Advances In Confocal Microscopy Research Articles

Advances in Confocal Microscopy and Selected Applications.

Over the last 30 years, confocal microscopy has emerged as a primary tool for biological investigation across many disciplines. The simplicity of use and widespread accessibility of confocal microscopy ensure that it will have a prominent place in biological imaging for many years to come, even with the recent advances in light sheet and field synthesis microscopy. Since these more advanced technologies still require significant expertise to effectively implement and carry through to analysis, confocal microscopy-based approaches still remain the easiest way for biologists with minimal imaging experience to address fundamental questions about how their systems are arranged through space and time. In this review, we discuss a number of advanced applications of confocal microscopy for probing the spatiotemporal dynamics of biological systems.

3D morphological analysis of Arabidopsis sepals.

How complicated cell activities produce characteristic tissue and organ morphologies is an important question in plant morphogenesis. To address this question, 3D morphometry of plant organs on multiscales is indispensable. In recent years, advances in confocal microscopy with fluorescent probes that mark the cell wall or plasma membrane enable the visualization of organ morphology with submicron precision. In parallel, new quantitative and correlative imaging pipelines realize 3D image processing on 2D curved surface, facilitating the study of cell and tissue behaviors in plant organogenesis. Here, we describe methods for 3D morphometry of Arabidopsis sepals, focusing on live imaging coupled with MorphoGraphX-based 3D image processing for cellular growth analysis.

Live Confocal Imaging of Developing Arabidopsis Flowers

The study of plant growth and development has long relied on experimental techniques using dead, fixed tissues and lacking proper cellular resolution. Recent advances in confocal microscopy, combined with the development of numerous fluorophores, have overcome these issues and opened the possibility to study the expression of several genes simultaneously, with a good cellular resolution, in live samples. Live confocal imaging provides plant biologists with a powerful tool to study development, and has been extensively used to study root growth and the formation of lateral organs on the flanks of the shoot apical meristem. However, it has not been widely applied to the study of flower development, in part due to challenges that are specific to imaging flowers, such as the sepals that grow over the flower meristem, and filter out the fluorescence from underlying tissues. Here, we present a detailed protocol to perform live confocal imaging on live, developing Arabidopsis flower buds, using either an upright or an inverted microscope.

Live Confocal Imaging of Developing Arabidopsis Flowers.

The study of plant growth and development has long relied on experimental techniques using dead, fixed tissues and lacking proper cellular resolution. Recent advances in confocal microscopy, combined with the development of numerous fluorophores, have overcome these issues and opened the possibility to study the expression of several genes simultaneously, with a good cellular resolution, in live samples. Live confocal imaging provides plant biologists with a powerful tool to study development, and has been extensively used to study root growth and the formation of lateral organs on the flanks of the shoot apical meristem. However, it has not been widely applied to the study of flower development, in part due to challenges that are specific to imaging flowers, such as the sepals that grow over the flower meristem, and filter out the fluorescence from underlying tissues. Here, we present a detailed protocol to perform live confocal imaging on live, developing Arabidopsis flower buds, using either an upright or an inverted microscope.

Live confocal imaging of Arabidopsis flower buds

Recent advances in confocal microscopy, coupled with the development of numerous fluorescent reporters, provide us with a powerful tool to study the development of plants. Live confocal imaging has been used extensively to further our understanding of the mechanisms underlying the formation of roots, shoots and leaves. However, it has not been widely applied to flowers, partly because of specific challenges associated with the imaging of flower buds. Here, we describe how to prepare and grow shoot apices of Arabidopsis in vitro, to perform both single-point and time-lapse imaging of live, developing flower buds with either an upright or an inverted confocal microscope.

Advances in Confocal Microscopy of the Eye.

This special issue focuses on the recent advances on in vivo confocal microscopy (IVCM), a technique used to investigate eye structures at the cellular level without tissue damage. In 1985 Lemp and coworkers were the first to study ex vivo corneas by confocal microscopy and to suggest a possible in vivo use [1]. In the early 1990s, the groups directed by Cavanagh and Kaufman published the first IVCM in humans; thereafter the scientific interest in ocular IVCM rapidly increased. In the last decade, IVCM progressively gained a relevant role in the clinical setting, being of help in the diagnosis and management of a number of conditions such as toxicity induced by preservatives [2, 3] and different eye treatments [4, 5], iatrogenic damage [6], infections [7], and dystrophies [8], pathology of the conjunctiva [9–11] and limbus [12], ocular surface tumors [13], and corneal deposits [14, 15]. IVCM is a valuable tool for enhancing our understanding of anterior segment physiology and pathology, as pointed out by Dr. V. Fasanella et al. in their paper investigating meibomian gland changes occurring with aging and a number of ocular surface diseases. Examples of the usefulness of IVCM in detecting early corneal changes can be found in the paper by Dr. D. Wang et al., who highlighted that a higher-than-normal immune activity may be observed in a subgroup of patients with clear corneal grafts in the absence of any other clinical signs. Dr. P. Song et al. studied the confocal and histopathological features of the stromal scar in keratoconus and suggested that IVCM is capable of detecting subtle keratocyte activation associated with fibrosis. In recent years, IVCM has become increasingly useful in evaluating corneal innervation (which is of key importance in regulating ocular surface homeostasis) [16] and corneal immune and inflammatory responses [17]. In this special issue, three papers explored the changes occurring in corneal innervation in both local and systemic diseases. The paper by Dr. E. F. Wang et al. reviewed the impact of various systemic diseases on corneal innervation and discussed the potential use of IVCM as a noninvasive marker of peripheral neuropathy. The reports by Dr. R. Shetty et al. and Dr. N. K. Pahuja et al. investigated nerve changes occurring, respectively, in dry eye disease and keratoconus. Finally, IVCM plays a role in studying the therapeutic effects of topical eye treatments, as shown in this issue by the paper of Dr. A. M. Fea et al. This special issue provides a useful update on the advances in the rapidly evolving field of IVCM and highlights the contribution of this technology to our understanding of the anterior segment in health and disease. Paolo Fogagnolo Michele Iester Hong Liang Dipika V. Patel

Erratum to: Application of advanced light microscopic techniques to gain deeper insights into cheese matrix physico-chemistry

Despite considerable advances in confocal microscopy and related technologies, advanced light microscopy techniques have been rarely used to study the microstructure of cheese matrices. Here, we demonstrate the potential of advanced microscopic techniques, namely fluorescence lifetime imaging (FLIM), confocal Raman spectroscopy and non-linear microscopy (two-photon excitation and second harmonic generation microscopy), to study localized physicochemical properties of cheese matrices. These methods allow for precise localization of individual chemical components and help to determine their spatial organization in three dimensions. We discuss both fluorescent labelling and label-free methods that provide valuable information about the localized environment. Overall, these new technologies will enable a greater understanding of the influence of manufacturing processes on cheese quality and consistency.

Application of advanced light microscopic techniques to gain deeper insights into cheese matrix physico-chemistry

Despite considerable advances in confocal microscopy and related technologies, advanced light microscopy techniques have been rarely used to study the microstructure of cheese matrices. Here, we demonstrate the potential of advanced microscopic techniques, namely fluorescence lifetime imaging (FLIM), confocal Raman spectroscopy and non-linear microscopy (two-photon excitation and second harmonic generation microscopy), to study localized physicochemical properties of cheese matrices. These methods allow for precise localization of individual chemical components and help to determine their spatial organization in three dimensions. We discuss both fluorescent labelling and label-free methods that provide valuable information about the localized environment. Overall, these new technologies will enable a greater understanding of the influence of manufacturing processes on cheese quality and consistency.

Insights into signaling and function of hematopoietic stem cells at the single-cell level

Development of a technique prospectively to isolate hematopoietic stem cells (HSCs) to near homogeneity has enabled clonal analysis and thus converted our understanding of HSCs from conceptual and qualitative to realistic and quantitative. Recent studies have revealed that despite their high proliferation potential, most HSCs are in G0 and enter cell cycle only after a long interval. This dormancy of HSCs, which seems to be important for long-term maintenance of 'stemness', appears to be regulated by the exchange of signals between HSCs and the bone marrow niche. Analysis of intersignaling and intrasignaling events in HSCs in and out of the bone marrow niche has begun. With the help of advances in confocal microscopy, laser scanning microscopy, and personal computer computational power over the last decade, it has become evident that thrombopoietin/c-Mpl signaling plays a role in HSC self-renewal and AKT-forkhead box O signaling in HSC dormancy. Furthermore, transforming growth factor-beta has been indicated as a candidate niche signal to induce hibernation in HSCs. Understanding of the signaling events between HSCs and niche is critical not only for stem cell biology in general and for transplantation medicine but also for the development of novel cancer therapy.

Imaging the cytoskeleton in live Xenopus laevis embryos.

Historically, much of our understanding of actin filaments, microtubules and intermediate filaments has come from the study of fixed cells and tissues. But the cytoskeleton is inherently dynamic, and so developing the means to image it in living cells has proved crucial. Advances in confocal microscopy and fluorescent protein technologies have allowed us to dynamically image the cytoskeleton at high resolution and so learn much more about its cellular functions. However, most of this work has been performed in cultured cells, and a critical next step is to understand how the cytoskeleton functions in the context of an intact organism. We, and others, have developed methods to image the cytoskeleton in living vertebrate embryos. Here, we describe an approach to image the cytoskeleton in embryos of the frog, Xenopus laevis, using mRNA to express fluorescently tagged cytoskeletal probes and confocal microscopy to visualize their dynamic behavior.

Toward Single‐metal‐ion Sensing by Förster Resonance Energy Transfer

Here we describe progress toward our objective of detecting single nonfluorescent hydrated metal ions. Single-ion detection represents detection and spectroscopy at the ultimate sensitivity level of approximately 1.6 x 10(-24) M. Achieving this goal would provide a breakthrough in analytical science and allow much more detailed insight into sensor-ion interaction than that available with conventional bulk detection methods. We combine recent advances in confocal microscopy with the sensitivity and the noninvasive nature of fluorescence by analyzing Förster resonance energy transfer between sensor fluorophores and transition metal ions.

A quantitative determination of multi-protein interactions by the analysis of confocal images using a pixel-by-pixel assessment algorithm

Recent advances in confocal microscopy have allowed scientists to assess the expression, and to some extent, the interaction/colocalization of multiple molecules within cells and tissues. In some instances, accurately quantifying the colocalization of two or more proteins may be critical. This can require the acquisition of multiple Z plane images (Z stacks) throughout a specimen and, as such, we report here the successful development of a freeware, open-source image analysis tool, IMAJIN_COLOC, developed in PERL (v. 5.8, build 806), using the PERLMagick libraries (ImageMagick). Using a pixel-by-pixel analysis algorithm, IMAJIN_COLOC can analyze images for antigen expression (any number of colors) and can measure all possible combinations of colocalization for up to three colors by analyzing a Z stack gallery acquired for each sample. The simultaneous (i.e. in a single pass) analysis of three-color colocalization, and batch analysis capabilities are distinctive features of this program. A control image, containing known individual and colocalized pixel counts, was used to validate the accuracy of IMAJIN_COLOC. As further validation, pixel counts and colocalization values from the control image were compared to those obtained with the software packaged with the Zeiss laser-scanning microscope (LSM AIM, version 3.2). The values from both programs were found to be identical. To demonstrate the applicability of this program in addressing novel biological questions, we examined the role of neurons in eliciting an immune reaction in response to viral infection. Specifically, we successfully examined expression of the chemokine RANTES in measles virus (MV) infected hippocampal neurons and quantified changes in RANTES production throughout the disease period. The resultant quantitative data were also evaluated visually, using a gif image created during the analysis. PERL (ActivePerl, version 5.8) is available at activestate.com; the PERLMagick libraries are available at imagemagick.org, and IMAJIN_COLOC, the source code and user documentation can be downloaded from http://www.fda.gov/cber/research/imaging/imageanalysis.htm.

PSAT vectors: a modular series of plasmids for autofluorescent protein tagging and expression of multiple genes in plants

Autofluorescent protein tags represent one of the major and, perhaps, most powerful tools in modern cell biology for visualization of various cellular processes in vivo. In addition, advances in confocal microscopy and the development of autofluorescent proteins with different excitation and emission spectra allowed their simultaneous use for detection of multiple events in the same cell. Nevertheless, while autofluorescent tags are widely used in plant research, the need for a versatile and comprehensive set of vectors specifically designed for fluorescent tagging and transient and stable expression of multiple proteins in plant cells from a single plasmid has not been met by either the industrial or the academic communities. Here, we describe a new modular satellite (SAT) vector system that supports N- and C-terminal fusions to five different autofluorescent tags, EGFP, EYFP, Citrine-YFP, ECFP, and DsRed2. These vectors carry an expanded multiple cloning site that allows easy exchange of the target genes between different autofluorescence tags, and expression of the tagged proteins is controlled by constitutive promoters, which can be easily replaced with virtually any other promoter of interest. In addition, a series of SAT vectors has been adapted for high throughput Gateway recombination cloning. Furthermore, individual expression cassettes can be assembled into Agrobacterium binary plasmids, allowing efficient transient and stable expression of multiple autofluorescently tagged proteins from a single vector following its biolistic delivery or Agrobacterium-mediated genetic transformation.

High-resolution confocal imaging and three-dimensional rendering

Intrinsic opacity and inhomeogeniety of most biological tissues have prevented the efficient light penetration and signal detection for high-resolution confocal imaging of thick tissues. Here, we summarize recent technical advances in high-resolution confocal imaging for visualization of cellular structures and gene expression within intact whole-mount thick tissues. First, we introduce features of the FocusClear technology that render biological tissue transparent and thus improve the light penetration and signal detection. Next, a universal fluorescence staining method that labels all nuclei and membranes is described. We then demonstrate the postrecording image processing techniques for 3D visualization. From these images, regions of interest in the whole-mount brain can be segmented and volume rendered. Together, these technical advances in confocal microscopy allow visualization of structures within whole-mount tissues up to 1 mm thick at a resolution similar to that of the observation of single cells in culture. Practical uses and limitations of these techniques are discussed.

4D confocal microscopy of Dictyostelium discoideum morphogenesis and its presentation on the Internet.

Methods to present three-dimensional (3D) and time series of 3D datasets (4D) are demonstrated using the recent advances in confocal microscopy and computer visualization. The process of cell sorting during tip formation in the slime mould Dictyostelium discoideum is examined as an example by in vivo confocal microscopy of spectrally different green fluorescent protein (GFP) variants as reporters of cell-type specific gene expression. Also, cell sorting of the co-aggregating slime mould species D. discoideum and D. mucoroides is observed using a GFP variant and a spectrally distinguishable fluorescent vital stain. The confocal data are handled as 3D and 4D datasets, their processing and the advantages of different methods of visualization are discussed step by step. Selected sequences of the experiments can be viewed on the Internet, giving a much better impression of the complex cellular movements during Dictyostelium morphogenesis than printed photographs.

Calcium channels in cardiac hypertrophy and heart failure

With the development of cardiac hypertrophy and heart failure, there are profound alterations in the ability of the cardiac cell to contract and relax. Despite several decades of intensive investigation, the precise cellular mechanisms responsible for this contractile dysfunction remain unknown. Recent advances in confocal microscopy and fluorescent calcium (Ca⊃2⊃+) indicators have allowed investigators to image the focal release of Ca⊃2⊃+ from the sarcoplasmic reticulum for the first time. The use of these techniques offers the unique opportunity to study the elemental events of excitation–contraction coupling in diseased myocardium. In this review, we focus on the role of calcium channels (L-type and T-type) and the TTX-sensitive Ca⊃2⊃+-permeable sodium (Na⊃+) channel in normal and abnormal cardiac function. Additionally, the role of the sodium–calcium exchanger and the voltage-activated process in cardiac excitation–contraction coupling is discussed. The application of newer microscopic techniques to study animal models that mimic human disease longitudinally will provide the means to identify precisely the abnormalities in calcium handling that occur with the development of cardiac hypertrophy and heart failure.