Everspark

The super-resolution microscopy buffer that offers multicolor, long-term and stable fluorescence imaging:
prepare your samples one day, and image them every day for weeks

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Overview

No more rushing your imaging. Prepare your slide, and image quietly whenever you want, even up to 10 weeks later.
Image with red, far red and green dyes (eg. AF488). Use different colors either at the same time or one after the other.

A technology designed by Karine Monier, Arnaud Favier and Christophe Place
Powered by the Eternity technology published in Scientific Reports

"We designed the Everspark buffer to facilitate sample preparation for dSTORM super-resolution imaging"

Karine Monier, Arnaud Favier and Christophe Place

John DOE • CEO of MyCompany

Citations

Nanoscale engagement and clusterization of Programmed death ligand 1 (PD-L1) in the membrane lipid rafts of Non-Small Cell Lung Cancer cells

Martina Ruglioni, Simone Civita, Tiziano Salvadori, Sofia Cristiani, Vittoria Carnicelli, Serena Barachini, Iacopo Petrini, Irene Nepita, Marco Castello, Alberto Diaspro, Paolo Bianchini, Barbara Storti, Ranieri Bizzarri, Stefano Fogli and Romano Danesi bioRxiv 2022.08.09.503318; doi: https://doi.org/10.1101/2022.08.09.503318

HIV-1 diverts actin debranching mechanisms for particle assembly and release in CD4 T lymphocytes
Rayane Dibsy, Erwan Bremaud, Johnson Mak, Cyril Favard, Delphine Muriaux
BioRxiv, December 16, 2022. doi. 10.1101/2022.12.15.520580

Fluorescent Polymer-AS1411-Aptamer Probe for dSTORM Super-Resolution Imaging of Endogenous Nucleolin
Fabre L, Rousset C, Monier K, Da Cruz-Boisson F, Bouvet P, Charreyre MT, Delair T, Fleury E, Favier A. Biomacromolecules. 2022 May 12. doi: 10.1021/acs.biomac.1c01706. PMID: 35549176

Comparative analysis of ChAdOx1 nCoV-19 and Ad26.COV2.S SARS-CoV-2 vector vaccines.
Michalik S, Siegerist F, Palankar R, Franzke K, Schindler M, Reder A, Seifert U, Cammann C, Wesche J, Steil L, Hentschker C, Gesell-Salazar M, Reisinger E, Beer M, Endlich N, Greinacher A, Völker U. Haematologica. 2022 Apr 1;107(4):947-957. doi: 10.3324/haematol.2021.280154. PMID: 35045692

Metabolic biorthogonal labeling and dSTORM imaging of peptidoglycan synthesis in Streptococcus pneumoniae
Jennyfer Trouve, Oleksandr Glushonkov and Cecile Morlot
Star Protocols, December 13, 2021. doi: 10.1016/j.xpro.2021.101006

Insights in ChAdOx1 nCoV-19 vaccine-induced immune thrombotic thrombocytopenia.
Greinacher A, Selleng K, Palankar R, Wesche J, Handtke S, Wolff M, Aurich K, Lalk M, Methling K, Völker U, Hentschker C, Michalik S, Steil L, Reder A, Schönborn L, Beer M, Franzke K, Büttner A, Fehse B, Stavrou EX, Rangaswamy C, Mailer RK, Englert H, Frye M, Thiele T, Kochanek S, Krutzke L, Siegerist F, Endlich N, Warkentin TE, Renné T.
Blood. 2021 Dec 2;138(22):2256-2268. doi: 10.1182/blood.2021013231. PMID: 34587242

Superresolution Microscopy of Drosophila Indirect Flight Muscle Sarcomeres.
Szikora S, Novák T, Gajdos T, Erdélyi M, Mihály J. Bio Protoc. 2020 Jun 20;10(12):e3654. doi: 10.21769/BioProtoc.3654. eCollection 2020 Jun 20. PMID: 33659324

Feedback from users

Everspark works with thick samples of 100µ (peripheral blinking).

Everspark does not work 100% efficiently with organs and tissues because there is most of the times a bit of remaining oxygen.

Feedback by Randy Stout

Everspark is a game changer: it is very, very useful for anyone doing dSTORM.

Randy Stout • New York Institute of Technology, 2023

Feedback diary

Images

Features

High blinking stability over time for repeated measurements

Once mounted, the sample is stable for up to 5 weeks and can be imaged multiple times

Multicolor (incl. green)

Compatible with green, red & far red dyes (JF646, JF549, AF647, CF647, DL550, CF568, DL650, CF680, SulfoCy5 and mEos2)

Ready to use

Can be used directly from the vial

Up to 6 months performance

Individualized packaging for optimal longevity

Techniques

PALM
dSTORM
HILO, TIRF pattern

Protocol

The protocol of use with a chambered coverslip
Sealing of all chambers with the lid

The protocol of use with a chambered coverslip
Sealing half of the chambers with a coverslip

The protocol of use with a glass-bottom dish

The protocol of use with a depression slide

A simplified protocol

Storage conditions:

Everspark has to be stored in the fridge at 4°C upon receiving the product. Do not freeze it.
Open the tube just before mounting to limit oxygenation
Store the slides at 4°C and in the dark before repeating dSTORM acquisition on the same slide.

Use method:

Rinse the coverslip with sterile water
Discard water in excess
Prepare sealant: 1 volume of sealant catalyst + 1 volume of sealant basis in a micro-dish – do not mix
Open the Everspark tube and pipette right away the desired volume onto a Wilco glass bottom dish
Flip the coverslips face towards the slide on the cavity filled with Everspark without making bubbles
Absorb excess of Everspark buffer on the sides with a kimwipe
Mix sealant
Seal quickly with the reconstituted sealant
Wait for the sealant to polymerize on the slide before moving the slide
Discard the open tube of Everspark buffer (cannot be used for another slide, once already opened)
Gently clean the coverslip with 70% ethanol
Perform regular dSTORM acquisition (best to wait a couple of hours)
Repeat dSTORM acquisition 24 hours up to 3 weeks on the same slide providing no air bubbles were present
Store the slides at 4°C and in the dark before repeating dSTORM acquisition on the same slide (up to 3 weeks)

Note 1: Blinking events have been validated with JF646, JF549, AF647, CF647, DL550, CF568, DL650, CF680, SulfoCy5 and mEos2

Note 2: Blinking is detected for several weeks without loss of events or photons per event

Note 3: Mounting can also be performed in various microdishes by adjusting the Everspark volume (Each vial contains 450 µl)

DOWNLOAD THE PROTOCOL

Recipe to prepare a good sealing

Technical information

Kit description:

10 vials with 450 µL of Everspark buffer at 100 mM MEA in Tris.
1 vial per experiment.

For use with: round glass-bottom dishes, standard or chambered glass coverslips.

Lifetime:

Up to 6 months (2 months after opening the pouch + 4 months storage in the pouch).

Resources:

Read the most Frequently Asked Questions
Everspark Safety Datasheet
Everspark Certificate of Analysis

Story

"We designed the Everspark buffer to facilitate sample preparation for dSTORM super-resolution imaging. Indeed, an oxygen-free buffer containing a thiol reducer is central to promote fluorophore blinking and thus single molecule detection, required for dSTORM imaging. With classical buffers, such as the Glox buffer, fluorophore blinking lifetime is limited to a few hours. With Everspark buffer, it is now possible to increase sample lifetime to several weeks. We demonstrated that a calibrated sample mounted once with the Everspark buffer can be submitted to several rounds of dSTORM imaging to acquire and reconstruct a structure reproducible in coverage and precision, over a period of two months."

Karine Monier - Christophe Place - Arnaud Favier

Odoo • Image and Text Esther Graudens
New projects at Idylle

"When I was working on long manipulations, I dreamed of being able to stop at certain crucial stages. Everspark is the ideal answer to the problem of many researchers who prepare their samples in a hurry simply because they need to image them right away. The Everspark buffer eliminates this tension: they take the time to image whenever they want, with complete peace of mind. They can also make acquisitions over several days. This will really change the way people prepare their experimentations, and that's exactly what I like about my job!
In terms of production, I am very proud of the way we have managed to set up a just-in-time production process in our labs in record time. And when researchers ask us to introduce variants, our production teams are able to build and produce very quickly their customized buffer to test it".

Everspark

The super-resolution microscopy buffer that offers multicolor, long-term and stable fluorescence imaging:
prepare your samples one day, and image them every day for weeks

Overview

Karine Monier, Arnaud Favier and Christophe Place

Citations