Modulated Illumination Microscopy Research Articles

Modulated illumination microscopy: Application perspectives in nuclear nanostructure analysis.

The structure of the cell nucleus of higher organisms has become a major topic of advanced light microscopy. So far, a variety of methods have been applied, including confocal laser scanning fluorescence microscopy, 4Pi, STED and localisation microscopy approaches, as well as different types of patterned illumination microscopy, modulated either laterally (in the object plane) or axially (along the optical axis). Based on our experience, we discuss here some application perspectives of Modulated Illumination Microscopy (MIM) and its combination with single-molecule localisation microscopy (SMLM). For example, spatially modulated illumination microscopy/SMI (illumination modulation along the optical axis) has been used to determine the axial extension (size) of small, optically isolated fluorescent objects between ≤ 200nm and ≥ 40nm diameter with a precision down to the few nm range; it also allows the axial positioning of such structures down to the 1nm scale; combined with laterally structured illumination/SIM, a 3D localisation precision of ≤1nm is expected using fluorescence yields typical for SMLM applications. Together with the nanosizing capability of SMI, this can be used to analyse macromolecular nuclear complexes with a resolution approaching that of cryoelectron microscopy.

Ultraviolet-Visible Multifunctional Vortex Metaplates by Breaking Conventional Rotational Symmetry.

Metasurfaces have shown remarkable potential to manipulate many of light's intrinsic properties, such as phase, amplitude, and polarization. Recent advancements in nanofabrication technologies and persistent efforts from the research community result in the realization of highly efficient, broadband, and multifunctional metasurfaces. Simultaneous control of these characteristics in a single-layered metasurface will be an apparent technological extension. Here, we demonstrate a broadband multifunctional metasurface platform with the unprecedented ability to independently control the phase profile for two orthogonal polarization states of incident light over dual-wavelength spectra (ultraviolet to visible). In this work, multiple single-layered metasurfaces composed of bandgap-engineered silicon nitride nanoantennas are designed, fabricated, and optically characterized to demonstrate broadband multifunctional light manipulation ability, including structured beam generation and meta-interferometer implementation. We envision the presented metasurface platform opening new avenues for broadband multifunctional applications including ultraviolet-visible spectroscopy, spatially modulated illumination microscopy, optical data storage, and information encoding.

Spatially modulated illumination microscopy: application perspectives in nuclear nanostructure analysis

Thousands of genes and the complex biochemical networks for their transcription are packed in the micrometer sized cell nucleus. To control biochemical processes, spatial organization plays a key role. Hence the structure of the cell nucleus of higher organisms has emerged as a main topic of advanced light microscopy. So far, a variety of methods have been applied for this, including confocal laser scanning fluorescence microscopy, 4Pi-, STED- and localization microscopy approaches, as well as (laterally) structured illumination microscopy (SIM). Here, we summarize the state of the art and discuss application perspectives for nuclear nanostructure analysis of spatially modulated illumination (SMI). SMI is a widefield-based approach to using axially structured illumination patterns to determine the axial extension (size) of small, optically isolated fluorescent objects between less than or equal to 200 nm and greater than or equal to 40 nm diameter with a precision down to the few nm range; in addition, it allows the axial positioning of such structures down to the 1 nm scale. Combined with SIM, a three-dimensional localization precision of less than or equal to 1 nm is expected to become feasible using fluorescence yields typical for single molecule localization microscopy applications. Together with its nanosizing capability, this may eventually be used to analyse macromolecular complexes and other nanostructures with a topological resolution, further narrowing the gap to Cryoelectron microscopy. This article is part of the Theo Murphy meeting issue ‘Super-resolution structured illumination microscopy (part 2)’.

Technological Innovation Leads to Fundamental Understanding in Cell Biology

Technological Innovation Leads to Fundamental Understanding in Cell Biology

Nanostructure analysis using spatially modulated illumination microscopy

We describe the usage of the spatially modulated illumination (SMI) microscope to estimate the sizes (and/or positions) of fluorescently labeled cellular nanostructures, including a brief introduction to the instrument and its handling. The principle setup of the SMI microscope will be introduced to explain the measures necessary for a successful nanostructure analysis, before the steps for sample preparation, data acquisition and evaluation are given. The protocol starts with cells already attached to the cover glass. The protocol and duration outlined here are typical for fixed specimens; however, considerably faster data acquisition and in vivo measurements are possible.

Spatially modulated illumination microscopy using one objective lens

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 cell nucleus taking centre stage. Workshop on the Functional Organization of the Cell Nucleus

The third European Molecular Biology Organization (EMBO) workshop on the Functional Organization of the Cell Nucleus was held in Prague between 5 and 8 May 2006, and was organized by U. Aebi, I. Raska and W. Earnshaw. ![][1] The cell nucleus is a highly complex organelle that hosts the chromosomes and many distinct macromolecular domains. Knowing the functional organization of the nucleus is essential for understanding many of the cell functions that are important for health and disease. This field has progressed considerably towards revealing the structure, dynamics and organization of the nucleus, as was evident from the excellent talks and discussions at the third European Molecular Biology Organization (EMBO) workshop on the Functional Organization of the Cell Nucleus held in the beautiful city of Prague. The workshop was attended by 115 participants, and included 31 speakers and 32 shorter talks chosen from the posters. The chromosomes form discrete domains within the nucleus. Outside these domains, a compartmentalization activity might dynamically organize nuclear bodies and speckles (Cremer & Cremer, 2001). T. Cremer (Munich, Germany) reported that increased osmolarity caused the condensation of interphase chromosomes and enlargement of the interchromatin compartment. A return to normal osmotic conditions caused chromosomes to occupy their previous positions, indicating a structural ‘memory’ of both chromosome positions and the interchromatin compartment containing the nuclear bodies. At present, it is not known which structural elements are engaged in, and necessary for, this remarkable preservation of the topography of the chromatin ‘network’. In particular, detailed structural information on the chromosome surface and the space between individual chromosome territories is missing. Therefore, new optical microscopy and cryo‐electron tomography methods are urgently needed to unravel the molecular organization of this part of the nuclear architecture. C. Cremer (Heidelberg, Germany) described an improvement in optical resolution by spatially modulated illumination microscopy, which … [1]: /embed/graphic-1.gif

Transillumination spatially modulated illumination microscopy

The diagnostic utility of a conventional transillumination microscope, the most common imaging modality in clinical use today, is limited by the microscope's resolution. It is, however, possible to achieve lateral resolution well beyond the classical limit by using laterally structured illumination in a wide-field, nonconfocal microscope. In this method, the spatially modulated illumination (SMI) makes high-resolution information that is normally inaccessible visible in the observed image. Previously presented SMI microscopy systems operated in epifluorescence mode. We describe the design, construction, and testing of a novel transillumination SMI microscope. As transillumination is necessary for most medical applications, such as histopathologic evaluation of biopsy tissue and chromosomal analysis, such a system should have a significant diagnostic effect.

Nano-Sizing of Specific Gene Domains in Intact Human Cell Nuclei by Spatially Modulated Illumination Light Microscopy

Although light microscopy and three-dimensional image analysis have made considerable progress during the last decade, it is still challenging to analyze the genome nano-architecture of specific gene domains in three-dimensional cell nuclei by fluorescence microscopy. Here, we present for the first time chromatin compaction measurements in human lymphocyte cell nuclei for three different, specific gene domains using a novel light microscopic approach called Spatially Modulated Illumination microscopy. Gene domains for p53, p58, and c-myc were labeled by fluorescence in situ hybridization and the sizes of the fluorescence in situ hybridization "spots" were measured. The mean diameters of the gene domains were determined to 103 nm (c-myc), 119 nm (p53), and 123 nm (p58) and did not correlate to the genomic, labeled sequence length. Assuming a spherical domain shape, these values would correspond to volumes of 5.7 x 10(-4) microm(3) (c-myc), 8.9 x 10(-4) microm(3) (p53), and 9.7 x 10(-4) microm(3) (p58). These volumes are approximately 2 orders of magnitude smaller than the diffraction limited illumination or observation volume, respectively, in a confocal laser scanning microscope using a high numerical aperture objective lens. By comparison of the labeled sequence length to the domain size, compaction ratios were estimated to 1:129 (p53), 1:235 (p58), and 1:396 (c-myc). The measurements demonstrate the advantage of the SMI technique for the analysis of gene domain nano-architecture in cell nuclei. The data indicate that chromatin compaction is subjected to a large variability which may be due to different states of genetic activity or reflect the cell cycle state.

Light Microscopy in Biological Research

Optical microscopy and particularly fluorescence confocal microscopy is used on a large scale in the studies of cell structure and function including nuclear arrangement of chromatin. Chromosome territories and subchromosomal domains in cell nuclei as well as other nuclear structures are systematically investigated by means of three-dimensional fluorescence in situ hybridization and immunostaining in different stages of the cell cycle. Dynamics of structural changes can be investigated using green fluorescent protein conjugated to any other protein in live cell experiments. In these studies, high speed in parallel with resolution are important characteristics of the microscopic system. Recent development of confocal heads with improved light throughput and reduced bleaching and phototoxicity, powerful lasers, and back-illuminated charge-coupled device cameras with on-chip amplification, together with optimized computer control, fulfill to a large extent the requirements of recent research providing excellent tools for in situ proteomics. The mentioned technological advances suffer from limited resolution given by the Abbe limit (∼200 nm), which imposes undesirable limitations on research activities. It can be supposed and the latest data actually show that gene regulation is related to changes of chromatin structure on the level of individual genes (Chambeyron and Bickmore, 2004) with dimensions beyond the Abbe limit. There are several solutions that overcome the Abbe limit of optical microscopy (scanning probe techniques or electron microscopy), however, they are inappropriate for structural studies of three-dimensional objects and cannot be used for in vivo imaging. The resolution requirements are fulfilled by spatially modulated illumination microscopy, a relatively novel technique, based on periodic excitation along the axial direction (Hildenbrand et al., 2005), which gives rise to a similarly periodic emission profile from a fluorescent object placed between the two objectives. The object causes variation of the emission profile in a size-dependent manner. The ratio between the maximum of this variation and the maximum emission intensity gives the “modulation contrast” (R), which is directly proportional to the object size (e.g., 30–120 nm for 488-nm excitation). The authors (Hildenbrand et al., 2005) demonstrated the feasibility of the measurements in fixed cells stained using fluorescence in situ hybridization, however, the spatially modulated illumination microscopy can be obviously applied to detect structures visualized by immunostaining. Further applications in both research and diagnostics can be expected. The importance of the new kind of microscopy can be documented by the fact that Leica Microsystems (Wetzlar, Germany) has recently introduced the so-called 4pi microscope system (Hell and Stelzer, 1992) that also uses wave fronts of two opposite objective lenses. The resolution in the z-direction is increased by a factor of 3–7. This new system allows imaging at or below 50-nm resolution. It becomes clear that progress in biological research in the postgenomic era will depend on new technologies that provide information on epigenetic mechanisms regulating gene expression. In particular, physicochemical states of chromatin play a very important role in gene transcription (Cremer and Cremer, 2001). To understand regulatory functions of chromatin it is necessary to analyze higher-order chromatin condensation and nuclear organization. In other words, gene regulation is a highly complex, spatiotemporal biochemical process that strongly depends on nuclear structures, particularly on the structure of chromatin. To predict gene expression quantitatively, detailed information on structural characteristics of the cell nucleus is needed. Owing to its descriptive nature that does not explain mechanisms, structural biology has been for quite a long time in the shadow of other biological disciplines such as biochemistry, genetics, or molecular biology. At present, structural studies are urgently needed to overcome the evident barrier on our way to understand the quantitative nature of biological processes. The article of Hildenbrand et al. (2005) in this issue provides evidence that recent progress in optical microscopy meets the needs of biological research.

Nanostructure Analysis Using Spatially Modulated Illumination Microscopy

For an improved understanding of cellular processes, it is highly desirable to develop light optical methods for the analysis of biological nanostructures and their dynamics in the interior of three-dimensionally (3D) conserved cells. Here, important structural parameters to be considered are the topology, i.e. the mutual positions and distances, as well as the sizes of the constituting subunits. This has become possible by the development of a novel method of far-field light fluorescence microscopy, spatially modulated illumination (SMI) microscopy. Using this approach, axial distances between fluorescence-labeled targets can be measured with an accuracy close to 1 nm; their sizes can be determined down to a few tens of nanometers. This approach can be extended to the determination of 3D positions and mutual 3D distances and sizes of any number of small objects/subunits that can be discriminated due to their spectral signatures. Consequently, the new approach allows an ‘in situ nanostructure elucidation, until now regarded to be beyond the possibilities of far-field light microscopy. Application examples discussed are: colocalization/nanosizing and topological analysis of large protein-protein complexes, of nucleic acid-protein complexes (such as transcription factories), or of the highly complex DNA-protein nanostructures of which active/ inactive gene regions in the eukaryotic cell nucleus are constituted.

Nanosizing of fluorescent objects by spatially modulated illumination microscopy.

A new approach to measuring the sizes of small fluorescent objects by use of spatially modulated illumination (SMI) far-field light microscopy is presented. This method is based on SME measurements combined with a new SMI virtual microscopy (VIM) data analysis calibration algorithm. Here, experimental SMI measurements of fluorescent objects with known diameter (size) were made. From the SMI data obtained, the size was determined in an independent way by use of the SMI VIM algorithm. The results showed that with SMI microscopy in combination with SMI VIM calibration, subwavelength object size measurements as small as 40 nm are experimentally feasible with high accuracy.

Spatially modulated illumination microscopy allows axial distance resolution in the nanometer range.

For an improved understanding of the structural basis of cellular mechanisms, it is highly desirable to develop methods for a detailed topological analysis of biological nanostructures and their dynamics in the interior of three-dimensionally conserved cells. We present a method of far-field laser fluorescence microscopy to measure relative axial positions of pointlike fluorescent targets and the distance between each target in the range of a few nanometers. The physical principle behind this approach can be extended to the determination of three-dimensional (3D) positions and 3D distances between any number of objects that can be discriminated owing to their spectral signature, thus allowing topological measurements so far regarded to be beyond the capabilities of light microscopy.

Spatially modulated illumination microscopy: online visualization of intensity distribution and prediction of nanometer precision of axial distance measurements by computer simulations.

During the last years, measurements considerably beyond the conventional "Abbe-Limit" of optical resolution in far field light microscopy were realized by several light microscopical approaches. Point spread function (PSF) engineering, spectral precision distance microscopy (SPDM), and related methods were used to demonstrate the feasibility of such measurements. SPDM allows the measurement of position and multiple distances between point-like fluorescent objects of different spectral signatures far below the optical resolution criterion as defined by the full width at half maximum of the PSF. Here, we report a software method to obtain online visualization of light distribution in the lateral and axial direction of any object detected in a spatially modulated illumination (SMI) microscope. This strongly facilitates routine application of SMI microscopy. The software was developed using Microsoft Visual C++ running on Windows NT. Furthermore, some aspects of the theoretical limits of the SPDM method were studied by virtual microscopy. For the case of SMI microscopy the precision of axial distance measurements was studied, taking into account photon statistics and image analysis procedures. The results indicate that even under low fluorescence intensity conditions typical for biological structure research, precise distance measurements in the nanometer range can be determined, and that axial distances in the order of 40 nm are detectable with such precision.