural regularization of the deconvolution process. Within the Bayesian approach to image restoration, we compare wavelet denoising with the use of a maximum entropy constraint as another natural regularization method. Numerical experiments performed with test images show a clear advantage of the wavelet denoising approach, allowing to `cool down' the image with respect to the signal, while suppressing much of the fine-scale artifacts appearing during deconvolution due to the presence of noise, incomplete knowledge of the point spread function, or undersampling problems. We further describe a natural development of this approach, which consists of performing the Bayesian inference directly in the wavelet domain.

Prior knowledge about the observed scene represents the key to recovering frequencies beyond the passband of an imaging system (super-resolution). With regard to microscopy mainly two super-resolution mechanisms have been reported: (1) analytic continuation of the frequency spectrum and (2) constrained image deconvolution. This paper describes an alternative super-resolution mechanism. Prior knowledge is introduced on a higher, more symbolic level of visual inference.

Spatial information that exceeds the classical resolution limit by a factor of two can be made visible in the widefield fluorescence microscope by illuminating the sample with spatially structured (patterned) excitation light. By computationally restoring this information to its proper location in reciprocal space, an image with twice the normal lateral resolution can be produced. The method can be applied in three dimensions, and yields an axial sectioning power equal to that of confocal microscopes. Unlike the case in confocal microscopy, however, both the lateral and axial resolution enhancements are achieved without any loss of emission light, resulting in uncompromised sensitivity. The method has been experimentally verified on both test objects and complex biological structures and performs in complete agreement with theoretical predictions. The resulting images possess a visual clarity that dramatically exceeds that of both conventional and confocal microscopes.

Using a white-light interference for high-precision surface structure analysis, 3D profilometry is realized. White-light surface profilers record the position of peak fringe contrast modulating the optical path difference of an imaging interferometer. This method is sensitive to random noise, such as spike or missing data points, which can be interpreted as positions of high fringe contrast. In order to overcome this problem, a median filter is employed for noise reduction of the acquired profile. Interferograms are generated simultaneously by scanning an object in a direction perpendicular to the object surface. These interferograms are filtered by the median filter, the surface height for each point of the image is obtained by finding the position of peak fringe contrast by extended depth from focus. It is shown in the experiment that the proposed method is able to cope with distortions of the fringe contrast envelope of noisy white-light interferograms without smoothing peak fringe contrast and distorting the position of peak. Moreover, the computation becomes fast and simple by using the advanced algorithm, which does not use complex spatial frequency domain processing.

We demonstrate that near-field optical and atomic force microscopy data can be used for super-resolution 3D image restoration in optical sectioning fluorescence microscopy. A crucial feature in this approach is the full integration of such data sets with digital far-field images. The scanned probe data is used to provide modalities for boundary constraints which define surface optical information and spatial domains of optical alterations in a sample with a spatial precision that has been unachievable in the past. A restoration algorithm that can use such a complex of data for 3D image deconvolution is presented. It uses a Tikhonov- Miller regularization scheme and allows for the imposition of different types of constraints to obtain super-resolution deconvolved images. Performance was tested by using simulated 3D imaging. An example is given of the restoration of a 3D wide field optical image of a biological specimen in the presence of atomic force constraints.

The 3D image formation technique using confocal microscopy has allows visualization of the 3D chemical structure in small parts of the bio-body. However, the large-scale 3D structure such as the distribution of chemical components throughout the whole body has not been shown. To allow such large scale visualization of the 3D internal analysis technique for bio-body has been developed.

In order to visualize sugar content of a melon, the relationship between sugar content and absorption spectra was investigated using a near-infrared (NIR) spectrometer. The absorbance at 676 nm, which is close to the chlorophyll absorption band, had a high inverse correlation with sugar content. A high-resolution cooled CCD imaging camera with the band-pass filter of 676 nm was used to capture the spectral absorption image. The calibration method for converting the absorbance on the image into Brix sugar content was developed in accordance with NIR techniques. Applying this method to each pixel of the absorption image, a color distribution map of the sugar content was constructed. In addition, a special slicing device that can cut a melon in each 5 mm thickness was developed in order to create a 3D image of sugar content distribution.

Using a new device which contains an array of microlenses whose focal lengths can be electrically varied, we have been able to control the input from one microlens to a single mode fiber using an applied voltage. For such a microlens array many closely-spaced focal spots can be generated in parallel, and electrically switched to address, potentially, an array of receiver fibers. We show how the particular switching characteristics of the device, whereby the lenses switch from diverging to converging, serves in turn to disperse light and to focus it into the fiber.