Imaging Of Biological Structures Research Articles

Analyzing Macromolecular Complexes in Situ Using Cellular Cryo‐Electron Microscopy

Cryo‐electron tomography (cryo‐ET) is a powerful technique for imaging biological structures in their native state and in an unperturbed cellular environment. We integrate high resolution imaging by either cryo‐ET and sub‐tomogram averaging or TYGRESS (Tomography‐Guided 3D Reconstruction of Subcellular Structures), with comparative genetics, biochemical methods and EM‐visible labeling to deconstruct the in situ 3D structure and functional organization of macromolecular complexes. We use cilia and flagella as model systems to advance techniques and approaches for high‐resolution imaging of complex cellular structures. Cilia and flagella are ubiquitous organelles with important biological roles in motility and sensation; defects in their assembly or function cause severe human diseases. The normal beating of cilia requires the precise coordination of thousands of dynein motors, but much remains to be learned about dynein's motility and regulation. We are in the process of generating a molecular blueprint of major ciliary complexes to gain a better understanding of the inner workings of these intriguing nano‐machines.

X-ray-induced shortwave infrared biomedical imaging using rare-earth nanoprobes.

Shortwave infrared (SWIR or NIR-II) light provides significant advantages for imaging biological structures due to reduced autofluorescence and photon scattering. Here, we report on the development of rare-earth nanoprobes that exhibit SWIR luminescence following X-ray irradiation. We demonstrate the ability of X-ray-induced SWIR luminescence (X-IR) to monitor biodistribution and map lymphatic drainage. Our results indicate X-IR imaging is a promising new modality for preclinical applications and has potential for dual-modality molecular disease imaging.

Silicon‐Vacancy Color Centers in Nanodiamonds: Cathodoluminescence Imaging Markers in the Near Infrared

We recently demonstrated [ 18 ] multi-color cathodoluminescence (CL) of nanodiamonds as a powerful tool for nanoscale imaging of biological structures. CL is the emission of light by matter as the result of electron bombardment. CL imaging of bulk matter is typically carried out in an electron microscope outfi tted with an optical detector, and is widely used in materials characterization. [ 19 ] However, application of CL to imaging biological structures has been hindered by low photon count rates and rapid signal degradation due to the destruction of biomolecules and organic fl uorophores under electron beam irradiation. [ 20 ] These problems may be overcome with correlated CL and secondary electron (SE) imaging of samples tagged with surface-functionalizable nanoparticles containing defects that are robust under electron beam illumination and emit stable, spectrally distinct CL: e.g., A-band defects and NV centers in nanodiamonds, as well as Ce:LuAG nanophosphors. [ 18 ] In this approach, the CLemitting particles function as color-distinguishable nanoscale markers of targeted epitopes, while the correlated SE image provides high-resolution information about the cellular structure. The combination of nanoscale molecular localization and structural imaging acquired simultaneously and in the same instrument constitutes a uniquely powerful new imaging modality. For applications with large intrinsic CL background, e.g., correlated CL and SE imaging of unfi xed/ living cells in an environmental chamber in a scanning electron microscope (SEM), [ 21 ] it is desirable to use spectrally narrow CL markers with emission peaks at wavelengths distinct from the CL background (such as from proteins, nucleic acid, and fl uorophore-conjugated antibodies, which usually emit CL at short optical wavelengths). [ 22 ] However, many of the nanoparticle species investigated to date have relatively broad (∼100 nm) CL emission spectra at room temperature. [ 20 , 23,24 ] Here, we show that silicon-vacancy (Si-V) color centers [ 25 ] in nanodiamonds provide a promising solution to this challenge. Specifi cally, we demonstrate experimentally that nanodiamonds fabricated to incorporate Si-V color centers provide bright, spectrally narrow (∼5 nm) CL emission in the near-infrared (∼740 nm), which lies within the near-infrared transmission window of biological tissue and is DOI: 10.1002/smll.201303582 Nanodiamonds

Synthesis and characterization of biocompatible nanodiamond-silk hybrid material

A new hybrid material consisting of nanodiamonds (NDs) and silk has been synthesized and investigated. NDs can contain bright fluorescence centers, important for bioprobes to image biological structures at the nanoscale and silk provides a transparent, robust matrix for these nanoparticles in-vivo or in-vitro. The ND-silk hybrid films were determined to be highly transparent in the visible to near infrared wavelength range. The NDs embedded in silk exhibited significant enhancement of emission relative to air, correlating with theoretical predictions. Furthermore, animal toxicity tests confirmed ND-silk films to be non-toxic in an in-vivo mice model.

Quantitativesuper-Resolution Imaging of Biological Processes with High Spatiotemporalresoltuion

In the last decade, far field optical microscopy has undergone a revolution, in which a number of methods have been developed that break the diffraction limit of spatial resolution. These methods allow imaging biological structures and protein organization at the nanometer length scales, opening the door for exciting new discoveries in biology. However, the super-resolution methods have certain limitations. One such limitation concerns live cell applications and the trade-off between spatial and temporal resolution, which makes it challenging to achieve high spatiotemporal resolution simultaneously. We have recently developed an all-optical correlative imaging platform to circumvent this problem and applied it to study how motor proteins overcome obstacles and roadblocks to move cargo from one place to other in the complex cellular environment.An additional complication in super-resolution microscopy is the ability to quantify the images to count proteins and extract information about protein stoichiometry. This challenge stems from the complex biophysics of fluorescent dyes and fluorescent proteins. We have also developed ways to characterize these photophysical properties in order to quantify super-resolution images.In this talk, I will explain the progress we have made in both correlated live-cell and super-resolution imaging as well as quantitative super-resolution imaging. I will also give examples of biological applications along both lines.

Atomic force microscopy as a tool to study Xenopus laevis embryo

Atomic force microscopy (AFM) has become a powerful tool for imaging biological structures (from single molecules to living cells) and carrying out measurements of their mechanical properties. AFM provides three-dimensional high-resolution images of the studied biological objects in physiological environment. However there are only few AFM investigations of fresh tissue explants and virtually no such research on a whole organism, since most researchers work with cell cultures. In the current work AFM was used to observe the surface of living and fixed embryos and to measure mechanical properties of naive embryos and embryos with overexpression of guanine nucleotide-binding protein G-alpha-13.

The Investigation of Lipid Membrane Deformation in Giant Unilamellar Vesicles using Microfluidic Technology

Membrane curvature is known to play an important role in cell growth, division and movement. Different lipid and membrane proteins can influence the cell's shape by changing the membrane curvature. Developing new technologies is vital for investigating and understanding the processes involved when membranes deform. We present an approach to study membrane curvature in giant unilamellar vesicles (GUVs) using microfluidic technology. Micron-sized channel networks in microfluidic devices are ideally suited for the manipulation and imaging of biological structures.

Sparse Statistical Deconvolution for High-Density Localization Microscopy

Super-resolution imaging based on single-molecule localization techniques can achieve image resolution of ∼20 nm but requires that the simultaneously activated fluorophores be relatively sparse in the field of view. Yet, both isolated and moderately crowded fluorophores provide valuable information about the image that is only partially degraded by overlap. Analyzing the complete fluorescence data set could accelerate super-resolution image estimation for dynamic biological samples. Whereas recent work has improved methods for labeling and imaging biological structures, less has been done to optimize the localization computation at the heart of these techniques to extract as much image information as possible from the data. We propose a novel computational analysis procedure for fluorescence imaging data based on iterative image deconvolution. Our algorithm exploits the sparse structure of data arising from dilute fluorophores, but does not require that molecules’ images are non-overlapping. We use the expected statistical properties of fluorescence imaging data, including the image sparseness and temporal correlations induced by intermittent activation of emitters, to constrain a deconvolution procedure that iteratively improves the estimated super-resolution image. We validated our approach and compared it with single- and multi-emitter fitting procedures using simulated data. We also applied our algorithm to stochastic optical reconstruction microscopy (STORM) data from immunohistochemically labeled microtubules. Our algorithm achieves accurate super-resolution image reconstruction even in moderately crowded conditions in which analysis by localization fails. This improved analysis allows a ∼5-fold speedup in imaging by permitting the use of a higher density of active emitters.

Synthesis and characterization of luminescent cadmium selenide/zinc selenide/zinc sulfide cholinomimetic quantum dots

Luminescent quantum dots conjugated with highly selective molecular recognition ligands are widely used for targeting and imaging biological structures. In this paper, water soluble cholinomimetic cadmium selenide (core), zinc selenide/zinc sulfide (shell) quantum dots were synthesized for targeting cholinergic sites. Cholinomimetic specificity was incorporated by conjugation of the quantum dots to an aminated analogue of hemicholinium-15, a well known competitive inhibitor of the high affinity choline uptake transporter. Detailed evaluation of the nanocrystal synthesis and characterization of the final product was conducted by (1)H and (31)P NMR, absorption and emission spectroscopy, as well as transmission electron microscopy.

Doppler encoded excitation pattern tomographic optical microscopy

Most far-field optical imaging systems rely on lenses and spatially resolved detection to probe distinct locations on the object. We describe and demonstrate a high-speed wide-field approach to imaging that instead measures the complex spatial Fourier transform of the object by detecting its spatially integrated response to dynamic acousto-optically synthesized structured illumination. Tomographic filtered backprojection is applied to reconstruct the object in two or three dimensions. This technique decouples depth of field and working distance from resolution, in contrast to conventional imaging, and can be used to image biological and synthetic structures in fluoresced or scattered light employing coherent or broadband illumination. We discuss the electronically programmable transfer function of the optical system and its implications for imaging dynamic processes. We also explore wide-field fluorescence imaging in scattering media by coherence gating. Finally, we present two-dimensional high-resolution tomographic image reconstructions in both scattered and fluoresced light demonstrating a thousandfold improvement in the depth of field compared to conventional lens-based microscopy.

Targeted Photoswitchable Probe for Nanoscopy of Biological Structures

We introduce a photoswitchable O6-benzylguanine derivative and demonstrate its use for super-resolution microscopy of SNAP-tagged proteins based on single fluorophore localization. Stochastic Optical Reconstruction Microscopy (STORM) reveals SNAP-tagged microtubule structures with 25 nm resolution. The described probe in combination with the versatile SNAP-tag labeling opens new possibilities for imaging biological structures at the nanoscale.

Biological imaging with 4D ultrafast electron microscopy

Advances in the imaging of biological structures with transmission electron microscopy continue to reveal information at the nanometer length scale and below. The images obtained are static, i.e., time-averaged over seconds, and the weak contrast is usually enhanced through sophisticated specimen preparation techniques and/or improvements in electron optics and methodologies. Here we report the application of the technique of photon-induced near-field electron microscopy (PINEM) to imaging of biological specimens with femtosecond (fs) temporal resolution. In PINEM, the biological structure is exposed to single-electron packets and simultaneously irradiated with fs laser pulses that are coincident with the electron pulses in space and time. By electron energy-filtering those electrons that gained photon energies, the contrast is enhanced only at the surface of the structures involved. This method is demonstrated here in imaging of protein vesicles and whole cells of Escherichia coli, both are not absorbing the photon energy, and both are of low-Z contrast. It is also shown that the spatial location of contrast enhancement can be controlled via laser polarization, time resolution, and tomographic tilting. The high-magnification PINEM imaging provides the nanometer scale and the fs temporal resolution. The potential of applications is discussed and includes the study of antibodies and immunolabeling within the cell.

Imaging biological structures with fluorescence photoactivation localization microscopy

Fluorescence photoactivation localization microscopy (FPALM) images biological structures with subdiffraction-limited resolution. With repeated cycles of activation, readout and bleaching, large numbers of photoactivatable probes can be precisely localized to obtain a map (image) of labeled molecules with an effective resolution of tens of nanometers. FPALM has been applied to a variety of biological imaging applications, including membrane, cytoskeletal and cytosolic proteins in fixed and living cells. Molecular motions can be quantified. FPALM can also be applied to nonbiological samples, which can be labeled with photoactivatable probes. With emphasis on cellular imaging, we describe here the adaptation of a conventional widefield fluorescence microscope for FPALM and present step-by-step procedures to successfully obtain and analyze FPALM images. The fundamentals of this protocol may also be applicable to users of similar imaging techniques that apply localization of photoactivatable probes to achieve super-resolution. Once alignment of the setup has been completed, data acquisitions can be obtained in approximately 1-30 min and analyzed in approximately 0.5-4 h.

Cell imaging by coherent backscattering microscopy using frequency-shifted optical feedback in a microchip laser

In this work, we present a new development of the laser optical feedback imaging technique for imaging biological structures with a high resolution. The first results obtained on human red blood cells and mice cerebral and muscular tissues slices are shown. The performances of the system and its future developments are also discussed.

Two-Photon Microscopy: Shedding Light on the Chemistry of Vision

Two-photon microscopy (TPM) has come to occupy a prominent place in modern biological research with its ability to resolve the three-dimensional distribution of molecules deep inside living tissue. TPM can employ two different types of signals, fluorescence and second harmonic generation, to image biological structures with subcellular resolution. Two-photon excited fluorescence imaging is a powerful technique with which to monitor the dynamic behavior of the chemical components of tissues, whereas second harmonic imaging provides novel ways to study their spatial organization. Using TPM, great strides have been made toward understanding the metabolism, structure, signal transduction, and signal transmission in the eye. These include the characterization of the spatial distribution, transport, and metabolism of the endogenous retinoids, molecules essential for the detection of light, as well as the elucidation of the architecture of the living cornea. In this review, we present and discuss the current applications of TPM for the chemical and structural imaging of the eye. In addition, we address what we see as the future potential of TPM for eye research. This relatively new method of microscopy has been the subject of numerous technical improvements in terms of the optics and indicators used, improvements that should lead to more detailed biochemical characterizations of the eyes of live animals and even to imaging of the human eye in vivo.

Imaging of biological structures by scanning probe microscopy

Imaging of biological structures by scanning probe microscopy

High-resolution imaging of bacteriorhodopsin by atomic force microscopy.

In the last years the atomic force microscope (AFM; ref. ) has become a powerful imaging tool for the biologist. The unique features like the possibility to image biological structures in their native environment (i.e., in buffer solution, at room temperature, and under normal pressure), the high lateral and vertical resolution, and the high signal-to-noise ratio of the topographs acquired by AFM make this instrument outstanding. It has made the observation of different single biomolecules at work and the monitoring of biomolecular interactions by time-lapse AFM possible (for recent reviews, see refs. , , ).

Imaging biological structures with the cryo atomic force microscope

It has long been recognized that one of the major limitations in biological atomic force microscopy (AFM) is the softness of most biological samples, which are easily deformed or damaged by the AFM tip, because of the high pressure in the contact area, especially from the very sharp tips required for high resolution. Another is the molecular motion present at room temperature due to thermal fluctuation. Using an AFM operated in liquid nitrogen vapor (cryo-AFM), we demonstrate that cryo-AFM can be applied to a large variety of biological samples, from immunoglobulins to DNA to cell surfaces. The resolution achieved with cryo-AFM is much improved when compared with AFM at room temperature with similar specimens, and is comparable to that of cryo-electron microscopy on randomly oriented macromolecules. We will also discuss the technical problems that remain to be solved for achieving even higher resolution with cryo-AFM and other possible applications of this novel technique.

To boldly glow ... applications of laser scanning confocal microscopy in developmental biology.

The laser scanning confocal microscope (LSCM) is now established as an invaluable tool in developmental biology for improved light microscope imaging of fluorescently labelled eggs, embryos and developing tissues. The universal application of the LSCM in biomedical research has stimulated improvements to the microscopes themselves and the synthesis of novel probes for imaging biological structures and physiological processes. Moreover the ability of the LSCM to produce an optical series in perfect register has made computer 3-D reconstruction and analysis of light microscope images a practical option.

Imaging of biological structures with the scanning transmission electron microscope

The scanning transmission electron microscope (STEM) is discussed in view of biological applications. Theoretical considerations are given, but the emphasis is directed to practical examples from a range of biological projects. The STEM is most efficiently used in elastic and inelastic dark-field modes providing information on the scattering power of the irradiated sample. Thus, the STEM is an ideal tool for quantitative measurements such as mass-mapping or element-mapping at high resolution. Limitations of such methods due to multiple scattering and quantum noise are briefly reviewed.