DOCSLIB.ORG

Death by Super-Resolution Imaging

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Death by Super-Resolution Imaging RESEARCH HIGHLIGHTS IMAGING Death by super-resolution imaging Researchers report that the illumination Sauer notes that just the 26-nm difference intensities used in super-resolution imag- between 488 nm and 514 nm can be the ing can irreversibly damage live cells. difference between life and death for cells Super-resolution imaging methods such being imaged. as direct stochastic optical reconstruction Sauer wants a take-home message from microscopy (dSTORM), photoactivated this work to be that “users should carefully localization microscopy (PALM) and stimu- dose the use of wavelengths shorter than 500 lated emission depletion (STED) microscopy nm for photoactivation or photoconversion, can provide highly detailed views of struc- even at very low intensities. The longer the tures within cells. However, these methods irradiation wavelength, the better the sur- require high doses of light, which can have vival chances of cells.” This may be particu- unwanted effects, known as photodamage larly relevant to PALM experiments, which and phototoxicity, on biological samples. use near-UV light to photoactivate fluoro- Markus Sauer at the University of Würzburg, phores over multiple rounds of imaging. along with two graduate students in his labo- The authors made other important ratory, Sina Wäldchen and Julian Lehmann, Image of a `frozen’ mammalian cell where the observations in the course of their experi- sought to carefully characterize such effects top portion was treated by light, destroying the ments. For example, they found that after in live cells. actin network. Reprinted from Wäldchen et al. irradiation, some cells underwent apopto- The research was motivated by con- (2015). sis before death, whereas others bypassed flicting reports regarding phototoxicity in dead and living cells. Some of their results that step. These latter, so-called frozen cells Nature America, Inc. All rights reserved. America, Inc. Nature the field of super-resolution fluorescence were unexpected. In particular, Sauer says were immediately killed and fixed in place 5 microscopy. “Some people claimed that he was surprised that “dyes contribute to by the light treatment. Further analysis © 201 very low light doses, even sunlight, can phototoxicity, but it is mainly the direct showed that, despite looking unchanged, seriously kill cells,” says Sauer, whereas interaction of the light with other intrinsic the membrane and cytoskeleton of frozen “other researchers use orders-of-magnitude cellular molecules which is responsible for cells were destroyed by the light treatment. higher light doses in living cells and even in damage.” They found that transfection can These results suggest that examination for npg living mouse brain and claim that they do increase cells’ photosensitivity relative to phototoxic effects should not be limited to not observe any signs of photodamage.” The that of untreated controls, as can the intro- phenotypic assessment immediately after team was interested in the effects of fluoro- duction of a fluorophore. They also discov- imaging. phores, which are known to generate harm- ered that cell lines differ in their photosen- These results have direct implications for ful reactive oxygen species, on phototoxic- sitivity, with the commonly used HeLa cell the design and implementation of live-cell ity. They were also interested in whether line being robust relative to the other tested super-resolution microscopy experiments, direct interaction of light with cells can lines. and they can also be used to guide the cause damage, which often goes unmen- The team also tested whether wavelength design of improved tools. Sauer believes that tioned. “We thought somebody should has a role in photodamage. They compared improved far-red dyes for super-resolution shed light on these obvious discrepancies several wavelengths of light at a range of microscopy and better tools for site-specific and remove the uncertainty,” recalls Sauer. light intensities and found striking results. labeling of proteins in live cells will be nec- The team systematically studied the They discovered that near-UV light is essary for live-cell imaging at the nanoscale effects of different light treatments on cells extremely phototoxic, even at low intensi- in the future. grown in culture. In one experiment, they ties, whereas far-red light causes minimal Rita Strack treated cells grown under different condi- phototoxicity, even at high intensities. A RESEARCH PAPERS tions with a range of light intensities. A day surprising result of this analysis was the Wäldchen, S. et al. Light-induced cell damage in later, they examined the effects of the light finding that a small change in wavelength live-cell super-resolution microscopy. Sci. Rep. 5, exposure by quantifying the numbers of can have a large effect on phototoxicity. 15348 (2015). NATURE METHODS | VOL.12 NO.12 | DECEMBER 2015 | 1111.
Load more

Recommended publications
  • STED Fluorescence Microscopy: a Method of Resolution Enhancement Submitted by David Biss and Jason Neiser
    STED Fluorescence Microscopy: A method of resolution enhancement Submitted by David Biss and Jason Neiser Introduction relaxed vibrational level of the ground electronic state. The microscope excitation light generates If geometrical aberrations are minimized in an a transition in the fluorophore from level L0 to optical system, the smallest spot size attainable is L1, a high vibrational level of the first excited the diffraction limited spot size. Confocal state. From here, the molecule undergoes a fast microscopy was the first method to extend vibrational decay from L1 to L2, and eventually resolution beyond the Abbe resolution limit and fluoresces to L3 by spontaneous emission. it added axial resolution to the system. This form of microscopy images a portion of the sample being investigated onto a confocal pinhole at the detection plane of the system. Since the invention of confocal microscopy other methods have been devised to reach beyond the standard diffraction limit. Some of these methods are 4π microscopy, two photon microscopy, near-field microscopy, and more recently, STimulated Emission Depletion (STED) fluorescence microscopy. [1, 2, 3] STED fluorescence microscopy takes standard fluorescence microscopy and introduces a technique to reduce the emitted spot size. STED microscopy uses stimulated emission to deplete fluorophores before they fluoresce. If this depletion occurs at the edges of the excited sample area the spot size (and volume) of the fluorescence can be reduced beyond the Fig. 1 Energy level diagram of a dye molecule. A short diffraction limit. wavelength pulse excites the molecule and it may be relaxed by either fluorescing or by stimulated emission via the STED Theory pulse.
    [Show full text]
  • Super-Resolution STED Microscopy and Its Application in Neuroscience
    Super-resolution STED microscopy and its application in neuroscience Katrin Willig 18th German-American Frontiers of Engineering Symposium Hamburg 20-23 March 2019 Nanoscale Microscopy and Molecular Physiology of the Brain MPI of Cluster of Excellence 171, Experimental DFG Research Center 103 Medicine 1 Resolution in far-field light microscopy diffraction limit: minimum resolvable distance l l a dmin d 2 nsina numerical aperture (NA) n: refractive index structure image “similar objects closer than about half the wavelength should not be distinguishable in a light microscope” Ernst Abbe 1873 2 Standard (confocal) vs. Superresolution (STED) 3 Confocal (fluorescence) microscopy x 200 nm Abbe‘s equation y λ d 2n sin a Excitation d Fluorescence Detection Dichroic Scanning Mirror 1 Device S1 Excitation Fluorescence S0 4 STED (STimulated Emission Depletion) microscopy x 200 nm Phase Plate y 0 2p STED Excitation d Fluorescence Detection Dichroic Dichroic Scanning Mirror 1 Mirror 2 Device Nobel Prize in Chemistry 2014 to Betzig, Hell & Moerner S1 "for the development of super-resolved fluorescence microscopy." . Excitation Stimul Emission STED beam: keeps molecules non-fluorescent Fluorescence S0 5 Diffraction limited resolution PSF 1 220 nm 0 -200 0 200 1.0 0.5 y 500 nm x Fluorescence 0.0 min max 0.0 0.5 1.0 1.5 I [GW/cm²] STED 6 Subdiffraction resolution Depletion distribution 1 132 nm 0 -200 0 200 1.0 0.5 y 500 nm Fluorescence x 0.0 min max 0.0 0.5 1.0 1.5 I [GW/cm²] STED 7 Subdiffraction resolution Depletion distribution 1 84 nm 0 -200 0
    [Show full text]
  • Super-Resolution STED Microscopy and Its Application in Neuroscience
    Super-resolution STED microscopy and its application in neuroscience Katrin Willig Fluorescence microscopy is a widely used technique, especially in biology. It combines staining specificity with relatively simple imaging capabilities. Especially if applied in the far-field it is almost non-invasive and therefore ideal to study protein assemblies or dynamics in living cells, tissues or animals. Until recently it was widely accepted that far-field optical microscopes cannot visualize details closer than about half the wavelength of light. Therefore, electron microscopy is needed to reveal structural details at exceptionally high resolution, down to the molecular level. EM, though, lacks the ability to image dynamic changes of the same morphological structures; temporal information is only gathered via comparative studies prepared at different time-points. However, to understand how and why the sub-structure of cells changes, and what functional consequence this change induces, we need to visualize cells or even whole, intact living organism over extended periods of time, i.e. in longitudinal studies. However, given the poor optical resolution small sub- cellular structures have still not been accurately assessed by standard light microscopy techniques available due to the diffraction limited resolution of far-field light microscopy being ~200-300 nm. With the 2014 Nobel Prize in Chemistry ‘for the development of superresolved fluorescence microscopy’ for Betzig, Hell and Moerner, a novel family of light microscopy techniques became widely recognized, which surpass the limited resolution of light microscopy: The general terms ‘superresolution’ microscopy or ‘nanoscopy’ encompass several techniques, which can be divided in coordinate-targeted approaches (e.g. stimulated emission depletion microscopy (STED), reversible saturable optical fluorescent transition microscopy (RESOLFT)), and coordinate-stochastic approaches (e.g.
    [Show full text]
  • Correlating STED and Synchrotron XRF Nano-Imaging Unveils
    TOOLS AND RESOURCES Correlating STED and synchrotron XRF nano-imaging unveils cosegregation of metals and cytoskeleton proteins in dendrites Florelle Domart1,2,3, Peter Cloetens4, Ste´ phane Roudeau1,2, Asuncion Carmona1,2, Emeline Verdier3, Daniel Choquet3,5†, Richard Ortega1,2†* 1Chemical Imaging and Speciation, CENBG, Univ. Bordeaux, Gradignan, France; 2CNRS, IN2P3, CENBG, UMR 5797, Gradignan, France; 3Univ. Bordeaux, CNRS, Interdisciplinary Institute for Neuroscience, IINS, UMR 5297, Bordeaux, France; 4ESRF, the European Synchrotron, Grenoble, France; 5Univ. Bordeaux, CNRS, INSERM, Bordeaux Imaging Center, BIC, UMS, Bordeaux, France Abstract Zinc and copper are involved in neuronal differentiation and synaptic plasticity but the molecular mechanisms behind these processes are still elusive due in part to the difficulty of imaging trace metals together with proteins at the synaptic level. We correlate stimulated- emission-depletion microscopy of proteins and synchrotron X-ray fluorescence imaging of trace metals, both performed with 40 nm spatial resolution, on primary rat hippocampal neurons. We reveal the co-localization at the nanoscale of zinc and tubulin in dendrites with a molecular ratio of about one zinc atom per tubulin-ab dimer. We observe the co-segregation of copper and F-actin within the nano-architecture of dendritic protrusions. In addition, zinc chelation causes a decrease in the expression of cytoskeleton proteins in dendrites and spines. Overall, these results indicate *For correspondence: new functions for zinc and copper in the modulation of the cytoskeleton morphology in dendrites, a [email protected] mechanism associated to neuronal plasticity and memory formation. †These authors contributed equally to this work Competing interests: The Introduction authors declare that no The neurobiology of copper and zinc is a matter of intense investigation since they have been competing interests exist.
    [Show full text]
  • Super-Resolution Deep Imaging with Hollow Bessel Beam STED Microscopy
    Laser & Photonics Review DOI: 10.1002/lpor.201500151 Super-resolution deep imaging with hollow Bessel beam STED microscopy Wentao Yu1, Ziheng Ji1, Dashan Dong1, Xusan Yang2, Yunfeng Xiao1, 3, Qihuang Gong1, 3, Peng Xi2*, and Kebin Shi1, 3* 1State Key Laboratory for Mesoscopic Physics, Collaborative Innovation Center of Quantum Matter, School of Physics, Peking University, Beijing 100871, China 2Department of Biomedical Engineering, College of Engineering, Peking University, Beijing 100871, China 3Collaborative Innovation Center of Extreme Optics, Shanxi University, Taiyuan, Shanxi 030006, China *E-mail: [email protected] [email protected] The achievable resolution in STED systems explicitly relies Abstract—Stimulated emission depletion (STED) microscopy on the fluorophore depletion efficiency, i.e. the efficient use of has become a powerful imaging and localized excitation method photons delivered by the designated depletion beam. As a result, breaking the diffraction barrier for improved lateral spatial it is challenging to maintain consistent resolution as the foci resolution in cellular imaging, lithography, etc. Due to specimen- moves from the surface to deep inside of the specimens due to induced aberrations and scattering distortion, it is a great challenge for STED to maintain consistent lateral resolution the spherical aberration, scattering distortion and loss.[12, 15, deeply inside the specimens. Here we report on a deep imaging 20] Experimental and theoretical efforts have been reported to STED microscopy by using Gaussian
    [Show full text]
  • STED Nanoscopy of Actin Dynamics in Synapses Deep Inside Living Brain Slices
    Biophysical Journal Volume 101 September 2011 1277–1284 1277 STED Nanoscopy of Actin Dynamics in Synapses Deep Inside Living Brain Slices Nicolai T. Urban,† Katrin I. Willig,† Stefan W. Hell,†* and U. Valentin Na¨gerl‡§* †Max Planck Institute for Biophysical Chemistry, Go¨ttingen, Germany; ‡Interdisciplinary Institute for Neuroscience, Universite´ de Bordeaux, Bordeaux, France; and §Interdisciplinary Institute for Neuroscience, Centre National de la Recherche Scientifique (CNRS), UMR 5297, Bordeaux, France ABSTRACT It is difficult to investigate the mechanisms that mediate long-term changes in synapse function because synapses are small and deeply embedded inside brain tissue. Although recent fluorescence nanoscopy techniques afford improved resolution, they have so far been restricted to dissociated cells or tissue surfaces. However, to study synapses under realistic conditions, one must image several cell layers deep inside more-intact, three-dimensional preparations that exhibit strong light scattering, such as brain slices or brains in vivo. Using aberration-reducing optics, we demonstrate that it is possible to achieve stimulated emission depletion superresolution imaging deep inside scattering biological tissue. To illustrate the power of this novel (to our knowledge) approach, we resolved distinct distributions of actin inside dendrites and spines with a resolution of 60–80 nm in living organotypic brain slices at depths up to 120 mm. In addition, time-lapse stimulated emission depletion imaging revealed changes in actin-based structures inside spines and spine necks, and showed that these dynamics can be modulated by neuronal activity. Our approach greatly facilitates investigations of actin dynamics at the nanoscale within func- tionally intact brain tissue. INTRODUCTION Understanding the structural and molecular mechanisms resolution usually deteriorates quickly with imaging depth that mediate synaptic plasticity is one of the central chal- because of scattering, absorption, and aberrations induced lenges for neurobiological research.
    [Show full text]
  • Lasers for Microscopy: Major Trends Marco Arrigoni, Nigel Gallaher, Darryl Mccoy, Volker Pfeufer and Matthias Schulze, Coherent Inc
    Lasers for Microscopy: Major Trends Marco Arrigoni, Nigel Gallaher, Darryl McCoy, Volker Pfeufer and Matthias Schulze, Coherent Inc. Laser development for the microscopy market continues to be driven by key trends in applications, which currently include superresolution techniques, multiphoton applications in optogenetics and other areas of neuroscience, and even a shift in multiphoton imaging toward preclinical and clinical usage. In spite of its long history, optical microscopy – particularly laser-based microscopy – is a very dynamic field. New techniques continue to be developed, while existing techniques are being applied to new applications. For biologists, drug developers, clinical lab professionals and other scientists to fully exploit these new techniques and applications, parallel developments in laser technology are often required. This article will take a broad overview of three important trends in laser-based microscopy and examine how laser manufacturers are responding with products optimized to match the needs of these applications. Superresolution microscopy – optical switching of fluorescent labels To better understand the details of processes such as signaling and the control of gene expression, biologists need to correlate molecular-level events with macroscopic structures and dynamics. This has fueled rapid growth in superresolution microscopy techniques, often referred to as nanoscopy, that go beyond the classical spatial resolution limit set by diffraction. This limit is about half the wavelength of light (i.e., for visible light, approximately 200-250 nm) in the X-Y plane and, in the case of confocal microscopy, about 500 nm in the Z direction. Most superresolution techniques use CW lasers, often with fast internal or external (on/off) modulation.
    [Show full text]
  • Development of a Two-Photon Excitation STED Microscope and Its Application to Neuroscience Philipp Bethge
    Development of a two-photon excitation STED microscope and its application to neuroscience Philipp Bethge To cite this version: Philipp Bethge. Development of a two-photon excitation STED microscope and its application to neuroscience. Neurons and Cognition [q-bio.NC]. Université de Bordeaux, 2014. English. NNT : 2014BORD0018. tel-01138959 HAL Id: tel-01138959 https://tel.archives-ouvertes.fr/tel-01138959 Submitted on 3 Apr 2015 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. THÈSE PRÉSENTÉE POUR OBTENIR LE GRADE DE DOCTEUR DE L’UNIVERSITÉ DE BORDEAUX ÉCOLE DOCTORALE Sciences de la Vie et de la Santé Philipp BETHGE Développement d'un microscope STED à excitation deux photons et son application aux neurosciences Sous la direction de : Prof. U. Valentin NÄGERL Soutenue le 27.03.2014 Membres du jury : MULLE, Christophe, Directeur de recherche (CE), CNRS UMR 5297 Bordeaux Président HOFER, Sonja, Professor, Universität Basel Rapporteur EMILIANI, Valentina, Directeur de recherche (DR2), CNRS UMR 8250 Paris Rapporteur MARSICANO, Giovanni, Directeur de recherche (DR2), INSERM U862 Bordeaux invité Développement d'un microscope STED à excitation deux photons et son application aux neurosciences L’avènement de la microscopie STED (Stimulated Emission Depletion) a bouleversé le domaine des neurosciences du au fait que beaucoup de structures neuronale, tels que les épines dendritiques, les axones ou les processus astrocytaires, ne peuvent pas être correctement résolu en microscopie photonique classique.
    [Show full text]
  • Two Approaches for a Simpler STED Microscope Using a Dual-Color Laser Or a Single Wavelength Ancuţa Teodora Şcheul
    Two approaches for a simpler STED microscope using a dual-color laser or a single wavelength Ancuţa Teodora Şcheul To cite this version: Ancuţa Teodora Şcheul. Two approaches for a simpler STED microscope using a dual-color laser or a single wavelength. Other [cond-mat.other]. Université de Grenoble, 2013. English. NNT : 2013GRENY040. tel-01558306 HAL Id: tel-01558306 https://tel.archives-ouvertes.fr/tel-01558306 Submitted on 7 Jul 2017 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. THESE` Pour obtenir le grade de DOCTEUR DE L’UNIVERSITE´ DE GRENOBLE Specialit´ e´ : Optique, Nanophysique Arretˆ e´ ministeriel´ : 7 aoutˆ 2006 Present´ ee´ par S¸cheul Ancut¸aTeodora These` dirigee´ par Jean-Claude Vial et codirigee´ par Irene` Wang prepar´ ee´ au sein du Laboratoire interdisciplinaire de Physique et de l’Ecole Doctorale de Physique, Grenoble Two approaches for a simpler STED microscope using a dual- color laser or a single wavelength These` soutenue publiquement le 22 Novembre 2013, devant le jury compose´ de : Dominique Bourgeois Directeur de Recherche
    [Show full text]
  • Label-Free Multiphoton Microscopy: Much More Than Fancy Images
    International Journal of Molecular Sciences Review Label-Free Multiphoton Microscopy: Much More than Fancy Images Giulia Borile 1,2,*,†, Deborah Sandrin 2,3,†, Andrea Filippi 2, Kurt I. Anderson 4 and Filippo Romanato 1,2,3 1 Laboratory of Optics and Bioimaging, Institute of Pediatric Research Città della Speranza, 35127 Padua, Italy; fi[email protected] 2 Department of Physics and Astronomy “G. Galilei”, University of Padua, 35131 Padua, Italy; [email protected] (D.S.); andrea.fi[email protected] (A.F.) 3 L.I.F.E.L.A.B. Program, Consorzio per la Ricerca Sanitaria (CORIS), Veneto Region, 35128 Padua, Italy 4 Crick Advanced Light Microscopy Facility (CALM), The Francis Crick Institute, London NW1 1AT, UK; [email protected] * Correspondence: [email protected] † These authors contributed equally. Abstract: Multiphoton microscopy has recently passed the milestone of its first 30 years of activity in biomedical research. The growing interest around this approach has led to a variety of applications from basic research to clinical practice. Moreover, this technique offers the advantage of label-free multiphoton imaging to analyze samples without staining processes and the need for a dedicated system. Here, we review the state of the art of label-free techniques; then, we focus on two-photon autofluorescence as well as second and third harmonic generation, describing physical and technical characteristics. We summarize some successful applications to a plethora of biomedical research fields and samples, underlying the versatility of this technique. A paragraph is dedicated to an overview of sample preparation, which is a crucial step in every microscopy experiment.
    [Show full text]
  • Super-Resolution Microscopy Demystified
    REVIEW ARTICLE | FOCUS https://doi.org/10.1038/s41556-018-0251-8REVIEW ARTICLE | FOCUS https://doi.org/10.1038/s41556-018-0251-8 Super-resolution microscopy demystified Lothar Schermelleh" "1*, Alexia Ferrand2, Thomas Huser" "3, Christian Eggeling4,5, Markus Sauer6, Oliver Biehlmaier2 and Gregor P. C. Drummen" "7,8* Super-resolution microscopy (SRM) bypasses the diffraction limit, a physical barrier that restricts the optical resolution to roughly 250 nm and was previously thought to be impenetrable. SRM techniques allow the visualization of subcellular orga- nization with unprecedented detail, but also confront biologists with the challenge of selecting the best-suited approach for their particular research question. Here, we provide guidance on how to use SRM techniques advantageously for investigating cellular structures and dynamics to promote new discoveries. n their pursuit of understanding cellular function, biologists seek (Fig. 1d–h; Box 1). One group of SRM techniques falls under super- to observe the processes that allow cells to maintain homeostasis resolution structured illumination microscopy (SR-SIM, reviewed and react dynamically to internal and external cues—on both a in7,8) and comprise traditional interference-based linear 2D and 3D I 9–11 molecular scale and inside structurally intact, ideally living speci- SIM (Fig. 1d), as well as more recently introduced point scanning mens. A pathway towards this goal was opened with the advent, SIM approaches12–15 (Fig. 1e). Even though they exceed the ‘classical’ and widespread application, of super-resolution microscopy (SRM) Abbe limit of resolution, SR-SIM approaches are still fundamentally techniques that manage to surpass the ‘classical’ diffraction limit bound by the laws of diffraction, at best doubling the spatial resolu- of optical resolution of about half the wavelength of the emitted tion in lateral (x,y) and axial (z) directions, equivalent to an ~8-fold light1.
    [Show full text]
  • What Can Stimulated Emission Do for Bioimaging?
    Ann. N.Y. Acad. Sci. ISSN 0077-8923 ANNALS OF THE NEW YORK ACADEMY OF SCIENCES Issue: Blavatnik Awards for Young Scientists What can stimulated emission do for bioimaging? Lu Wei and Wei Min Department of Chemistry, Columbia University, New York, New York Address for correspondence: Wei Min, Department of Chemistry, Columbia University, 3000 Broadway, New York, NY 10027. [email protected] Advances in bioimaging have revolutionized our ability to study life phenomena at a microscopic scale. In particular, the stimulated emission process, a universal mechanism that competes with spontaneous emission, has emerged as a powerful driving force for advancing light microscopy. The present review summarizes and compares three related techniques that each measure a different physical quantity involved in the stimulated emission process in order to tackle various challenges in light microscopy. Stimulated emission depletion microscopy, which detects the residual fluorescence after quenching, can break the diffraction-limited resolution barrier in fluorescence microscopy. Stimulated emission microscopy is capable of imaging nonfluorescent but absorbing chromophores by detecting the intensity gain of the stimulated emission beam. Very recently, stimulated emission reduced fluorescence microscopy has been proposed, in which the reduced fluorescence due to focal stimulation is measured to extend the fundamental imaging-depth limit of two-photon microscopy. Thus, through ingenious spectroscopy design in distinct microscopy contexts,stimulatedemissionhasopenedupseveralnewterritoriesforbioimaging,allowingexaminationofbiological structures that are ever smaller, darker, and deeper. Keywords: stimulated emission; superresolution; nonfluorescent chromophore; pump-probe microscopy; deep tissue imaging; imaging-depth limit quency, polarization, and direction as the incident one. Figure 1 illustrates the competition between Introduction stimulated emission and spontaneous emission (i.e., Light microscopy, since its invention several cen- fluorescence) processes.
    [Show full text]