creating the navigation map for clem and preparation of the ultrathin sections for electron microscopy

Ny protokol til samtidig fluorescens og ultrastrukturbevarelse

Fluorescence microscopy (FM) can be used to obtain the localization and distribution of the target protein. However, the context that surrounds the target protein is lost, which is crucial for investigating the target protein thoroughly. Electron microscopy (EM) has the highest imaging resolution, providing several subcellular details. Nevertheless, EM lacks target labeling. By accurately merging the fluorescence image taken by FM with the gray image acquired by EM, correlative light and electron microscopy (CLEM) can combine the information obtained by these two imaging modes1,2,3,4.
CLEM is a trade-off between fluorescence and the ultrastructure1. Because of the limitations of current fluorescent proteins and the traditional EM sample preparation procedures, especially the use of osmic acid (OsO4) and hydrophobic resins such as Epon, the ultrastructure always compromises fluorescence5. OsO4 is an indispensable reagent in EM sample preparation, which is used to improve the contrast of EM images. Compared with other embedding resins, Epon is superior in ultrastructure preservation and sectioning properties5. However, no fluorescent proteins can retain the fluorescence signal after the treatment of OsO4 and Epon embedding6. To overcome the limitations of fluorescent proteins, pre embedding CLEM was developed, in which FM imaging is done before EM sample preparation6. However, the drawback of pre embedding CLEM is the imprecise registration between the FM and the EM images5.
On the contrary, post embedding CLEM method performs the FM imaging after the EM sample preparation, the registration accuracy of which can reach 6-7 nm5,6. To retain the fluorescence of fluorescent proteins, very low concentrations of OsO4 (0.001%)3 or the high-pressure frozen (HPF) and freeze substitution (FS) EM preparation methods4,7 have been used at the expense of compromised ultrastructure or the contrast of the EM image. The development of mEos4b greatly promotes the progress of post embedding CLEM, although glycidyl methacrylate is used as the embedding resin5. With the development of mEosEM, which can survive the OsO4 staining and Epon embedding, Epon post embedding super-resolution CLEM was achieved for the first time, maintaining the fluorescence and ultrastructure simultaneously6. After mEosEM, several fluorescent proteins that can survive the OsO4 staining and the Epon embedding were developed8,9,10,11. This greatly promotes the development of CLEM.
There are three key aspects to Epon post-embedding CLEM. The first is the fluorescent protein, which should maintain the fluorescent signal after EM sample preparation. According to our experience, mScarlet is superior to other reported fluorescent proteins. The second is how to find the same cell imaged by FM imaging in EM imaging. To solve this problem, we improve the procedure for this step so that one can readily find the targeted cell. The last is the method to align the FM image with the EM image. Here, we detail the registration between the FM and the EM images. In this protocol, we express mScarlet in VGLUT2 neurons and demonstrate that mScarlet can target secondary lysosomes using Epon post-embedding CLEM. We provide step-by-step details for Epon post-embedding CLEM, without compromising the fluorescence and the ultrastructure.

Forfatter Spotlight: Fremskridt inden for korrelativ lys- og elektronmikroskopi med fluorescerende proteinkonservering

Epon Post indlejring korrelativt lys og elektronmikroskopi

Correlative light and electron microscopy is a completely stable microscopy technique, which can combine the localization information provided by fluorescent microscopy and is the ultra structure by electron microscopy. However, the mentality of this technique is to preserve the fluorescence of after you have a simple preparation. Using this fluorescent protein, we can achieve correlative light and the electron microscope which can maintain fluorescence and ultra structure simultaneously.

Oprettelse af navigationskort til CLEM og forberedelse af de ultratynde sektioner til elektronmikroskopi 

Til at begynde med skal du tage en ultratynd sektion af musehjernevæv, der udtrykker mScarlet. Tilsæt 10 mikroliter monteringsbuffer på den ultratynde sektion på dækglasset, og læg et nyt rent dækglas på monteringsbufferen. Sæt de to dækglas med den ultratynde sektion i et billedkammer.Find sektionen ved hjælp af annulusmarkøren i den lyse feltbilleddannelsestilstand i et inverteret fluorescensmikroskop, og flyt til et hjørne af sektionen. Tænd for det hvide lys, og tag det lyse feltbillede. Sluk derefter det hvide lys.Aktivér laseren, og tag fluorescerende billeder af det samme synsfelt. Deaktiver laseren, og flyt til det tilstødende synsfelt med en 10%overlapning. Tag både det lyse felt og det fluorescerende billede af det andet synsfelt, og optag billedstien for at sy synsfeltet sammen.Brug nu navigationskortet til at målrette celler af interesse. Aktivér laseren og tag 300 rammer fluorescerende billeder af cellen af interesse. Deaktiver derefter laseren.Derefter skal du aktivere det hvide lys og tage ca. 100 sekventielle lyse feltbilleder. For at forberede sektionerne til elektronmikroskopi skal du fylde en glasburk med dobbeltdestilleret vand. Fjern dækbrillerne, der holder de ultratynde sektioner fra billedkammeret, og adskil dem med pincet.Klem dækglasset med sektionen. Vask monteringsbufferen af med dobbeltdestilleret vand, og lad den tørre. Brug et enkeltsidet blad til at score en hash omkring den ultratynde sektion.Drop 10 mikroliter 12% flussyre i hvert hjørne af scoren. Placer langsomt dækglasset i vandet. Flyd derefter Pioloform-filmen og den ultratynde sektion på vandoverfladen.Placer et ubelagt slotgitter på sektionen for at fange det i gittermidten. Dæk et glasglas med paraform. Tag gitteret op med Pioloform-filmen, og lad den lufttørre ved stuetemperatur.

Research

JoVE Journal

Biology

Creating the Navigation Map for CLEM and Preparation of the Ultrathin Sections for Electron Microscopy

Epon Post Embedding Correlative Light and Electron Microscopy

Correlative light and electron microscopy (CLEM) is a comprehensive microscopy that combines the localization information provided by fluorescence ...

Ultrathin Sectioning for Epon Post-Embedding Correlative Light and Electron Microscopy (CLEM)

Ultratynd sektionering til epon post-embedding korrelativt lys og elektronmikroskopi (CLEM)

Image Analysis of Brightfield and Fluorescence Images of the Mouse Brain Sections for Epon Post-Embedding Correlative Light and Electron Microscopy

Billedanalyse af Brightfield og fluorescens Billeder af musehjernesektionerne til epon post-indlejring korrelativt lys og elektronmikroskopi

Registration of the Fluorescence Microscopy Image with the Electron Microscopy Image for Correlative Light and Electron Microscopy

Registrering af fluorescensmikroskopibilledet med elektronmikroskopibilledet for korrelativt lys og elektronmikroskopi