Towards 3D modelling and imaging of infection scenarios at the single cell level using holographic optical tweezers and digital holographic microscopy

The analysis of dynamic interactions of microorganisms with a host cell is of utmost importance for understanding infection processes. We present a biophotonic holographic workstation that allows optical manipulation of bacteria by holographic optical tweezers and simultaneously monitoring of dynamic processes with quantitative multi‐focus phase imaging based on self‐interference digital holographic microscopy. Our results show that several bacterial cells, even with non‐spherical shape, can be aligned precisely on the surface of living host cells and localized reproducibly in three dimensions. In this way a new label‐free multipurpose device for modelling and quantitative analysis of infection scenarios at the single cell level is provided. (© 2013 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Cell and nucleus refractive‐index mapping by interferometric phase microscopy and rapid confocal fluorescence microscopy

We present a multimodal technique for measuring the integral refractive index and the thickness of biological cells and their organelles by integrating interferometric phase microscopy (IPM) and rapid confocal fluorescence microscopy. First, the actual thickness maps of the cellular compartments are reconstructed using the confocal fluorescent sections, and then the optical path difference (OPD) map of the same cell is reconstructed using IPM. Based on the co‐registered data, the integral refractive index maps of the cell and its organelles are calculated. This technique enables rapidly measuring refractive index of live, dynamic cells, where IPM provides quantitative imaging capabilities and confocal fluorescence microscopy provides molecular specificity of the cell organelles. We acquire human colorectal adenocarcinoma cells and show that the integral refractive index values are similar for the whole cell, the cytoplasm and the nucleus on the population level, but significantly different on the single cell level.     

Optical‐mechanical signatures of cancer cells based on fluctuation profiles measured by interferometry

We propose to establish a cancer biomarker based on the unique optical‐mechanical signatures of cancer cells measured in a noncontact, label‐free manner by optical interferometry. Using wide‐field interferometric phase microscopy (IPM), implemented by a portable, off‐axis, common‐path and low‐coherence interferometric module, we quantitatively measured the time‐dependent, nanometer‐scale optical thickness fluctuation maps of live cells in vitro. We found that cancer cells fluctuate significantly more than healthy cells, and that metastatic cancer cells fluctuate significantly more than primary cancer cells. Atomic force microscopy (AFM) measurements validated the results. Our study shows the potential of IPM as a simple clinical tool for aiding in diagnosis and monitoring of cancer. (© 2014 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Quantitative phase imaging of cells in a flow cytometry arrangement utilizing Michelson interferometer‐based off‐axis digital holographic microscopy

We combined Michelson‐interferometer‐based off‐axis digital holographic microscopy (DHM) with a common flow cytometry (FCM) arrangement. Utilizing object recognition procedures and holographic autofocusing during the numerical reconstruction of the acquired off‐axis holograms, sharply focused quantitative phase images of suspended cells in flow were retrieved without labeling, from which biophysical cellular features of distinct cells, such as cell radius, refractive index and dry mass, can be subsequently retrieved in an automated manner. The performance of the proposed concept was first characterized by investigations on microspheres that were utilized as test standards. Then, we analyzed two types of pancreatic tumor cells with different morphology to further verify the applicability of the proposed method for quantitative live cell imaging. The retrieved biophysical datasets from cells in flow are found in good agreement with results from comparative investigations with previously developed DHM methods under static conditions, which demonstrates the effectiveness and reliability of our approach. Our results contribute to the establishment of DHM in imaging FCM and prospect to broaden the application spectrum of FCM by providing complementary quantitative imaging as well as additional biophysical cell parameters which are not accessible in current high‐throughput FCM measurements.     

Volume scanning electron microscopy for imaging biological ultrastructure

Electron microscopy (EM) has been a key imaging method to investigate biological ultrastructure for over six decades. In recent years, novel volume EM techniques have significantly advanced nanometre‐scale imaging of cells and tissues in three dimensions. Previously, this had depended on the slow and error‐prone manual tasks of cutting and handling large numbers of sections, and imaging them one‐by‐one with transmission EM. Now, automated volume imaging methods mostly based on scanning EM (SEM) allow faster and more reliable acquisition of serial images through tissue volumes and achieve higher z‐resolution. Various software tools have been developed to manipulate the acquired image stacks and facilitate quantitative analysis. Here, we introduce three volume SEM methods: serial block‐face electron microscopy (SBEM), focused ion beam SEM (FIB‐SEM) and automated tape‐collecting ultramicrotome SEM (ATUM‐SEM). We discuss and compare their capabilities, provide an overview of the full volume SEM workflow for obtaining 3D datasets and showcase different applications for biological research.     

An optical study of drug resistance detection in endometrial cancer cells by dynamic and quantitative phase imaging

Platinum chemosensitivity detection plays a vital role during endometrial cancer treatment because chemotherapy responses have profound influences on patient's prognosis. Although several methods can be used to detect drug resistance characteristics, studies on detecting drug sensitivity based on dynamic and quantitative phase imaging of cancer cells are rare. In this study, digital holographic microscopy was applied to distinguish drug‐resistant and nondrug‐resistant endometrial cancer cells. Based on the reconstructed phase images, temporal evolutions of cell height (CH), cell projected area (CPA) and cell volume were quantitatively measured. The results show that change rates of CH and CPA were significantly different between drug‐resistant and nondrug‐resistant endometrial cancer cells. Furthermore, the results demonstrate that morphological characteristics have the potential to be utilized to distinguish the drug sensitivity of endometrial cancer cells, and it may provide new perspectives to establish optical methods to detect drug sensitivity and guide chemotherapy in endometrial cancer.      

High‐resolution analysis of neuronal growth cone morphology by comparative atomic force and optical microscopy

Neuronal growth cones are motile sensory structures at the tip of axons, transducing guidance information into directional movements towards target cells. The morphology and dynamics of neuronal growth cones have been well characterized with optical techniques; however, very little quantitative information is available on the three‐dimensional structure and mechanical properties of distinct subregions. In the present study, we imaged the large Aplysia growth cones after chemical fixation with the atomic force microscope (AFM) and directly compared our data with images acquired by light microscopy methods. Constant force imaging in contact mode in combination with force‐distant measurements revealed an average height of 200 nm for the peripheral (P) domain, 800 nm for the transition (T) zone, and 1200 nm for the central (C) domain, respectively. The AFM images show that the filopodial F‐actin bundles are stiffer than surrounding F‐actin networks. Enlarged filopodia tips are 60 nm higher than the corresponding shafts. Measurements of the mechanical properties of the specific growth cone regions with the AFM revealed that the T zone is stiffer than the P and the C domain. Direct comparison of AFM and optical data acquired by differential interference contrast and fluorescence microscopy revealed a good correlation between these imaging methods. However, the AFM provides height and volume information at higher resolution than fluorescence methods frequently used to estimate the volume of cellular compartments. These findings suggest that AFM measurements on live growth cones will provide a quantitative understanding of how proteins can move between different growth cone regions. © 2006 Wiley Periodicals, Inc. J Neurobiol, 2006     

Nonlinear‐optical stain‐free stereoimaging of astrocytes and gliovascular interfaces

Methods of nonlinear optics provide a vast arsenal of tools for label‐free brain imaging, offering a unique combination of chemical specificity, the ability to detect fine morphological features, and an unprecedentedly high, subdiffraction spatial resolution. While these techniques provide a rapidly growing platform for the microscopy of neurons and fine intraneural structures, optical imaging of astroglia still largely relies on filament‐protein‐antibody staining, subject to limitations and difficulties especially severe in live‐brain studies. Once viewed as an ancillary, inert brain scaffold, astroglia are being promoted, as a part of an ongoing paradigm shift in neurosciences, into the role of a key active agent of intercellular communication and information processing, playing a significant role in brain functioning under normal and pathological conditions. Here, we show that methods of nonlinear optics provide a unique resource to address long‐standing challenges in label‐free astroglia imaging. We demonstrate that, with a suitable beam‐focusing geometry and careful driver‐pulse compression, microscopy of second‐harmonic generation (SHG) can enable a high‐resolution label‐free imaging of fibrillar structures of astrocytes, most notably astrocyte processes and their endfeet. SHG microscopy of astrocytes is integrated in our approach with nonlinear‐optical imaging of red blood cells based on third‐harmonic generation (THG) enhanced by a three‐photon resonance with the Soret band of hemoglobin. With astroglia and red blood cells providing two physically distinct imaging contrasts in SHG and THG channels, a parallel detection of the second and third harmonics enables a high‐contrast, high‐resolution, stain‐free stereoimaging of gliovascular interfaces in the central nervous system. Transverse scans of the second and third harmonics are shown to resolve an ultrafine texture of blood‐vessel walls and astrocyte‐process endfeet on gliovascular interfaces with a spatial resolution within 1 μm at focusing depths up to 20 μm inside a brain.     

The lipid raft hypothesis revisited - New insights on raft composition and function from super-resolution fluorescence microscopy

Recently developed super‐resolution microscopy techniques are changing our understanding of lipid rafts and membrane organisation in general. The lipid raft hypothesis postulates that cholesterol can drive the formation of ordered domains within the plasma membrane of cells, which may serve as platforms for cell signalling and membrane trafficking. There is now a wealth of evidence for these domains. However, their study has hitherto been hampered by the resolution limit of optical microscopy, making the definition of their properties problematic and contentious. New microscopy techniques circumvent the resolution limit and, for the first time, allow the fluorescence imaging of structures on length scales below 200 nm. This review describes such techniques, particularly as applied to the study of membrane organisation, synthesising newly emerging facets of lipid raft biology into a state‐of‐the art model. Editor's suggested further reading in BioEssays: Super‐resolution imaging prompts re‐thinking of cell biology mechanisms Abstract and Quantitative analysis of photoactivated localization microscopy (PALM) datasets using pair‐correlation analysis Abstract     

Quantifying molecular colocalization in live cell fluorescence microscopy

One of the most challenging tasks in microscopy is the quantitative identification and characterization of molecular interactions. In living cells this task is typically performed by fluorescent labeling of the interaction partners with spectrally distinct fluorophores and imaging in different color channels. Current methods for determining colocalization of molecules result in outcomes that can vary greatly depending on signal‐to‐noise ratios, threshold and background levels, or differences in intensity between channels. Here, we present a novel and quantitative method for determining the degree of colocalization in live‐cell fluorescence microscopy images for two and more data channels. Moreover, our method enables the construction of images that directly classify areas of high colocalization. (© 2013 WILEY‐VCH Verlag GmbH &Co. KGaA, Weinheim)     

Atomic force microscopy study of the antigen‐antibody binding force on patient cancer cells based on ROR1 fluorescence recognition

Knowledge of drug–target interaction is critical to our understanding of drug action and can help design better drugs. Due to the lack of adequate single‐molecule techniques, the information of individual interactions between ligand‐receptors is scarce until the advent of atomic force microscopy (AFM) that can be used to directly measure the individual ligand‐receptor forces under near‐physiological conditions by linking ligands onto the surface of the AFM tip and then obtaining force curves on cells. Most of the current AFM single‐molecule force spectroscopy experiments were performed on cells grown in vitro (cell lines) that are quite different from the human cells in vivo. From the view of clinical practice, investigating the drug–target interactions directly on the patient cancer cells will bring more valuable knowledge that may potentially serve as an important parameter in personalized treatment. Here, we demonstrate the capability of AFM to measure the binding force between target (CD20) and drug (rituximab, an anti‐CD20 monoclonal antibody targeted drug) directly on lymphoma patient cancer cells under the assistance of ROR1 fluorescence recognition. ROR1 is a receptor expressed on some B‐cell lymphomas but not on normal cells. First, B‐cell lymphoma Raji cells (a cell line) were used for ROR1 fluorescence labeling and subsequent measurement of CD20‐rituximab binding force. The results showed that Raji cells expressed ROR1, and the labeling of ROR1 did not influence the measurement of CD20‐rituximab binding force. Then the established experimental procedures were performed on the pathological samples prepared from the bone marrow of a follicular lymphoma patient. Cancer cells were recognized by ROR1 fluorescence. Under the guidance of fluorescence, with the use of a rituximab‐conjugated tip, the cellular topography was visualized by using AFM imaging and the CD20‐Rituximab binding force was measured by single‐molecule force spectroscopy. Copyright © 2013 John Wiley & Sons, Ltd.     

Cell imaging and manipulation by nonlinear optical microscopy

Advances in the technologies for labeling and imaging biological samples drive a constant progress in our capability of studying structures and their dynamics within cells and tissues. In the last decade, the development of numerous nonlinear optical microscopies has led to a new prospective both in basic research and in the potential development of very powerful noninvasive diagnostic tools. These techniques offer large advantages over conventional linear microscopy with regard to penetration depth, spatial resolution, three-dimensional optical sectioning, and lower photobleaching. Additionally, some of these techniques offer the opportunity for optically probing biological functions directly in living cells, as highlighted, for example, by the application of second-harmonic generation to the optical measurement of electrical potential and activity in excitable cells. In parallel with imaging techniques, nonlinear microscopy has been developed into a new area for the selective disruption and manipulation of intracellular structures, providing an extremely useful tool of investigation in cell biology. In this review we present some basic features of nonlinear microscopy with regard both to imaging and manipulation, and show some examples to illustrate the advantages offered by these novel methodologies.     

Increasing fluorescence lifetime for resolution improvement in stimulated emission depletion nanoscopy

Super‐resolution microscopy (SRM) has had a substantial impact on the biological sciences due to its ability to observe tiny objects less than 200 nm in size. Stimulated emission depletion (STED) microscopy represents a major category of these SRM techniques that can achieve diffraction‐unlimited resolution based on a purely optical modulation of fluorescence behaviors. Here, we investigated how the laser beams affect fluorescence lifetime in both confocal and STED imaging modes. The results showed that with increasing illumination time, the fluorescence lifetime in two kinds of fluorescent microspheres had an obvious change in STED imaging mode, compared with that in confocal imaging mode. As a result, the reduction of saturation intensity induced by the increase of fluorescence lifetime can improve the STED imaging resolution at the same depletion power. The phenomenon was also observed in Star635P‐labeled human Nup153 in fixed HeLa cells, which can be treated as a reference for the synthesis of fluorescent labels with the sensitivity to the surrounding environment for resolution improvement in STED nanoscopy.      

Multicomposite super‐resolution microscopy: Enhanced Airyscan resolution with radial fluctuation and sample expansions

Either modulated illumination or temporal fluctuation analysis can assist super‐resolution techniques in overcoming the diffraction limit of conventional optical microscopy. As they are not contradictory to each other, an effective combination of spatial and temporal super‐resolution mechanisms would further improve the resolution of fluorescent images. Here, a super‐resolution imaging method called fluctuation‐enhanced Airyscan technology (FEAST) is proposed, which achieves ~40 nm lateral imaging resolution and is useful for a range of fluorescent proteins and organic dyes. It was demonstrated not only to obtain different subcellular super‐resolution images, but also to improve the accuracy of counting the average human epidermal growth factor receptor 2 (HER2) copy number for diagnosis in breast cancer. Furthermore, the combination of FEAST and sample expansion microscopy (Ex‐FEAST) improves the lateral resolution to ~26 nm.     

Comparison between high definition FT‐IR,Raman and AFM‐IR for subcellular chemical imaging of cholesteryl esters in prostate cancer cells

The family of vibrational spectroscopic imaging techniques grows every few years and there is a need to compare and contrast new modalities with the better understood ones, especially in the case of demanding biological samples. Three vibrational spectroscopy techniques (high definition Fourier‐transform infrared [FT‐IR], Raman and atomic force microscopy infrared [AFM‐IR]) were applied for subcellular chemical imaging of cholesteryl esters in PC‐3 prostate cancer cells. The techniques were compared and contrasted in terms of image quality, spectral pattern and chemical information. All tested techniques were found to be useful in chemical imaging of cholesterol derivatives in cancer cells. The results obtained from FT‐IR and Raman imaging showed to be comparable, whereas those achieved from AFM‐IR study exhibited higher spectral heterogeneity. It confirms AFM‐IR method as a powerful tool in local chemical imaging of cells at the nanoscale level. Furthermore, due to polarization effect, p‐polarized AFM‐IR spectra showed strong enhancement of lipid bands when compared to FT‐IR.     

In situ Imaging of Pea Starch in Seeds

A method has been developed for the in situ imaging of starch in dry seeds by exploiting the tight packing of the starch and protein storage reserves within the cells of the embryo. The method can be adapted to prepare seed samples which are suitable for light microscopy (birefringence and iodine staining), scanning electron microscopy and atomic force microscopy. Its potential for imaging the internal structure of starch granules without any prior isolation process is demonstrated for round smooth peas. Using a standard ultramicrotome, thin sections were cut directly from selected regions of dry pea seeds and examined by light microscopy before and after hydration. The sectioning procedure left a planed surface with the internal structure of the starch granules exposed. This material was examined by scanning electron microscopy and atomic force microscopy directly or after controlled hydration. In the hydrated pea samples, the growth ring structure and blocklet sub-structure of individual starch granules within the seed were visualised directly by atomic force microscopy. Furthermore, the effects of hydration and staining were monitored and have been used to introduce contrast into the images. The observations have revealed new information on the blocklet distribution within pea starch granules and the physical origins of the growth ring structure of the granules: the blocklet distribution suggests that the granules contain alternating bands with different levels of crystallinity, rather than alternating amorphous and crystalline growth rings.     

Direct imaging electron microscopy (EM) methods in modern structural biology: Overview and comparison with X‐ray crystallography and single‐particle cryo‐EM reconstruction in the studies of large macromolecules

Determining the structure of macromolecules is important for understanding their function. The fine structure of large macromolecules is currently studied primarily by X‐ray crystallography and single‐particle cryo‐electron microscopy (EM) reconstruction. Before the development of these techniques, macromolecular structure was often examined by negative‐staining, rotary‐shadowing and freeze‐etching EM, which are categorised here as ‘direct imaging EM methods’. In this review, the results are summarised by each of the above techniques and compared with respect to four macromolecules: the ryanodine receptor, cadherin, rhodopsin and the ribosome–translocon complex (RTC). The results of structural analysis of the ryanodine receptor and cadherin are consistent between each technique. The results obtained for rhodopsin vary to some extent within each technique and between the different techniques. Finally, the results for RTC are inconsistent between direct imaging EM and other analytical techniques, especially with respect to the space within RTC, the reasons for which are discussed. Then, the role of direct imaging EM methods in modern structural biology is discussed. Direct imaging methods should support and verify the results obtained by other analytical methods capable of solving three‐dimensional molecular architecture, and they should still be used as a primary tool for studying macromolecule structure in vivo.     

Multiple microscopic approaches demonstrate linkage between chromoplast architecture and carotenoid composition in diverse Capsicum annuum fruit

Increased accumulation of specific carotenoids in plastids through plant breeding or genetic engineering requires an understanding of the limitations that storage sites for these compounds may impose on that accumulation. Here, using Capsicum annuum L. fruit, we demonstrate directly the unique sub‐organellar accumulation sites of specific carotenoids using live cell hyperspectral confocal Raman microscopy. Further, we show that chromoplasts from specific cultivars vary in shape and size, and these structural variations are associated with carotenoid compositional differences. Live‐cell imaging utilizing laser scanning confocal (LSCM) and confocal Raman microscopy, as well as fixed tissue imaging by scanning and transmission electron microscopy (SEM and TEM), all demonstrated morphological differences with high concordance for the measurements across the multiple imaging modalities. These results reveal additional opportunities for genetic controls on fruit color and carotenoid‐based phenotypes.     

Investigation of HIV‐1 Assembly and Release Using Modern Fluorescence Imaging Techniques

The replication of HIV‐1, like that of all viruses, is intimately connected with cellular structures and pathways. For many years, bulk biochemical and cell biological methods were the main approaches employed to investigate interactions between HIV‐1 and its host cell. However, during the past decade advancements in fluorescence imaging technologies opened new possibilities for the direct visualization of individual steps occurring throughout the viral replication cycle. Electron microscopy (EM) methods, which have traditionally been employed for the study of viruses, are complemented by fluorescence microscopy (FM) techniques that allow us to follow the dynamics of virus–cell interaction. Subdiffraction fluorescence microscopy, as well as correlative EM/FM approaches, are narrowing the fundamental gap between the high structural resolution provided by EM and the high temporal resolution and throughput accomplished by FM. The application of modern microscopy to the study of HIV‐1–host cell interactions has provided insights into the biology of the virus which could not easily, or not at all, have been gained by other methods. Here, we review how modern fluorescence imaging techniques enhanced our knowledge of the dynamic and structural changes involved in HIV‐1 particle formation.      

Correlation of histology and linear and nonlinear microscopy of the living human cornea

The morphology and the function of cellular and non‐cellular structures in the living human cornea can be determined with modern correlative linear and nonlinear optical microscopic techniques and histology. Correlative microscopy is based on the use of different optical techniques to study the same specimen, ideally at the same location within the specimen, in order to increase the functional and/or morphological understanding of the specimen. A case study to assess the effect of overnight lid‐closure on in vivo human corneal morphology is presented to illustrate correlative linear microscopy and optical low‐coherence reflectometry. Nonlinear multiphoton excitation microscopy provides functional information on cellular metabolism based on the intrinsic fluorescence from the reduced pyridine nucleotides and the oxidized flavoproteins. Second‐harmonic generation microscopy, a scattering process that does not deposit net energy into the tissue, provides structural information on corneal collagen organization. Molecular third‐harmonic generation microscopy generates a signal in all materials and it an emerging technique. Coherent anti‐Stokes Raman scattering microscopy provides chemical imaging for biology and medicine. The comparison and limitations of these microscopic modalities, linear and nonlinear microscopy applied to the cornea, and a review of some key findings is analyzed. A correlative integration and correlation of linear and nonlinear microscopies to study corneal function and structure is proposed to validate the clinical interpretation of microscopic images of the cornea. (© 2009 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)