Name: Gold Nanoparticle Synthesis for Mammalian Cells Using Sapphire Discs
Uploaded: 2023-04-28T12:20:00
Description: Watch this Scientific Journal Video about Gold Nanoparticle Synthesis for Mammalian Cells Using Sapphire Discs at JoVE.com

The video outlines a detailed protocol for preparing sapphire discs with HeLa cells for imaging and analysis of protein distribution. It involves several steps, including washing, incubation with reducing and gold precursor solutions, polymerization in epoxy resin, and freeze-thaw techniques, leading to the visualization of proteins in cellular structures.

gold nanoparticle synthesis for mammalian cells using sapphire discs

Direkt syntes av guldnanopartiklar för analys av proteinlokalisering

In the postgenomic era, the development of green fluorescent protein (GFP) as a single-molecule reporter for light microscopy has revolutionized the field of modern life science research1,2. For decades, electron microscopy (EM) has been a powerful tool for intuitively observing the cellular ultrastructure with nanoscale resolution3; however, the precise identification and localization of protein molecules remain challenging.
The most commonly used EM labeling technique is the immunoelectron microscopy (IEM) labeling technique, which is based on the antigen-antibody reaction. However, although many techniques have been developed in the field of IEM labeling, including pre-embedding IEM and post-embedding IEM (on resin sections or hydrated cryosections), it still suffers from low labeling efficiency (&#60;10%)4,5, which is related to the sample preparation and antibody quality. To overcome these limitations, developing genetically encoded tags has great application potential.
Two main types of EM tags have been thoroughly explored in recent years. One type is the DAB staining method, which utilizes tags such as APEX2 to oxidize 3,3&#39;-diaminobenzidine (DAB) to osmiophilic polymers for EM visualization6,7,8,9,10,11,12. It enables the labeling of high-abundance proteins in subcellular regions but is not suitable for single-molecule counting. The other type utilizes metal-binding proteins, such as ferritin13 and metallothionein (MT)14,15,16,17,18,19,20,21,22,23,24,25,26, to generate electron-dense metal deposits in situ for EM visualization. Only the latter has real potential for single-molecule visualization and counting. The molecular size of ferritin is too large (~450 kD) for it to be used as a promising tag, whereas the small size (~5 kD) of MT and its ability to bind various ions through its 20 cysteines have attracted great attention. Several labs have tried to label purified MT-fusion proteins or MT-expressing cells by incubating directly with Au+. These attempts have initially proved that MT tags can bind gold ions to form high-contrast signals, but none have really achieved the effective identification of individual proteins in cells, and they are not widely applicable14,15,16,17,18,19,20,21,22,23.
The ANSM-based AuNP synthesis technique, which involves synthesizing 2-6 nm-sized AuNPs directly on cysteine-rich tags (e.g., MT, the MT variants MTn and MT&#945;, AFP) as electron-dense labels for EM visualization, is the first reliable and applicable approach for protein labeling and single molecule detection in cells24,25,26. It allows for the specific synthesis of AuNPs on isolated tag fusion proteins and has achieved an unprecedented labeling efficiency in nonfixed or chemically fixed prokaryotic (E. coli) and eukaryotic (S. pombe) cells. However, implementing the same protocol in more advanced systems such as mammalian cells or even tissues involves additional challenges, such as the more complex intracellular redox homeostasis and more fragile cellular structure.
This study presents a clonable EM labeling technology, which combines the novel ANSM-based AuNP synthesis technique for labeling genetically encoded cysteine-rich tags (MT) with the HPF/FSF-rehydration-HPF/FSF sample preparation method, enables the unambiguous single-molecule identification of tagged proteins in the ER membrane, ER lumen, and mitochondrial matrix in HeLa cells. The current method combines the characteristics of high labeling efficiency, a high signal-to-noise ratio, single-molecule labeling, and strong universality, and this method has broad application prospects in life science research.

Författare Spotlight: Direkt syntes av EM-synliga guldnanopartiklar i celler för proteinlokaliseringsanalys med välbevarad ultrastruktur  

Direkt syntes av EM-synliga guldnanopartiklar i celler för proteinlokaliseringsanalys med välbevarad ultrastruktur

Protein detection and localization are essential in biological research. Our protocol describes a novel EM-labeling technology using genetically-encoded tags which allows single-molecule visualization in situ with well-preserved ultrastructure. It is crucial to avoid over-fixation.The labeling method is based on the activity of cysteines in the tags, which are highly sensitive to aldehyde cross-linking. Our protocol avoids use of aldehyde fixatives and preserves the tag activity and ultrastructure. at the same time.EM labeling has always been challenging, suffering from poor morphology or inefficient labeling. Our protocols synthesizes in situ nanoparticles directly on individual proteins to provide clear and precise localization of the target proteins, with a high signal-to-noise ratio, high efficiency, and specificity. We have so far achieved excellent labeling in isolated proteins, E.coli cells, yeast cells, and HeLa cells.We will explore and optimize the method for application to tissue samples. Combining the prevailing cryo-FIB and cryo-EM technologies will allow us to address important biological questions.

Watch this Scientific Journal Video about Gold Nanoparticle Synthesis for Mammalian Cells Using Sapphire Discs at JoVE.com

Gold Nanoparticle Synthesis for Mammalian Cells Using Sapphire Discs  

Guldnanopartikelsyntes för däggdjursceller med safirskivor

Ta en förberedd HPF- och FSF-behandlad safirskiva som innehåller stabila HeLa-celler, tvätta den med PBS-A-buffert i fem minuter och upprepa denna process tre gånger. Överför safirskivorna till en 35-millimeters odlingsskål som innehåller en milliliter PBS-A-buffert vid rumstemperatur. Byt ut PBS-A-bufferten i odlingsskålen med en milliliter reducerande lösning och inkubera i en timme vid rumstemperatur.Förbered guldprekursorn i en milliliter reducerande lösning och blanda omedelbart genom virvelning. Tillsätt 80 mikroliter 500 millimolar D-penicillamin i dubbeldestillerat vatten till guldprekursorlösningen. Och omedelbart virvel.Byt ut lösningen i den odlade skålen med en milliliter guldprekursorlösning och inkubera i två timmar vid fyra grader Celsius. Tillsätt 20 till 100 mikroliter av den nyberedda natriumborhydridlösningen till guldprekursorlösningen, skaka omedelbart för att blanda väl och inkubera i fem minuter vid rumstemperatur. Byt ut lösningen med två milliliter PBS-A-buffert för att stoppa reaktionen.Förbered sedan epoxihartsblandningen enligt tillverkarens instruktioner, överför safirskivorna till inbäddningskapslar med platt botten och infiltrera med harts i minst 30 minuter vid rumstemperatur. Polymerisera provet vid 60 grader Celsius i en ugn i minst 18 timmar. Förbered ultratunna sektioner av det polymeriserade provet genom att trimma provblocken försiktigt med en rakhyvel för att exponera safirskivorna.Doppa blockspetsarna med safirskivor i flytande kväve i flera sekunder och tina rumstemperatur. Upprepa frys-tina åtgärden flera gånger för att lossa skivorna från blocken. EGFP-MTn-KDEL-proteinet uppträdde som två till fem nanometer stora guldnanopartiklar exklusivt fördelade i perifer ER-lumen och i kärnhöljets perinukleära utrymme.Den välbevarade ultrastrukturen möjliggjorde identifiering av enskilda molekyler av märkta proteiner och underlättade analysen av organellinteraktioner, såsom ER-mitokondrierinteraktioner. Nanopartiklar av Ost4-EGFP-MTn-proteinet avgränsade ER-membranet i kärnhöljets yttre membran. På samma sätt uppvisade Mito-acGFP-MTn-uttryckande celler specifik märkning i mitokondriell matris.