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Carmichael’s Concise Review


Coming Events 2017


International Frontiers of Electron Microscopy in Materials Science September 10–15, 2017


Johannesburg, South Africa http://femms2017.org CIASEM 2017


September 25–29, 2017


Varadero Beach, Cuba www.ciasem2017.sld.cu


MS&T17: Materials Science & Technology October 8–12, 2017


Pittsburgh, PA www.matscitech.org


American Vacuum Society October 29–November 3, 2017


Tampa, FL www.avs.org


Testing and Failure Analysis November 5–9, 2017 Pasadena, CA


www.asminternational.org/web/istfa-2017/home


Neuroscience 2017 November 11–15, 2017


Washington, DC www.sfn.org


2017 MRS Fall Meeting November 26–December 1, 2017 Boston, MA


www.mrs.org/fall2017


American Society for Cell Biology December 2–6, 2017 Philadelphia, PA


http://ascb.org/future-ascb-annual-meetings 2018


Microscopy & Microanalysis 2018 August 5–9, 2018


Baltimore, MD www.microscopy.org 2019


Microscopy & Microanalysis 2019 August 4–8, 2019


Portland, OR www.microscopy.org 2020


Microscopy & Microanalysis 2020 August 2–6, 2020


Milwaukee, WI www.microscopy.org 2021


Microscopy & Microanalysis 2021 August 1–5, 2021


Pittsburgh, PA www.microscopy.org 2022


Microscopy & Microanalysis 2022 July 31–August 4, 2022


Portland, OR www.microscopy.org


More Meetings and Courses Check the complete calendar near the back of this magazine.


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Figure 1 : Super-multiplex optical imaging for investigation of proteome turnover dynamics in hippocampal neuronal cultures. (a) Pulse-chase (HPG-AHA) experimental design. MG132: proteasome inhibitor. (b–e) Cytosolic aggregations (indicated by arrowheads) are mostly from proteins synthesized in the chase period (b-c) and appear mostly in astrocytes (d) but not in neurons (e). HPG: 1-homopropargylglyceine. AHA: 1-azidohomoalanine. GFAP: glial fi brillary acidic protein. MBP: myelin basic protein. Scale bars = 10 μ m.


doi: 10.1017/S1551929517000852 2017 September


Developing a Bigger Palette to Elucidate Intricate Biologic Interactions Stephen W. Carmichael


Mayo Clinic , Rochester , MN 55905 carmichael.stephen@mayo.edu


Fluorescence microscopy has proven to be a valuable method to localize molecules of biologic interest. However, the nature of broad fl uorescence spectra limits the number of resolvable colors to about 5 (or with complicated instrumentation and analysis up to 9). Stimulated Raman scattering (SRS) microscopy avoids this limitation and provides peaks about 100 times narrower than fl uorescence by relying on the interaction between the vibrational motion from the chemical bonds in the molecule of interest and laser beams. Lu Wei, Wei Min, and their colleagues have developed a methodology and an assortment of dyes to create a palette of 24 resolvable colors, with the potential for further expansion [ 1 ].


In SRS microscopy two laser beams (referred to as Stokes photons and pump photons) with diff erent energies are collinearly focused onto cell samples. When the energy diff erences between the two beams matches the vibrational frequency of the targeted chemical bonds, the chemical bonds are effi ciently excited to the vibrational excited state. A method to detect the intensity loss of the pump beam was used to spatially and temporally resolve molecules with selected chemical bonds. All previous SRS imaging is in the non-resonance region where the laser wavelengths are far away from molecular absorptions and thus have inadequate sensitivity for general applica- bility. Wei et al. exploited and determined a special region by carefully adjusting the two laser beams to be close to the electronic absorption of the molecules that could obtain signal enhancement with optimal contrast between vibrational signal and electronic background. T ey termed this electronic pre-resonance SRS (epr-SRS). Combining epr-SRS with selected dyes provided an improvement in sensitivity of about 1,000 over other non-resonance SRS imaging, which was comparable to confocal fl uorescence microscopy, while retaining distinct vibrational contrast. Wei et al. created a palette of triple-bond-conjugated (with, for example, alkynes) near-infrared dyes. Each displays a single peak in the Raman spectral window where background noise is non-existent (cell-silent). Combining this palette with available fl uorescent probes provided 24 resolvable colors. T ey called this super-multiplex vibrational imaging.


Proof-of-principle experiments on both fi xed and living cells demonstrated several successful applications. For example, imaging of proteome turnover dynamics


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