Expansion microscopy

Last updated

Expansion microscopy (ExM) is a sample preparation tool for biological samples that allows investigators to identify small structures by expanding them using a polymer system. [1] The premise is to introduce a polymer network into cellular or tissue samples, and then physically expand that polymer network using chemical reactions to increase the size of the biological structures. Among other benefits, ExM allows those small structures to be imaged with a wider range of microscopy techniques. It was first proposed in a 2015 article by Fei Chen, Paul W. Tillberg, and Edward Boyden. [2] Current research allows for the expansion of samples up to 16x larger than their initial size. [3] This technique has been found useful in various laboratory settings, such as analyzing biological molecules. ExM allows researchers to use standard equipment in identifying small structures, but requires following of procedures in order to ensure clear results.

Contents

Principles

Purpose

4-step process of ExM ExM Steps.jpg
4-step process of ExM

Traditional light microscopy has limits of resolution that prevent it from reliably distinguishing small structures that are important to biological function, and must instead be imaged by a higher-resolution technique, such as electron microscopy. For example, synaptic vesicles are 40-50 nanometers in diameter, which is below the commonly quoted resolution limit of 200 nanometers for light microscopy. [4] Expansion microscopy solves this problem by expanding the underlying tissue sample. One key advantage of samples prepared using expansion microscopy and light microscopy over conventional electron microscopy is that it also allows investigators to stain for and visualize particular molecules in the sample, such as specific proteins or RNA to identify their density and distribution in relation to the biological structures of interest. The most beneficial principle of expansion microscopy is that it requires no specialized equipment; [5] the materials for expansion are worth little to nothing compared to the price of a microscope that could get the same resolution.

Stages

Expansion microscopy is a multistep process that, depending on the protocol, has different requirements for gelation and expansion. The sequence of steps are stain, link, digest, and expand. [5] The staining process can take many different forms, and only requires that the fluorophores used can attach to the polymer in the next step. Linking is the process of adding a polymer gel to the cells, which permeabilize through the cell. The linking step also includes, as the name suggests, the process of linking the fluorophores to the gel. The digestion step involves adding a solution that digests the cell, removing the structure from the cell. If this step fails, the gel will not expand uniformly because the cell will try to stay together. Failure of this step can also cause cracking or fractures in the cell. [6] Lastly, expansion causes the gel to be physically expanded in all directions, which causes the fluorophores that are attached to the gel to expand as well.

History

In 2015, Chen, Tillberg, and Boyden, all of MIT first described expansion microscopy as a method to enhance microscopy resolution by swelling a sample rather than using higher resolution equipment. [2] Since then, the use of ExM has continued to grow. The novelty of the technique means that few applications have been developed. The most common use is in biological samples.

In 2016, several papers were published detailing workarounds for ExM's traditional limitation of labeling probes. These changes proposed a way to use ExM with conventional microscopy probes, allowing wider use. In 2016, these new labeling methods were applied to allow fluorescence microscopy of RNA molecules, which in turn led to spatially precise in situ sequencing, namely ExSeq (expansion sequencing), in 2021. [7]

Even with expansion microscopy, Alzheimer’s disease-related amyloid-beta plaques cannot be resolved. Boyden devised "expansion-revealing microscopy" in 2022, adding fluorescent markers after expansion instead of before. He replaced enzymes with heat. This enabled an up to 20-fold expansion, wiithodamaging proteins. It has been used to reveal synapse details, and to shed light on Alzheimer’s disease, revealing occasional spirals of amyloid-beta protein around axons, which are the threadlike parts of nerve cells that carry electrical impulses. [8]

Theory

Expansion microscopy is achieved by synthesizing a polymer system within a specimen. By then swelling this polymer network, the sample is expanded to be examined under conventional microscopic analysis tools without degrading the integrity of the sample. This allows a sample to be analyzed with a less powerful microscope than would be needed without expansion, and makes analysis of microscopic biological samples more accessible to labs that would otherwise have not been available to afford or obtain necessary powerful microscopy technology. [4]

Applications

Use

Expansion microscopy is a method which improves the final image resolution during regular microscopy by physically enlarging the organism, tissue, or molecule itself. After the enlargement of the organism, tissue, or molecule, more standard microscopes can achieve higher resolution imaging of smaller physiological properties. The primary fields this method is used in are those involved in analyzing biological samples with the addition of immunostaining or fluorescent dyes. [9] Fluorescent labels are applied after expansion microscopy to make visible dense clusters of proteins and molecules. [10] However, this technique has since been adopted into many different fields of research and continues to grow and be applied in more and more laboratory settings.

Disease and diagnoses

Before the discovery of expansion microscopy, examination of cellular structures and biomolecules were done using diffraction-limited microscopy. They were mainly used to diagnose or investigate the pathogenesis of a wide variety of predisease and disease states. However, biomolecules are nanoscale in dimension and are located with nanoscale precision throughout cells and tissues. Several techniques such as super-resolution microscopy were used, but these required complex hardware and were difficult to apply to human tissues. Thus, expansion microscopy was developed. This method physically magnified the tissue samples rather than optically, and as a result was able to produce images with high resolution. These high quality images of tissues served as a turning point in diagnostic and medical expansion microscopy. [11]

Like many other techniques, expansion microscopy also possesses many potentials in the medical and diagnostic fields. Expansion microscopy improves the resolution of light microscopy by physically expanding the specimens. When this technique is applied to the clinical tissue samples nanoscale imaging of human tissue specimens. First, expansion pathology is used to convert clinical samples into a compatible state for the expansion microscopy. This process can be used for optical diagnosis of kidney minimal-change disease, early breast neoplastic lesions and to spot the difference of normal human tissue specimens to cancer tissue specimens, enabling a routine use of clinical research. [12] The use of pathogenic expansion microscopy enabled clear images of tissue. Applying expansion microscopy on microarrays containing specimens from various organs, such as breast, prostate, lung, colon, pancreas, kidney, liver, and ovary, including normal and cancer-containing tissues, enabled the diagnostic and the examination of cellular network of diseased state tissues. This imaging reveals sub-diffraction limit sized features of the intermediate filaments keratin and vimentin, critical in the epithelial mesenchymal transition, cancer progression and initiation of metastasis. [11]

In the future, after further development of this technique, observation of nanoscale morphology of biomolecules and samples from wide range of human organs is anticipated to be provided.

Neuroscience

Many of the questions surrounding neuroscience attempt to answer and understand molecules and wiring in neural circuits. However, mapping these structures across the large scales of neural circuits is difficult. In these cases, ExM magnifies biological specimens such as brain circuits and allows them to be more easily mapped. Biomolecules, such as proteins and nucleic acids, are anchored to the polymer, which is then swelled in order to expand the biomolecules. Due to the increased distance between the biomolecules, ordinary microscopes can then perform nanoscale resolution imaging. Through the use of ExM technique, neuroscientists can more easily map images of synapses, cells, and neural circuits. [13]

Subsets

With the development of expansion microscopy, scientists have begun to create subsets of the technique, including scanning Joule expansion microscopy, or SJEM. SJEM utilizes a thermal imaging technique which measures the thermal expansion of Joule-heated elements. One of the largest advantages of SJEM over older submicron thermal imaging techniques is that SJEM does not require the nanofabrication of specialized probes. Rather, SJEM only requires a standard atomic force microscope and simple electronics. [14]

Advantages

One of the most significant advantages of expansion microscopy versus other forms of microscopy is that it does not require a stronger optical equipment to perform high-resolution imaging. Because ExM enlarges the physical sample, it relieves researchers from the need to purchase an expensive microscopy equipment such as electron microscopes for super-resolution studies. By expanding the sample, it becomes more easily examinable as the larger structures can then be examined using traditional microscopy techniques, such as light microscopy.

Limitations

Each of the four preparation steps of ExM must fully complete, or the cell will not yield a bright and clear stain. Failure to complete these steps can result in the breaking of the cell or uneven expansion, distorting the image beyond use. ExM struggles in those procedures that use fluorophore markers, as the polymerization process can bleach these fluorophores, rendering them unusable. There are some, such as Alexa 488 and Atto 565 that are still effective after polymerization, however their efficacy is greatly decreased to about 50%. The conjugation of DNA with another antibody is often very costly and difficult. These two issues are the primary limitations to using ExM in biological samples. [15] It is important to note that while rebinding new antibodies can be costly and time consuming, it is sometimes made possible, post expansion, in cases where the antibody struggles to bind in dense tissue. After expansion, the tissue is far less dense and often allows for better reception of fluorescent antibodies.

Related Research Articles

<span class="mw-page-title-main">Microscopy</span> Viewing of objects which are too small to be seen with the naked eye

Microscopy is the technical field of using microscopes to view objects and areas of objects that cannot be seen with the naked eye. There are three well-known branches of microscopy: optical, electron, and scanning probe microscopy, along with the emerging field of X-ray microscopy.

<span class="mw-page-title-main">Microscope</span> Scientific instrument

A microscope is a laboratory instrument used to examine objects that are too small to be seen by the naked eye. Microscopy is the science of investigating small objects and structures using a microscope. Microscopic means being invisible to the eye unless aided by a microscope.

<span class="mw-page-title-main">Optical microscope</span> Microscope that uses visible light

The optical microscope, also referred to as a light microscope, is a type of microscope that commonly uses visible light and a system of lenses to generate magnified images of small objects. Optical microscopes are the oldest design of microscope and were possibly invented in their present compound form in the 17th century. Basic optical microscopes can be very simple, although many complex designs aim to improve resolution and sample contrast.

<span class="mw-page-title-main">Fluorescent tag</span>

In molecular biology and biotechnology, a fluorescent tag, also known as a fluorescent label or fluorescent probe, is a molecule that is attached chemically to aid in the detection of a biomolecule such as a protein, antibody, or amino acid. Generally, fluorescent tagging, or labeling, uses a reactive derivative of a fluorescent molecule known as a fluorophore. The fluorophore selectively binds to a specific region or functional group on the target molecule and can be attached chemically or biologically. Various labeling techniques such as enzymatic labeling, protein labeling, and genetic labeling are widely utilized. Ethidium bromide, fluorescein and green fluorescent protein are common tags. The most commonly labelled molecules are antibodies, proteins, amino acids and peptides which are then used as specific probes for detection of a particular target.

<span class="mw-page-title-main">Immunostaining</span>

In biochemistry, immunostaining is any use of an antibody-based method to detect a specific protein in a sample. The term "immunostaining" was originally used to refer to the immunohistochemical staining of tissue sections, as first described by Albert Coons in 1941. However, immunostaining now encompasses a broad range of techniques used in histology, cell biology, and molecular biology that use antibody-based staining methods.

<span class="mw-page-title-main">Immunofluorescence</span> Technique used for light microscopy

Immunofluorescence is a technique used for light microscopy with a fluorescence microscope and is used primarily on microbiological samples. This technique uses the specificity of antibodies to their antigen to target fluorescent dyes to specific biomolecule targets within a cell, and therefore allows visualization of the distribution of the target molecule through the sample. The specific region an antibody recognizes on an antigen is called an epitope. There have been efforts in epitope mapping since many antibodies can bind the same epitope and levels of binding between antibodies that recognize the same epitope can vary. Additionally, the binding of the fluorophore to the antibody itself cannot interfere with the immunological specificity of the antibody or the binding capacity of its antigen. Immunofluorescence is a widely used example of immunostaining and is a specific example of immunohistochemistry. This technique primarily makes use of fluorophores to visualise the location of the antibodies.

A total internal reflection fluorescence microscope (TIRFM) is a type of microscope with which a thin region of a specimen, usually less than 200 nanometers can be observed.

<span class="mw-page-title-main">Fluorescence microscope</span> Optical microscope that uses fluorescence and phosphorescence

A fluorescence microscope is an optical microscope that uses fluorescence instead of, or in addition to, scattering, reflection, and attenuation or absorption, to study the properties of organic or inorganic substances. "Fluorescence microscope" refers to any microscope that uses fluorescence to generate an image, whether it is a simple set up like an epifluorescence microscope or a more complicated design such as a confocal microscope, which uses optical sectioning to get better resolution of the fluorescence image.

<span class="mw-page-title-main">Confocal microscopy</span> Optical imaging technique

Confocal microscopy, most frequently confocal laser scanning microscopy (CLSM) or laser confocal scanning microscopy (LCSM), is an optical imaging technique for increasing optical resolution and contrast of a micrograph by means of using a spatial pinhole to block out-of-focus light in image formation. Capturing multiple two-dimensional images at different depths in a sample enables the reconstruction of three-dimensional structures within an object. This technique is used extensively in the scientific and industrial communities and typical applications are in life sciences, semiconductor inspection and materials science.

<span class="mw-page-title-main">Autofluorescence</span>

Autofluorescence is the natural emission of light by biological structures such as mitochondria and lysosomes when they have absorbed light, and is used to distinguish the light originating from artificially added fluorescent markers (fluorophores).

<span class="mw-page-title-main">Single-molecule experiment</span>

A single-molecule experiment is an experiment that investigates the properties of individual molecules. Single-molecule studies may be contrasted with measurements on an ensemble or bulk collection of molecules, where the individual behavior of molecules cannot be distinguished, and only average characteristics can be measured. Since many measurement techniques in biology, chemistry, and physics are not sensitive enough to observe single molecules, single-molecule fluorescence techniques caused a lot of excitement, since these supplied many new details on the measured processes that were not accessible in the past. Indeed, since the 1990s, many techniques for probing individual molecules have been developed.

<span class="mw-page-title-main">STED microscopy</span>

Stimulated emission depletion (STED) microscopy is one of the techniques that make up super-resolution microscopy. It creates super-resolution images by the selective deactivation of fluorophores, minimizing the area of illumination at the focal point, and thus enhancing the achievable resolution for a given system. It was developed by Stefan W. Hell and Jan Wichmann in 1994, and was first experimentally demonstrated by Hell and Thomas Klar in 1999. Hell was awarded the Nobel Prize in Chemistry in 2014 for its development. In 1986, V.A. Okhonin had patented the STED idea. This patent was unknown to Hell and Wichmann in 1994.

<span class="mw-page-title-main">Immunocytochemistry</span>

Immunocytochemistry (ICC) is a common laboratory technique that is used to anatomically visualize the localization of a specific protein or antigen in cells by use of a specific primary antibody that binds to it. The primary antibody allows visualization of the protein under a fluorescence microscope when it is bound by a secondary antibody that has a conjugated fluorophore. ICC allows researchers to evaluate whether or not cells in a particular sample express the antigen in question. In cases where an immunopositive signal is found, ICC also allows researchers to determine which sub-cellular compartments are expressing the antigen.

<span class="mw-page-title-main">Fluorescence in the life sciences</span> Scientific investigative technique

Fluorescence is used in the life sciences generally as a non-destructive way of tracking or analysing biological molecules. Some proteins or small molecules in cells are naturally fluorescent, which is called intrinsic fluorescence or autofluorescence. Alternatively, specific or general proteins, nucleic acids, lipids or small molecules can be "labelled" with an extrinsic fluorophore, a fluorescent dye which can be a small molecule, protein or quantum dot. Several techniques exist to exploit additional properties of fluorophores, such as fluorescence resonance energy transfer, where the energy is passed non-radiatively to a particular neighbouring dye, allowing proximity or protein activation to be detected; another is the change in properties, such as intensity, of certain dyes depending on their environment allowing their use in structural studies.

Super-resolution microscopy is a series of techniques in optical microscopy that allow such images to have resolutions higher than those imposed by the diffraction limit, which is due to the diffraction of light. Super-resolution imaging techniques rely on the near-field or on the far-field. Among techniques that rely on the latter are those that improve the resolution only modestly beyond the diffraction-limit, such as confocal microscopy with closed pinhole or aided by computational methods such as deconvolution or detector-based pixel reassignment, the 4Pi microscope, and structured-illumination microscopy technologies such as SIM and SMI.

The following outline is provided as an overview of and topical guide to biophysics:

Photo-activated localization microscopy and stochastic optical reconstruction microscopy (STORM) are widefield fluorescence microscopy imaging methods that allow obtaining images with a resolution beyond the diffraction limit. The methods were proposed in 2006 in the wake of a general emergence of optical super-resolution microscopy methods, and were featured as Methods of the Year for 2008 by the Nature Methods journal. The development of PALM as a targeted biophysical imaging method was largely prompted by the discovery of new species and the engineering of mutants of fluorescent proteins displaying a controllable photochromism, such as photo-activatible GFP. However, the concomitant development of STORM, sharing the same fundamental principle, originally made use of paired cyanine dyes. One molecule of the pair, when excited near its absorption maximum, serves to reactivate the other molecule to the fluorescent state.

<span class="mw-page-title-main">Live-cell imaging</span> Study of living cells using time-lapse microscopy

Live-cell imaging is the study of living cells using time-lapse microscopy. It is used by scientists to obtain a better understanding of biological function through the study of cellular dynamics. Live-cell imaging was pioneered in the first decade of the 21st century. One of the first time-lapse microcinematographic films of cells ever made was made by Julius Ries, showing the fertilization and development of the sea urchin egg. Since then, several microscopy methods have been developed to study living cells in greater detail with less effort. A newer type of imaging using quantum dots have been used, as they are shown to be more stable. The development of holotomographic microscopy has disregarded phototoxicity and other staining-derived disadvantages by implementing digital staining based on cells’ refractive index.

Super-resolution dipole orientation mapping (SDOM) is a form of fluorescence polarization microscopy (FPM) that achieved super resolution through polarization demodulation. It was first described by Karl Zhanghao and others in 2016. Fluorescence polarization (FP) is related to the dipole orientation of chromophores, making fluorescence polarization microscopy possible to reveal structures and functions of tagged cellular organelles and biological macromolecules. In addition to fluorescence intensity, wavelength, and lifetime, the fourth dimension of fluorescence—polarization—can also provide intensity modulation without the restriction to specific fluorophores; its investigation in super-resolution microscopy is still in its infancy.

<span class="mw-page-title-main">Fluorescence imaging</span> Type of non-invasive imaging technique

Fluorescence imaging is a type of non-invasive imaging technique that can help visualize biological processes taking place in a living organism. Images can be produced from a variety of methods including: microscopy, imaging probes, and spectroscopy.

References

  1. ↑ Markoff J (2015-01-19). "Expansion Microscopy Stretches Limits of Conventional Microscopes". New York Times. Retrieved 21 October 2015.
  2. 1 2 Chen F, Tillberg PW, Boyden ES (January 2015). "Optical imaging. Expansion microscopy". Science. 347 (6221): 543–8. Bibcode:2015Sci...347..543C. doi:10.1126/science.1260088. PMC   4312537 . PMID   25592419.
  3. ↑ "Larger than Life: Monique Copeland and Paul Tillberg Explain Expansion Microscopy | Janelia Research Campus". www.janelia.org. Retrieved 2019-05-01.
  4. 1 2 Fagan T. "Kiss and Tell—STED Microscopy Resolves Vesicle Recycling Question". AlzForum. Retrieved 21 October 2015.
  5. 1 2 Chozinski TJ, Halpern AR, Okawa H, Kim HJ, Tremel GJ, Wong RO, Vaughan JC (June 2016). "Expansion microscopy with conventional antibodies and fluorescent proteins". Nature Methods. 13 (6): 485–8. doi:10.1038/nmeth.3833. PMC   4929147 . PMID   27064647.
  6. ↑ Wassie AT, Zhao Y, Boyden ES (January 2019). "Expansion microscopy: principles and uses in biological research". Nature Methods. 16 (1): 33–41. doi:10.1038/s41592-018-0219-4. PMC   6373868 . PMID   30573813.
  7. ↑ Alon, Shahar; Goodwin, Daniel R.; Sinha, Anubhav; Wassie, Asmamaw T.; Chen, Fei; Daugharthy, Evan R.; Bando, Yosuke; Kajita, Atsushi; Xue, Andrew G.; Marrett, Karl; Prior, Robert; Cui, Yi; Payne, Andrew C.; Yao, Chun-Chen; Suk, Ho-Jun; Wang, Ru; Yu, Chih-Chieh (Jay); Tillberg, Paul; Reginato, Paul; Pak, Nikita; Liu, Songlei; Punthambaker, Sukanya; Iyer, Eswar P. R.; Kohman, Richie E.; Miller, Jeremy A.; Lein, Ed S.; Lako, Ana; Cullen, Nicole; Rodig, Scott; Helvie, Karla; Abravanel, Daniel L.; Wagle, Nikhil; Johnson, Bruce E.; Klughammer, Johanna; Slyper, Michal; Waldman, Julia; Jané-Valbuena, Judit; Rozenblatt-Rosen, Orit; Regev, Aviv; Church, George M.; Marblestone, Adam H.; Boyden, Edward S. (2021). "Expansion sequencing: Spatially precise in situ transcriptomics in intact biological systems". Science. 371 (6528): eaax2656. doi:10.1126/science.aax2656. ISSN   0036-8075. PMC   7900882 .
  8. ↑ "Making the invisible visible". The Economist. September 7, 2022. ISSN   0013-0613 . Retrieved 2022-09-19.
  9. ↑ Chozinski, T.; Halpertn, A.; Okawa, H.; Kim, H.; Tremel, G.; Wong, R.; Vaughan, J. Expansion microscopy with conventional antibodies and fluorescent proteins. Nature Methods, 2016, 13, 485-488.
  10. ↑ "Microscopy technique reveals hidden nanostructures in cells and tissues". August 29, 2022.
  11. 1 2 Zhao Y, Bucur O, Irshad H, Chen F, Weins A, Stancu AL, Oh EY, DiStasio M, Torous V, Glass B, Stillman IE, Schnitt SJ, Beck AH, Boyden ES (August 2017). "Nanoscale imaging of clinical specimens using pathology-optimized expansion microscopy". Nature Biotechnology. 35 (8): 757–764. doi:10.1038/nbt.3892. PMC   5548617 . PMID   28714966.
  12. ↑ "Synthetic Neurobiology Group: Ed Boyden, Principal Investigator". syntheticneurobiology.org. Retrieved 2019-05-03.
  13. ↑ Karagiannis ED, Boyden ES (June 2018). "Expansion microscopy: development and neuroscience applications". Current Opinion in Neurobiology. 50: 56–63. doi:10.1016/j.conb.2017.12.012. PMC   5984670 . PMID   29316506.
  14. ↑ Majumdar A, Varesi J (1998). "Nanoscale Temperature Distributions Measured by Scanning Joule Expansion Microscopy". Journal of Heat Transfer. 120 (2): 297. doi:10.1115/1.2824245.
  15. ↑ Cho, I.; Seo, J. Y.; Chang, J. (2018). "Expansion microscopy". Journal of Microscopy. 271 (2): 123–128. doi:10.1111/jmi.12712. ISSN   1365-2818. PMID   29782656.
This page is based on this Wikipedia article
Text is available under the CC BY-SA 4.0 license; additional terms may apply.
Images, videos and audio are available under their respective licenses.