A miniaturized setup for sample rotation on a microscope stage has been developed, combined with light sheet, confocal or structured illumination microscopy and applied to living cells as well as to small organisms. This setup permits axial tomography with improved visualization of single cells or small cell clusters as well as an enhanced effective 3D resolution upon sample rotation.
Single cell microscopy in a three-dimensional (3-D) environment is reported. Cells are grown in an agarose culture gel, located within microcapillaries and observed from different sides after adaptation of an innovative device for sample rotation. Thus, z-stacks can be recorded by confocal microscopy in different directions and used for illustration in 3-D. This gives additional information, since cells or organelles that appear superimposed in one direction, may be well resolved in another one. The method is tested and validated with single cells expressing a membrane or a mitochondrially associated green fluorescent protein, or cells accumulating fluorescent quantum dots. In addition, axial tomography supports measurements of cellular uptake and distribution of the anticancer drug doxorubicin in the nucleus (2 to 6 h after incubation) or the cytoplasm (24 h). This paper discusses that upon cell rotation an enhanced optical resolution in lateral direction compared to axial direction can be utilized to obtain an improved effective 3-D resolution, which represents an important step toward super-resolution microscopy of living cells.
For a reliable understanding of cellular processes, high resolution 3D images of the investigated cells are necessary.
Unfortunately, the ability of fluorescence microscopes to image a cell in 3D is limited since the resolution along
the optical axis is by a factor of two to three worse than the transversal resolution. Standard microscopy image
deblurring algorithms like the Total Variation regularized Richardson Lucy algorithm are able to improve the
resolution but the problem of a lower resolution in direction along the optical axis remains. However, it is possible
to overcome this problem using Axial Tomography providing tilted views of the object by rotating it under the
microscope. The rotated images contain additional information about the objects and an advanced method to
reconstruct a 3D image with an isotropic resolution is presented here. First, bleaching has to be corrected in
order to allow a valid registration correcting translational and rotational shifts. Hereby, a multi-resolution rigid
registration method is used in our method. A single high-resolution image can be reconstructed on basis of
all aligned images using an extended Richardson Lucy method. In addition, a Total Variation regularization is
applied in order to guarantee a stable reconstruction result. The results for both simulated and real data show
a considerable improvement of the resolution in direction of the optical axis.
Novel methods of visible light microscopy have overcome the limits of resolution hitherto thought to be insurmountable. The localization microscopy technique presented here based on the principles of Spectral Precision Distance Microscopy (SPDM) with conventional fluorophores under special physical conditions allows to measure the spatial distribution of single fluorescence labeled molecules in entire cells with macromolecular precision which is comparable to a macromolecular effective optical resolution. Based on detection of single molecules, in a novel combination of SPDM and Spatially Modulated Illumination (SMI) microscopy, a lateral (2D) effective optical resolution of cellular nanostructures around 10 - 20 nm (about 1/50th of the exciting wavelength) and a three dimensional (3D) effective optical resolution in the range of 40 - 50 nm are achieved.
The spatially modulated illumination (SMI) microscope is a wide-field fluorescence microscope featuring axially structured illumination, through which access to information about subresolution object structures is obtained. We present a simplified setup where the interference pattern is generated by reflecting the laser beam with a mirror. We characterize our setup by presenting measurements on fluorescent microspheres with diameters ranging from 44 to 200 nm. The results agree well with the sizes provided by the manufacturer. Furthermore, the spheres are analyzed with 458-, 514-, 488-, and 568-nm excitation wavelengths, giving good agreement of the sizes determined at the respective wavelengths. A measurement of the same objects using different excitation wavelengths leads to a size difference of a few nanometers only. The potential of SMI microscopy for the fast analysis of many fluorescent objects is also addressed. In addition, the applicability to biological specimens is shown on fluorescently labeled, specific chromatin domains. The results obtained using the presented mirror geometry agree well with data obtained using a standard SMI microscope setup.
The method of Spectral Precision Distance Microscopy (SPDM) has been used to determine distances between
two FISH (Fluorescence-in situ-Hybridization)-labeled gene regions on chromosome 9. To this end we applied
methods to correct for chromatic aberrations of the microscope optics alone and also of the sample induced
aberrations due to mismatch of the refractive indices. Using a confocal microscope and a threshold based position
determination algorithm, positions could be measured with an accuracy of about 65 nm inside of fixed cell nuclei.
Distances obtained from the measurements have been verified using a 3D computer model of the cell nucleus.
In principle, this SPDM approach could be combined with novel fluorescence microscopes to obtain structural
information well below the optical resolution. At present the precision limit of the distance measurements is set
by variations of the refractive index throughout the specimens.
Spatially Modulated Illumination (SMI) microscopy was applied to determine changes of the local refractive index at discrete fluorescently labeled sites within the cell nucleus. We present measurements on polymerase II complexes, where we found a variation of the local refractive index of 1.38 - 1.55 (standard deviation interval) throughout the nucleus. This variability is not correlated to the accumulations and the extensions of the polymerase II complexes, which have been determined in a previous experiment.1 Local protein accumulations such as adherent transcriptionally active proteins could possibly contribute to such variations, as could also different compactions of the DNA fiber. Altogether, we present a method to precisely obtain a map of the local refractive index inside of cell nuclei, which provides another contrasting mechanism for visualizing sub cellular structures.
Recently, several light microscopical approaches, such as point spread function (PSF) engineering, Spectral Precision Distance Microscopy (SPDM) and related methods have demonstrated the feasibility of measurements considerably beyond the conventional Abbe-Limit of optical resolution in far field light microscopy. The Heidelberg Spatially Modulated Illumination (SMI) microscope is based on the generation of a standing wave light field in the direction of the optical axis of the system. The theoretical PSF is obtained by the manipulation of the sin2-shaped illumination PSF and the epifluorescent detection PSF. Here, we report a software method to obtain online visualization of the axial light distribution of any object detected, developed using Microsoft Visual C++ and based on Windows NT. This strongly facilitates routine application of SMI Microscopy.
Far field optical light microscopy with its unique capability for contactless, non destructive imaging inside thick transparent specimen such as cell nuclei has contributed widely to the present knowledge of the three- dimensional (3D-) architecture of the interphase nucleus. A serious drawback, however, is the limited optical resolution. A recently introduced light microscopical approach, Spectral Precision Distance Microscopy (SPDM) allows the measurement of distances between point-like fluorescent objects of different spectral signature far below the optical resolution criterion as defined by the Full Width at Half Maximum (FWHM) of the point spread function (PSF). Here, an aspect of the theoretical limits of this method was studied by virtual microscopy. The precision of the axial distance measurements was studied, taking into account photon statistics and image analysis. The results indicate that even under low fluorescence intensity conditions typical for biological structure research, a precision of distance measurements in the nanometer range can be determined.
Fluorescence in situ hybridization (FISH) offers an appropriate technique to specifically label any given chromatin region by multi spectrally labeled, specific DNA probes. Using confocal laser scanning microscopy, quantitative measurements on the spatial distribution of labeling sites can be performed in 3D conserved cell nuclei. Recently, 'Spectral Precision Distance Microscopy' has been developed that allows 3D distance measurements between point-like fluorescence objects of different spectral signatures far beyond the diffraction limited resolution. In a well characterized and sequenced DNA region, the Prader- Willi/Angelman region q11-13 on chromosome 15, geometric distances between the fluorescence intensity bary centers of four different 'point-like' labeling sites were measured. More than 300 cell nuclei were evaluated with a 3D resolution equivalent better than 100 nm. The geometric bary center distances in nanometers were compared with the genomic bary center distance in kilobases (kb). A direct correlation, for instance linear correlation between geometric and genomic distances was not observed. From the measured values, a local compaction factor for the high order chromatin folding in the analyzed genome region was calculated. Along the 1000 kb chromatin segment analyzed, which spans nearly the compete Prader-Willi/Angelman region, different compaction factors were found. The compaction factor 40 typical for a straight 30 nm chromatin fiber was not observed. This shows that chromatin folding and compaction in intact nuclei may be more complex. With SPDM, however, a microscopical technique is available that can sensitively analyze chromatin organization in the 100 nm range in 3D conserved cell nuclei.
The determination of the 3D nanostructure of specific chromatic regions is highly relevant for an improved understanding of the functional topology of the genome. The use of different spectral signatures for the labeling and high accuracy nanodistance measurements in the spectral precision distance microscopy mode allows the investigation of the topology of such targets in 3D conserved nuclei. To obtain the required high-accuracy nanolocalization of small targets, interferometric illumination is a well established and reliable tool. New approaches use spatially modulated illumination (SMI) in various ways. In our laboratory a stage controlled optical sectioning through the object was applied. In this case the SMI point spread function is the product of the axial illumination modulation and of the conventional PSF of the microscope objective. Using this approach and an appropriate analysis algorithm, the position of 'point-like' fluorescent objects was determined with an axial localization precision in the range of 2nm. To provide a reliable high precision localization performance also for long time measurements, thermally invariant mounting devices have been developed for the SMI-system. Using this improved system, it was possible to measure the thermal shift induced by the microscope objective itself.
In recent years, confocal Laser-Scanning microscopy became the most sophisticated microscopic technique for 3D-imaging in biomedical microstructure research. Experimental evidence, however, showed that under optical conditions relevant e.g. for nuclear genome analysis, the resolution of such an instrument as given by the Full Width at Half Maximum of the confocal point-spread function (PSF) is limited to about >= 0.3 micrometers laterally and to about >= 0.7 micrometers axially. A recently introduced light- microscopic approach, termed Spectral Precision Distance Microscopy (SPDM), allows the precise measurement of distances and angles between specifically labeled target sites far below the above mentioned resolution limit. SPDM is based on the use of 'point-like' objects labeled with different spectral signatures. Since in most cases, spectral signature differences have been realized by variation of excitation/fluorescence emission spectra, the calibration of chromatic aberrations is of utmost importance. Here, an improved procedure for the correction of chromatic shifts is presented. Statistical errors introduced by the localization accuracy can be minimized by the multiple measurement of the 3D-distances between the same specifically labeled target sites in a number of cases and subsequent averaging. However, the thus obtained mean distance, the estimate for the 'true' distance may be biased and therefore limited by the localization accuracy. Virtual microscopy simulations of test objects using an experimentally obtained PSF, showed that a few thousand detected fluorescence photons are sufficient to measure reliably distances down to about 20 nm, if other sources of error apart from voxelization, digitization and photon noise are negligible.
Confocal laser scanning fluorescence microscopy is presently being used widely in biomedical research. A severe limitation for its use is its often still insufficient resolution. In situ measurements in 3D conserved human cell nuclei showed that distance measurements between fluorescent targets located in the interior of such objects are limited a resolution regime of appr. greater than or equal to 0.3 micrometer in lateral and appr. greater than or equal to 0.7 micrometer in axial direction. A technique to overcome these restrictions is the recently developed Spectral Precision Distance Microscopy (SPM). This approach allows the determination of distances between targets which carry different spectral signatures with high precision. In situ measurements revealed that the SPM approach allows the determination of distances in 3D intact cell nuclei with a 'Resolution Equivalent' better than 50 nm. Here we present an improved chromatic shift calibration method for Spectral Precision Distance Microscopy. Furthermore, micro axial tomography allows the tilting of objects perpendicular to the optical axis; thus two objects can always be tilted in such a way that they can be recorded in the same focal plane. Therefore the error in distance determination is minimized. Here we present some preliminary data for the applicability of spectral precision distance microscopy (SPM) to micro axial microscopy.
High spatial frequencies in the illuminating light of microscopes lead to a shift of the object spatial frequencies detectable through the objective lens. If a suitable procedure is found for evaluation of the measured data, a microscopic image with a higher resolution than under flat illumination can be obtained. A simple method for generation of a laterally modulated illumination pattern is discussed here. A specially constructed diffraction grating was inserted in the illumination beam path at the conjugate object plane (position of the adjustable aperture) and projected through the objective into the object. Microscopic beads were imaged with this method and evaluated with an algorithm based on the structure of the Fourier space. The results indicate an improvement of resolution.
In biological dosimetry after radiation or chemical exposure, it has been well established to estimate exposure doses from the relative rate of aberrant chromosomes, especially dicentric chromosomes in a given number of cells. For this purpose, dose-efficiency curves depending on laboratory parameters (e.g. preparation technique, analysis procedure etc.) have to be measured under standard conditions. For statistical reasons, a high number of chromosomes or cells, respectively, has to be evaluated. For a Chinese hamster cell line (CO60) as a typical model system in mutation research, a dose efficiency relation after H2O2/L-histidine treatment of the cells was determined using the Heidelberg slit-scan flow fluorometer. This technique has the advantage that several thousand chromosomes can be automatically analyzed in a very short time. As expected, for low doses of H2O2/L-histidine exposure, a nearly linear dependence of the relative number of dicentric chromosomes to the concentration of H2O2 was obtained. In order to correlate the relative number of dicentric chromosomes to the relative number of double strand breaks, the cells were analyzed by the technique of the neutral comet assay. The dose dependent `tail moment' obtained from the comet assay also showed a linear behavior. This confirmed the results obtained by slit-scan flow fluorometry. Furthermore, the linear dependence of the dose efficiency curve was well compatible to results obtained by visual counting by means of a fluorescence microscope. In this case chromosome 1 of the Chinese hamster cell line DON was specifically labelled by fluorescence in situ hybridization.
To study the 3D-organization and 3D-pathology of the genome in intact cell nuclei, precise and accurate 3D-object localizations and 3D-distance measurements of fluorescent labelled chromatin-nanostructures are required. For this purpose, a high precision fluorescence microscope setup with spatially modulated excitation (SME) has been built up combining advantages of an epifluorescence microscope with interferometric laser illumination in the direction of the optical axis and optical sectioning. The SME allows high precision localization in the nanometer range resulting in a considerable increase of the axial resolution equivalent. This is shown for a configuration of five fluorescent microspheres of 100 nm diameter. Since the optical sections are acquired in an epifluorescent mode, image analysis procedures of high precision spectral distance microscopy were applied to determine the lateral particle localization by the position of the bary center of intensity. From these data, relative axial distances as well as relative 3D-distances were calculated. The results indicate that distance measurements between fluorescent objects can be performed with an accuracy of more than one order of magnitude better than the lateral epifluorescent optical resolution given the full width half maximum of the central peak of the effective point spread function. Since the definition of the optical resolution in refraction limited optical systems is based on distance measurement, the measure for the accuracy of our results in precision distance calculation is called resolution equivalent.
KEYWORDS: Tomography, Confocal microscopy, 3D metrology, Microscopes, Luminescence, Microscopy, 3D acquisition, Point spread functions, Distance measurement, 3D image processing
For many biological applications, precise and accurate 3D object localizations and 3D-distance measurements are necessary. Point spread functions of artificial objects of subwavelength dimensions have been measured in order to characterize the image forming properties as well as to localize extended objects in both conventional and confocal florescence light microscopy with and without the axial tomographic technique. With the axial tomographic technique it is possible to tilt the object in such a way, that substructures are located in the same focal plane. The distance of two points measured under this optimal perspective fits best to the real 3D-distance. In this case, optical sectioning is unnecessary, if only distance measurements have to be performed.
In a slit-scan flow cytometer particles specifically labelled by fluorochromes (e.g., cells, chromosomes) are aligned coaxially in a flow stream. One by another they pass a ribbon-like shaped laser beam with a diameter smaller than the particle length. Although several slit-scan flow systems have been developed during the last two decades, a complete description of the theory of optical resolution under the real experimental conditions used as well as a description how to overcome experimental limitations are missing. Often, resolution values are estimated under the assumption of ideal Gaussian beam propagation. These estimates suffer from a discrepancy to practical implementation, Here, some of these effects in slit-scan optics are discussed from a more theoretical point of view. In order to obtain an acceptable depth of field, a focal width around 2 micrometer appears to be an optimum under the regime of Gaussian beam propagation. However, in practice, effects due to thick lenses, finite apertures, chromatic aberrations, or the ellipticity of the laser beam overshadow this result and influence the laser beam shape. To further improve the resolution with a high depth of field, new concepts are required. Therefore, a combination of an interference fringe pattern of two coherent laser beams for excitation (fringe-scanning) with a slit-scan detection of the incoherent fluorescence light is introduced. Preliminary experiences of the first experimental realization are discussed.
Quantitative measurements in far field light microscopy are complicated by the different lateral and axial resolutions. For principle reasons the spatial resolution in the direction of the optical axis is lower than in the focal plane. To overcome these limitations, we have developed a 2(pi) -tilting device for full specimen rotation perpendicular to the optical axis. Due to the influence of specimen and mounting media on the spatial resolution of a CLSM, the focal shift increases with the refractive index mismatch and the depth of the investigated region. Attenuation and absorption effects of excitation and emitted light due to layer thickness and refractive index mismatches have to be considered. By means of a capillary with a square shaped cavity in combination with the tilting device, it may become possible to directly calibrate the confocal system in the direction of the optical axis. With this technique it is possible to test 3D deconvolution and segmentation procedures applied to the same object acquired under different perspectives.
Chromosomes play a fundamental role in heredity. This reasons the interest in new highly resolving microscopical techniques for their analysis. New preparation techniques have offered a direct approach to detect specific nucleic acid sequences by in situ hybridization. Labelled DNA probes detected by fluorochrome conjugates make it possible to visualize regions down to single genes by light microscopy. Scanning force microscopy (SFM) provides the possibility to image surfaces of biological objects with a resolution one to two orders of magnitude better than a classical fluorescence light microscope. Here, air dried human metaphase chromosomes were examined by SFM before and after in situ hybridization. Different hybridization protocols were compared in their influence on chromosomal morphology. An immunogold technique was introduced for topographic labeling detection by SFM. By propidium iodide staining, identically the same chromosomes which were already examined by SFM were visualized by high resolution confocal light microscopy. The SFM results suggest that the hybridization procedure induced alterations in the overall chromosomal morphology which were not directly detectable by the fluorescence image in light microscopy. Using the immunogold labelling technique and silver enhancement, it was possible to study hybridization features and chromosomal morphology at high resolution simultaneously by SFM. The application of this approach may offer possibilities to investigate the hybridization mechanisms and to develop new hybridization protocols inducing minimal ultrastructural effects on the chromosomes.
The spatial resolution of a conventional light microscope or a confocal laser scanning microscope can be determined by calculating the point spread function for the objective used. Normally, ideal conditions are assumed for these calculations. Such conditions, however, are often not fulfilled in biological applications especially in those cases where biochemical requirements (e.g. buffer conditions) influence the specimen preparation on the microscope slide (i.e. 'practical' light microscopy). It has been shown that the problem of a reduced z- resolution in 3D-microscopy (optical sectioning) can be overcome by a capillary in a 2(pi) - tilting device that allows object rotation into an optimal perspective. The application of the glass capillary instead of a standard slide has an additional influence on the imaging properties of the microscope. Therefore, another 2(pi) -tilting device was developed, using a glass fiber for object fixation and rotation. Such a fiber could be covered by standard cover glasses. To estimate the resolution of this setup, point spread functions were measured under different conditions using fluorescent microspheres of subwavelength dimensions. Results obtained from standard slide setups were compared to the glass fiber setup. These results showed that in practice rotation leads to an overall 3D-resolution improvement.
Slit-scan flow fluorometry is a laser-technological approach for accelerated screening and sorting of fluorescence labelled metaphase chromosomes. Details of the optics of the Heidelberg slit-scan sorter are presented. In a fluid stream the fluorescence labelled chromosomes rapidly pass one at a time by a scanning laser beam. The laser can be focused by a less complex optic consisting of only a few commercially available lenses. The laser intensity distribution around the focus was measured for 488 nm for two lens configurations. Although the light distribution obtained by such an optic is normally not aberration free, the requirements of a 'ribbonlike' shape in the center of the fluid stream can be fulfilled. Since the chromosomes are oriented perpendicularly to the laser beam by hydrodynamic focusing of the fluid stream, the fluorescence intensity along the chromosome axis can be measured time (equals spatially) resolved. According to their intensity profiles the chromosomes can be classified. Signal processing of the profiles can be performed in less than 600 microseconds, so that in the order of hundred chromosomes per second can be sorted out by a computer controlled electro-acoustic sorting unit. The final spatial resolution of a slit-scan flow sorter is not only affected by the focusing optics of the laser but also by the fluid stream, the detection optics and electronics, as well as by the computer analysis algorithm. Calculations often consider only the optics under ideal conditions. Here, a method is shown how to estimate the overall resolution of a slit-scan flow fluorometer experimentally. According to this criterion the resolution of the Heidelberg slit-scan sorter for 488 nm fluorescence excitation was estimated to be 2.4 micrometer in its basic optical configuration and 1.7 micrometer with additional correction of chromatic aberration effects.
Laser fluorescence activated slit-scan flow cytometry offers an approach to a fast, quantitative characterization of chromosomes due to morphological features. It can be applied for screening of chromosomal abnormalities. We give a preliminary report on the development of the Heidelberg slit-scan flow cytometer. Time-resolved measurements of the fluorescence intensity along the chromosome axis can be registered simultaneously for two parameters when the chromosome passes perpendicularly through a narrowly focused laser beam combined by a detection slit in the image plane. So far automated data analysis has been performed off-line on a PC. In its final performance, the Heidelberg slit-scan flow cytometer will achieve on-line data analysis that allows an electroacoustical sorting of chromosomes of interest. Interest is high in the agriculture field to study chromosome aberrations that influence the size of litters in pig (Sus scrofa domestica) breeding. Slit-scan measurements have been performed to characterize chromosomes of pigs; we present results for chromosome 1 and a translocation chromosome 6/15.
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