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Tomographic diffraction microscopy (TDM) is a generalisation of digital holographic microscopy (DHM), for which the illumination angle onto the sample is fully controlled, which has become a tool of choice for 3D, high-resolution imaging of unlabelled samples. TDM makes it possible to obtain the optical field in both amplitude and phase for each illumination angle. Proper information reallocation eventually allows for 3D reconstruction of the complex refractive index map. On the other hand, polarisation array sensors (PAS) paves new way for TDM, as vectorial information assessment about the investigated sample. In this contribution, we show an alternative use of this polarisation information based on the phase sensitive nature of TDM. Here, we demonstrated that TDM coupled with PAS can lead to a 3D differential interference contrast (DIC) microscope with almost no experimental configuration modification., (© 2022 The Authors. Journal of Microscopy published by John Wiley & Sons Ltd on behalf of Royal Microscopical Society.)

Adaptive optics is being applied widely to a range of microscopies in order to improve imaging quality in the presence of specimen-induced aberrations. We present here the first implementation of wavefront-sensorless adaptive optics for a laser-free, aperture correlation, spinning disk microscope. This widefield method provides confocal-like optical sectioning through use of a patterned disk in the illumination and detection paths. Like other high-resolution microscopes, its operation is compromised by aberrations due to refractive index mismatch and variations within the specimen. Correction of such aberrations shows improved signal level, contrast and resolution., (© 2020 The Authors. Journal of Microscopy published by John Wiley & Sons Ltd on behalf of Royal Microscopical Society.)

The aim of this study is to extend, for a larger range of mineral volume ( V1) and different aqueous immersion media, the model for calculating the refractive index of the non-mineral phase of the human dental enamel proposed by Sousa et al. Published experimental birefringence data of carious and developing human enamel in air and aqueous immersion media with different refractive indexes were interpreted. For 48%≤ V1≤ 95%, quantitative estimates on the non-mineral contents consistent with the values described in the literature were derived. New polynomial relationships of the non-mineral contents as a function of the mineral content (48%≤ V1≤ 95%) are introduced, which allows computing the theoretical mean enamel birefringence in water and air for most of the points of developing, carious and mature enamel with a difference of one order of magnitude in relation to the classical model for the non-mineral content's refractive index and closer to the experimental data. For enamel birefringence in immersion media other than water and air, a less expressive improvement was obtained. Perspectives for future studies are discussed. [ABSTRACT FROM AUTHOR]

The main problem in interpreting birefringence of dental enamel under polarizing microscopy is the lack of physical constants able to allow the Wiener equation to be applied directly to the composition of such tissue. The present study introduces a new approach to circumvent this constraint. Because the nonmineral phase of enamel is heterogeneous, its refractive index can be computed in terms of its components (namely, water, which is partially replaced by the immersion medium, and organic matter), thereby providing a more acceptable refractive index to be used in the Wiener equation. Furthermore, the enamel mineral volume is ordinarily calculated on the basis of the density 3.15 g cm−3. The density 2.99 g cm−3 has been, however, reported to be more accurate for enamel hydroxyapatite, so enamel mineral volumes from selected published data were converted using such a density. The birefringence of mature enamel computed by the Wiener equation, taking into account the above refinements, matched, for the first time, published experimental birefringence values. The theoretical water and organic contents were also consistent with published experimental data. Thus, a direct application of the Wiener equation to the enamel composition has now been achieved. It is speculated that quantitative data on the mineral, the water and the organic contents of mature dental enamel can be derived from interpretation of birefringence in two immersion media (obtained before and after extraction of the organic matter) with this new approach. [ABSTRACT FROM AUTHOR]

Observations using conventional microscopy often lack information related to the fine structures of an object, such as intensity and phase, because of limitations in the lens aperture. In this study, the intensity and phase information are appropriately converted into an electrical signal using laser scanning and photodetectors. Intensity and phase are completely separated, and the missing information is restored based on the frequency of the electrical signal. Using this method, the original intensity and phase information of the object to be observed can be correctly restored. Therefore, we propose a novel method to calculate the degree of intensity and phase modulation by calculating the direct and alternating current components obtained from the output of the sum and difference of the two photodetectors. The degree of spatial frequency modulation is corrected according to the electrical signal frequency to detect transparent or unstained cells. We first performed laser scanning of an object. Then, signals were detected using two photodetectors placed in the far-field, separated by the optical axis as the boundary. The output signals of the photodetectors were processed and the intensity and phase were unambiguously separated, thus allowing the visualization of the phase information of the transparent bodies and unstained cells. Spatial frequency correction was performed to correct the modulation. Our method successfully separated the information related to the intensity and optical path difference (OPD). In future work, by accurately correcting the intensity and OPD, it will be possible to separate the absorption rate from the ratio of the irradiation light intensity to the observed intensity and to separate the OPD into the refractive index and the thickness information. This method allows the accurate determination of these parameters in a noninvasive manner., (© 2021 Royal Microscopical Society.)

The interface of mediums with refractive indices discontinuous, for example air-glass and glass-water, are inevitable in microscopic imaging. In this work, the aberration of oblique interface with refractive index discontinuous on the laser scanning microscope was investigated theoretically with numerical simulations. It was found that the position, shape and FWHM of focal spots, were all significantly affected by the aberration due to oblique interface. The aberration can cause serious shifting of focal spots in the axial direction of beam during z -scanning and lead to an inaccurate reconstruction of three-dimensional (3D) targets. The aberration can also lead to a decreasing spatial resolution. To correct the influence of the aberration, a pure-phase modulation method has been proposed. By applying a phase compensation map into a spatial light modulator (SLM), the oblique interface aberration had been corrected experimentally in a laser scanning microscope. We hope this research can attract the attention of researchers when using scanning microscope, especially for reconstructing 3D biological and material structures., (© 2021 Royal Microscopical Society.)

The x-z cross-sectional profiles of fluorescent objects can be distorted in confocal microscopy, in large part due to mismatch between the refractive index of the immersion medium of typical high numerical aperture objectives and the refractive index of the medium in which the sample is present. Here, we introduce a method to mount fluorescent samples parallel to the optical axis. This mounting allows direct imaging of what would normally be an x-z cross-section of the object, in the x-y plane of the microscope. With this approach, the x-y cross-sections of fluorescent beads were seen to have significantly lower shape-distortions as compared to x-z cross-sections reconstructed from confocal z-stacks. We further tested the method for imaging of nuclear and cellular heights in cultured cells, and found that they are significantly flatter than previously reported. This approach allows improved imaging of the x-z cross-section of fluorescent samples. LAY DESCRIPTION: Optical distortions are common in confocal microscopy. In particular, the mismatch between the refractive index of the immersion medium of the microscope objective and the refractive index of the sample medium distorts the shapes of fluorescent objects in the x-z plane of the microscope. Here, we introduced a method to eliminate the shape-distortion in the x-z cross-sections. This was achieved by mounting fluorescent samples on vertical glass slides such that the cross-sections orthogonal to the glass surface could be imaged in the x-y plane of the microscope. Our method successfully improved the imaging of nuclear and cellular heights in cultured cells and revealed that the heights were significantly flatter than previously reported with conventional approaches., (© 2020 Royal Microscopical Society.)

Correct interpretation of image contrast obtained with full-field optical coherence tomography (FFOCT) technique is required for accurate medical diagnosis applications. In this work, first, the characteristics of microscopic structures of tissue that generate the contrast in en-face tomographic image obtained with FFOCT are discussed. Then an overview is given of the parameters that affect image contrast. Finally, the contrast correction factor for correct image interpretation and the contrast limits to practical FFOCT systems are outlined., (© 2015 The Authors Journal of Microscopy © 2015 Royal Microscopical Society.)

Localization microscopy relies on computationally efficient Gaussian approximations of the point spread function for the calculation of fluorophore positions. Theoretical predictions show that under specific experimental conditions, localization accuracy is significantly improved when the localization is performed using a more realistic model. Here, we show how this can be achieved by considering three-dimensional (3-D) point spread function models for the wide field microscope. We introduce a least-squares point spread function fitting framework that utilizes the Gibson and Lanni model and propose a computationally efficient way for evaluating its derivative functions. We demonstrate the usefulness of the proposed approach with algorithms for particle localization and defocus estimation, both implemented as plugins for ImageJ., (© 2012 The Authors Journal of Microscopy © 2012 Royal Microscopical Society.)

This study proposes a method for measuring the refractive index of articular cartilage within a thin and small specimen slice. The cartilage specimen, with a thickness of about 50 μm, was put next to a thin film of immersion oil of similar thickness. Both the articular cartilage and immersion oil were scanned along the depth direction using a confocal microscope. The refractive index mismatch between the cartilage and the immersion oil induced a slight axial deformation in the confocal images of the cartilage specimen that was accurately measured by a subpixel edge-detection-based technique. A theoretical model was built to quantify the focal shift of confocal microscopy caused by the refractive index mismatch. With the quantitative deformations of cartilage images and the quantified function of focal shift, the refractive index of articular cartilage was accurately interpolated. At 561 nm, 0.1 MPa and 20 °C, the overall refractive index of the six cartilage plugs was 1.3975 ± 0.0156. The overall coefficient of variation of all cartilage specimens was 0.68%, which indicated the high repeatability of our method. The verification experiments using distilled water showed a minimal relative error of 0.02%., (© 2012 The Authors Journal of Microscopy © 2012 Royal Microscopical Society.)

Microscopic fluorescent samples of interest to cell and molecular biology are commonly embedded in an aqueous medium near a solid surface that is coated with a thin film such as a lipid multilayer, collagen, acrylamide, or a cell wall. Both excitation and emission of fluorescent single molecules near film-coated surfaces are strongly affected by the proximity of the coated surface, the film thickness, its refractive index and the fluorophore's orientation. For total internal reflection excitation, multiple reflections in the film can lead to resonance peaks in the evanescent intensity versus incidence angle curve. For emission, multiple reflections arising from the fluorophore's near field emission can create a distinct intensity pattern in both the back focal plane and the image plane of a high aperture objective. This theoretical analysis discusses how these features can be used to report film thickness and refractive index, and fluorophore axial position and orientation., (© 2012 The Author Journal of Microscopy © 2012 Royal Microscopical Society.)

This study examined the action of anisosmotic media on the volume of nucleated erythrocytes isolated from Rana temporaria. Elevation of medium osmolarity from 100 to 345 mOsm resulted in attenuation of mean cell volume by more than 3-fold, estimated by hematocrit measurement. By contrast to this 'classic' erythrocyte volume evaluation technique, we did not observe any significant cell volume modulation by examining the 3D reconstruction of erythrocyte interference images obtained by laser interference microscopy. Comparative analysis of mean cell volume, phase height and cell area appraised by laser interference microscopy showed that the lack of visible alterations of phase image geometry was caused by sharp elevation of the average refractive index of the cytoplasm in shrunken cells. Thus, our results show for the first time that laser interference microscopy in combination with a direct method for cell volume measurement may be employed for estimation of the refractory index of intracellular milieu and for assessment of changes of physical chemical properties of the cytoplasm evoked by diverse stimuli including osmotic stress., (© 2011 The Authors Journal of Microscopy © 2011 Royal Microscopical Society.)

Multiphoton fluorescence excitation microscopy is almost invariably conducted with samples whose refractive index differ from that of the objective immersion medium, conditions that cause spherical aberration. Due to the quadratic nature of multiphoton fluorescence excitation, spherical aberration is expected to profoundly affect the depth dependence of fluorescence excitation. In order to determine the effect of refractive index mismatch in multiphoton fluorescence excitation microscopy, we measured signal attenuation, photobleaching rates and resolution degradation with depth in homogeneous samples with minimal light scattering and absorption over a range of refractive indices. These studies demonstrate that signal levels and resolution both rapidly decline with depth into refractive index mismatched samples. Analyses of photobleaching rates indicate that the preponderance of signal attenuation with depth results from decreased rates of fluorescence excitation, even in a system with a descanned emission collection pathway. Similar results were obtained in analyses of fluorescence microspheres embedded in rat kidney tissue, demonstrating that spherical aberration is an important limiting factor in multiphoton fluorescence excitation microscopy of biological samples., (© 2010 The Authors Journal of Microscopy © 2010 Royal Microscopical Society.)

Although multiphoton fluorescence excitation microscopy has improved the depth at which useful fluorescence images can be collected in biological tissues, the reach of multiphoton fluorescence excitation microscopy is nonetheless limited by tissue scattering and spherical aberration. Scattering can be reduced in fixed samples by mounting in a medium whose refractive index closely matches that of the fixed material. Using optical 'clearing', the effects of refractive index heterogeneity on signal attenuation with depth are investigated. Quantitative measurements show that by mounting kidney tissue in a high refractive index medium, less than 50% of signal attenuates in 100 μm of depth., (© 2010 The Authors Journal of Microscopy © 2010 The Royal Microscopical Society.)

We present a novel approach for deconvolution of 3D image stacks of cortical tissue taken by mosaic/optical-sectioning technology, using a transmitted light brightfield microscope. Mosaic/optical-sectioning offers the possibility of imaging large volumes (e.g. from cortical sections) on a millimetre scale at sub-micrometre resolution. However, a blurred contribution from out-of-focus light results in an image quality that usually prohibits 3D quantitative analysis. Such quantitative analysis is only possible after deblurring by deconvolution. The resulting image quality is strongly dependent on how accurate the point spread function used for deconvolution resembles the properties of the imaging system. Since direct measurement of the true point spread function is laborious and modelled point spread functions usually deviate from measured ones, we present a method of optimizing the microscope until it meets almost ideal imaging conditions. These conditions are validated by measuring the aberration function of the microscope and tissue using a Shack-Hartmann sensor. The analysis shows that cortical tissue from rat brains embedded in Mowiol and imaged by an oil-immersion objective can be regarded as having a homogeneous index of refraction. In addition, the amount of spherical aberration that is caused by the optics or the specimen is relatively low. Consequently the image formation is simplified to refraction between the embedding and immersion medium and to 3D diffraction at the finite entrance pupil of the objective. The resulting model point spread function is applied to the image stacks by linear or iterative deconvolution algorithms. For the presented dataset of large 3D images the linear approach proves to be superior. The linear deconvolution yields a significant improvement in signal-to-noise ratio and resolution. This novel approach allows a quantitative analysis of the cortical image stacks such as the reconstruction of biocytin-stained neuronal dendrites and axons.

We propose a method to image the surface topography of transparent objects. The space between the object and the opposite closely positioned surface (such as a cover glass or a slide) is filled with a strongly absorbing dye. The contrast is generated by recording a transmission image at a wavelength where the dye absorbs. Since the transmitted intensity depends on the depth of the dye layer, it carries information about the relief of the tested surface. With sufficiently concentrated dyes, nanometre unevenness of a surface can be detected. By using less-concentrated solutions, it is possible to image and measure larger objects, such as biological cells. At the present stage, biological applications of the method are only semi-quantitative, but the method still provides detailed information about cell shapes that is not readily obtainable with other imaging techniques. Conversion of the image grey scale into the units of vertical distance requires knowledge of the absorption coefficient of the dye. The same method that is used for imaging can be adapted to measure the absorption coefficient of concentrated dyes. The solution to be analyzed is placed between a glass slide and a spherical lens of known radius. The absorption coefficient is determined from attenuation of transmitted intensity as a function of the distance to the centre. At the same time, the interference pattern in the reflected image allows measurement of the refractive index of the dye.

Confocal or multiphoton microscopes, which deliver optical sections and three-dimensional (3D) images of thick specimens, are widely used in biology. These techniques, however, are sensitive to aberrations that may originate from the refractive index structure of the specimen itself. The aberrations cause reduced signal intensity and the 3D resolution of the instrument is compromised. It has been suggested to correct for aberrations in confocal microscopes using adaptive optics. In order to define the design specifications for such adaptive optics systems, one has to know the amount of aberrations present for typical applications such as with biological samples. We have built a phase stepping interferometer microscope that directly measures the aberration of the wavefront. The modal content of the wavefront is extracted by employing Zernike mode decomposition. Results for typical biological specimens are presented. It was found for all samples investigated that higher order Zernike modes give only a small contribution to the overall aberration. Therefore, these higher order modes can be neglected in future adaptive optics sensing and correction schemes implemented into confocal or multiphoton microscopes, leading to more efficient designs.

Total internal reflection fluorescence (TIRF) microscopy is finding increasing application for selectively detecting molecules at or near a glass-water surface. As with all fluorescence methods, the efficiency of excitation of a fluorophore is potentially sensitive to the polarization state of the source. In TIRF, s-polarized excitation produces an evanescent field that is perpendicular to the incident plane (y direction), whereas p-polarized light generates a more complex pattern but one dominated by a field that is vertical to the surface (z direction). Thus, fluorophores whose absorption dipoles are fixed in the x direction are not favourably aligned for excitation. Here we describe a beam-splitting prism arrangement that allows excitation by two orthogonal beams, thus giving isotropic excitation in the x-y plane with s-polarized light. With linearly polarized light at the magic angle, near isotropic excitation in three dimensions should be achieved. This prism design should find application in polarized fluorescence microscopy to investigate the rotational motions of macromolecules or to minimize flickering of fluorescence emission arising from molecular rotations in single molecule studies.

We consider various strategies for confocal imaging of human skin which seek to reduce the effects of the specimen-induced aberrations. We calculate the spherical aberration introduced by the stratified structure of skin and show how the confocal signal is affected when attempting to image at various depths within the dermis. Using simple methods it is shown how images might be improved by compensating for the induced aberration. The methods include the use of an iris to reduce the pupil area, changing the refractive index of the immersion medium and using a lens with variable coverglass correction.

The method of using immersion medium to correct spherical aberration for water immersion objectives when the samples are not water is investigated. Spherical aberration is measured by an interferometer converted from a confocal microscope for samples with different refractive indices. When the proper refractive index of the immersion medium and thickness of cover slip are selected, the measured spherical aberration approaches zero. A theoretical model can be used for prediction of the immersion medium to correct spherical aberration for various samples. Using the thinnest available cover slip (100 microm), the zero spherical aberration condition can be applied to samples with refractive index as high as 1.40. Confocal images in the condition of almost no spherical aberration are included to demonstrate the improvement of axial resolution due to this correction.

The wavelength dependence of the incoherent point spread function in a wide-field microscope was investigated experimentally. Dispersion in the sample and optics can lead to significant changes in the point spread function as wavelength is varied over the range commonly used in fluorescence microscopy. For a given sample, optical conditions can generally be optimized to produce a point spread function largely free of spherical aberration at a given wavelength. Unfortunately, deviations in wavelength from this value will result in spherically aberrated point spread functions. Therefore, when multiple fluorophores are used to localize different components in the same sample, the image of the distribution of at least one of the fluorophores will be spherically aberrated. This aberration causes a loss of intensity and resolution, thereby complicating the localization and analysis of multiple components in a multi-wavelength image. We show that optimal resolution can be restored to a spherically aberrated image by constrained, iterative deconvolution, as long as the spherical aberration in the point spread function used for deconvolution matches the aberration in the image reasonably well. The success of this method is essentially independent of the initial degree of spherical aberration in the image. Deconvolution of many biological images can be achieved by collecting a small library of spherically aberrated and unaberrated point spread functions, and then choosing a point spread function appropriate for deconvolving each image. The co-localization and relative intensities of multiple components can then be accurately studied in a multi-wavelength image.

In optical microscopic measurement of internal blood-vessel diameters, the effect of refraction must be taken into account to ensure accuracy of the result. This effect is discussed and an analytical correction formula derived. Phantom blood vessels with known internal and external diameters were used to test the validity of the correction formula. The errors obtained prior to correction were reduced significantly after correction.

Using a Jamin-Lebedeff-type interference microscope an analysis of frozen sections of human first trimester chorionic villi reveals regional differences down to subcellular resolution. The evidence indicates compositional differences between villus cell types and shows that the syncytiotrophoblast is differentiated into at least three layers, one of which corresponds positionally to the previously described syncytioskeletal layer. Quantitative measurements have been made of specimen thickness, refractive index and dry mass of regions in the tissue. Local differences in syncytiotrophoblast have been noted with respect to the content of a population of organelles with distinctive optical properties. These may correspond to stored forms of steroid hormone or their precursors.

An experimental setup has been built to correct the errors caused by overlapping 'ghost' images when scanning and integrating measurements are carried out with the Jamin-Libedef interference microscope. First a reference field with the object, producing the 'ghost' image, is scanned and the values of the optical path difference (OPD) of each point in the reference field are stored in a computer. Subsequently, the OPD of the object to be measured is calculated for each point by adding the measuring result to the average OPD of five points, located around the corresponding point in the reference field. The applicability of the setup has been tested by measurements with Sepharose beads, Hela cells and the Zeiss microinterference refractometer. The difference between measurements on the same object with and without overlapping 'ghost' image was about 10%. The reproducibility was tested by repeated measurements on the same cell whereby a standard deviation of 1.1% was found.

The method of Duc de Chaulnes was employed to determine the mechanical and optical thickness, as well as the refractive index, of transparent tissue layers in living specimens. To this end the reproducible accuracy of the method and its dependence on the adjustment in focusing and on the numerical aperture of the objective was evaluated on test specimens using the procedures of transmitted light, phase-contrast (PC), and differential interference contrast (DIC) microscopy. The best working conditions were then applied to the actual measurements.