The power of correlative microscopy: multi-modal, multi-scale, multi-dimensional
Highlights
► Correlative microscopy combines multiple imaging platforms for targeted analysis. ► Sample preparation is critical for successful correlation. ► Probes which contrast at light and electron levels are discussed. ► Improved software and hardware tools for automation facilitate throughput. ► Value of correlative microscopy is enhanced by broader application and data integration.
Introduction
In this era of genetic manipulation, the widespread adoption of fluorescent protein chimeras has a prominent role in enhancing our understanding of cellular and molecular phenomena. As new molecules of interest are discovered and known molecules reassessed, fluorescent protein fusions have become a critical tool to analyze their functions from the whole organism to sub-cellular machinery to single molecules. However, it is clear that no single imaging technology can reveal all the details of a biological sample. This readily can be appreciated when one considers while transmission electron microscopy (TEM) has the best resolution, live cell imaging is not possible and has restricted field-of-view and severe limitations in three-dimensionality. Conversely, photon-based optical microscopy, enables live-cell, large field-of-view and is far more amenable to extended 3D imaging, but spatial resolution often prevents definitive localization. It is thus a natural extension of the various imaging tools, each with inherent capabilities and limitations, to expand the overall information content by correlative microscopy.
The concept of correlative microscopy is not new. Virtually all classically trained electron microscopists screen semi-thick resin sections from a microtome and by light microscopy (LM) locate a desired region of the sample for ultrathin sections and TEM. Although the same exact region is not viewed by both LM and TEM, this simple approach exploits four fundamental features of correlative microscopy: first, identifying a specific target (structure, region, cell type, tissue or biological phenomenon); second, putting the target into context of a much larger region; third, unique information content from different probes, stains or contrasting reagents; and fourth, resolution improvements by EM that provide ultrastructural detail. Any one of these features justifies the appropriate use of correlative microscopy. Indeed, this basic approach has naturally evolved into the most commonly applied correlative combination referred to as Correlative Light and Electron Microscopy (CLEM).
Although the merits of basic CLEM are well documented, there are numerous other correlative combinations that can broaden the range of data content from individual targets. However, more sophisticated correlative methods require the precise relocation of the same target using very different sample preparation protocols, support structures, contrasting mechanisms and physical sample size. Here, we describe major advances the key areas of sample preparation, correlative probes [1, 2], automation and image alignment that will have the biggest impact on driving the development and mainstream application of correlative microscopy. Detailed protocols are beyond the scope of this opinion; however, references to excellent reviews [3] and methods are provided to guide those interested in exploring recent advanced adaptations of correlative microscopy.
Section snippets
Sample preparation
Careful consideration of fixation is paramount to minimize artifacts that lead to data misinterpretation. While conventional fixation has its place (and in some cases is the only way to preserve samples) physical fixation using cryogenic methods is the gold-standard for ultrastructural preservation. This is attributed to the rapid cessation of cellular activity within milliseconds as opposed to minutes with conventional chemical fixation. Cryo-fixation technologies such as high-pressure
Correlative probes
A current limitation of correlative microscopy is the lack of compatibility between probes used in LM and the standard preparation protocols that are typically employed on another imaging platform. Many organic dyes and FPs quench when moved from an aqueous to a solvent or dehydrated environment. For FPs, addition of 5% water in solvent or resin steps enables preservation en bloc [8]. Fortunately, even if fluorescence cannot be maintained en bloc, we can garner much information by overlaying
Hybrid versus automated approach
The single most significant bottleneck in correlative microscopy is the slow and laborious process of sample relocation between imaging platforms, hence the appeal for hybrid systems that do not rely on separate microscopes. The most widely used hybrid system is an AFM microscope integrated with an inverted light microscope. In this case, the light microscope serves as an excellent tool for identifying cells or other structures using transmitted light and fluorescence microscopy combined with a
Image alignment
Image alignment is a significant challenge for correlative microscopy and this is compounded by the fact that not only can non-linear distortions occur because of the inherent differences between various scanning and imaging systems, but also numerous processing steps leave them prone to physical distortions (i.e., shrinkage, sectioning artifacts). Furthermore, because data are often near or beyond the boundaries of what is measurable, each type of microscopy introduces artifacts: AFMs dilate
The future of correlative microscopy
The most significant near term impact on correlative microscopy will come from the rapid development and availability of suitable commercial solutions putting this approach at the fingertips of many more scientists. Furthermore, it must be stressed that the scope of correlative microscopy must be considerably broadened to include any combinations of the numerous more advanced tools that exist such as: Förster Resonance Energy Transfer (FRET) [38, 39], Fluorescence Correlation Spectroscopy (FCS)
Conclusions
Recent correlative efforts have placed emphasis on new probes, compatible sample preparation methods, automation, standardized cell and tissue support and transfer devices, fiducial markers and data alignment/integration. The clear need for monitoring multi-parameters for individual events maximizing information content cannot be overstated. However, in order to meet the demands of rigorous scientific protocol, the power of statistics must be a driving factor in this endeavor. Thus, a concerted
Disclosure statement
Russell Taylor is the president of and a stockholder in nano Manipulator, Incorporated, which is a company that sells a commercial version of the software used to develop the AFM and fluorescence overlay technology. Kirk Czymmek occasionally serves as a paid consultant for Carl Zeiss to train new users and sales and applications representatives on the basics of confocal or correlative microscopy. He also provides Zeiss feedback for enhancing existing and future correlative products as it
References and recommended reading
Papers of particular interest, published within the period of review, have been highlighted as:
• of special interest
•• of outstanding interest
Acknowledgement
The work presented here was partly supported by NIH 5-P41-RR02170.
References (55)
- et al.
Studying intracellular transport using high-pressure freezing and Correlative Light Electron Microscopy
Semin Cell Dev Biol
(2009) - et al.
Identification of neuromuscular junctions by correlative confocal and transmission electron microscopy
J Neurosci Methods
(2010) - et al.
Visualizing macromolecules with fluoronanogold: from photon microscopy to electron tomography
Methods Cell Biol
(2007) - et al.
Correlative fluorescence microscopy and scanning transmission electron microscopy of quantum-dot-labeled proteins in whole cells in liquid
ACS Nano
(2010) - et al.
A fluorescence scanning electron microscope
Ultramicroscopy
(2009) - et al.
Integrated fluorescence and transmission electron microscopy
J Struct Biol
(2008) - et al.
Correlative fluorescence and transmission electron microscopy: an elegant tool to study the actin cytoskeleton of whole-mount (breast) cancer cells
J Microsc
(2009) Correlative microscopy
G. I. T. Imaging Microsc
(2010)- et al.
Integration of confocal and atomic force microscopy images
J Neurosci Methods
(2009) - et al.
Protein localization in electron micrographs using fluorescence nanoscopy
Nat Methods
(2011)