https://www.cytometry.me/blog daily 0.75 2021-02-25 https://www.cytometry.me/blog/thelightinside monthly 0.5 2020-07-13 https://images.squarespace-cdn.com/content/v1/5efa95517826cc473e77a4b4/1594408093629-TBI7WR9HFRD3RJ8ATJ9Z/Screen+Shot+2020-07-07+at+9.18.11+PM.png Blog - The Light Inside (literally) https://images.squarespace-cdn.com/content/v1/5efa95517826cc473e77a4b4/1594428552728-47EZSJU0S9HLPUNW46K0/Screen%2BShot%2B2020-06-29%2Bat%2B10.23.02%2BAM.jpg Blog - The Light Inside (literally) - Bovine Mesenteric Lymphatic Vessel Dr. Kenton Arkill Second Harmonic generation image (left) and 2-photon autofluorescence image (right) of a bovine mesenteric collecting lymphatic vessel, stretched with an transmural pressure head. The wavelike collagen fibers can be seen to cross over one another in a “wicker basket” type pattern. The stretched elastin fibers are clearly visible as bright straight lines, mostly in the direction of the vessel (left/right). The dimmer green background is the spillover of the collagen fluorescence. https://images.squarespace-cdn.com/content/v1/5efa95517826cc473e77a4b4/1594405648377-1KTMFL7FGNZZZVXWMWDB/Screen+Shot+2020-06-29+at+10.08.55+AM.png Blog - The Light Inside (literally) Croce and Bottiroli, European Journal of Histochemistry, 2014. https://images.squarespace-cdn.com/content/v1/5efa95517826cc473e77a4b4/1594407063687-E4QYOI3WQOMI4WED0LT8/VitaminA.png Blog - The Light Inside (literally) https://images.squarespace-cdn.com/content/v1/5efa95517826cc473e77a4b4/1594407467944-HKAFOASDM3L4T4HZV4N3/Vitamin+Autofluor.png Blog - The Light Inside (literally) https://www.cytometry.me/blog/fluorpersonalities monthly 0.5 2020-07-13 https://images.squarespace-cdn.com/content/v1/5efa95517826cc473e77a4b4/1594612378880-L0TFHLMY1BY66TF386KB/Screen+Shot+2020-07-12+at+8.52.32+PM.png Blog - The different Personalities of Fluorophores (no one is perfect) - One of my pet peeves in marketing is how fluorophores are named. It gives this illusion that all members of that named family are equal and not still defined by their individual strengths and weaknesses. https://images.squarespace-cdn.com/content/v1/5efa95517826cc473e77a4b4/1594614002802-TTNPCNVYUA8WDPB4BP7Y/AF488%2Bneurons.jpg Blog - The different Personalities of Fluorophores (no one is perfect) - If the application were to label an antibody or a protein that will be suspended and imaged in an aqueous environment, Bodipy quantum yield would become less efficient and the antibody would not likely be able to sustain the same degree of labeling (fluorophore:protein ratio). Another undesirable characteristic of conjugating an antibody with a non-polar fluor is that the conjugation chemistry most often involves occupying a charged primary amine side chain of the protein (like a lysine residue). As a result, proteins and antibodies are more likely to precipitate out of solution. Alexa Fluors and some equivalent DY dyes from Dyomics on the other hand are very desirable for protein conjugation to maintain solubility. Although the conjugation chemistry remains the same in neutralizing the primary amine, these fluors are sulfonated conferring additional polarity to the fluorophore. My business brain wants to add that the patent covering the sulfonation of fluors like Alexa 488 expired in 2019, which leaves this area open for further development. The rest of the sulfonated Alexa Fluors will come off patent in 2021. https://images.squarespace-cdn.com/content/v1/5efa95517826cc473e77a4b4/1594613729823-XVCL3F2PV554WB7CWLQJ/airy+disc.png Blog - The different Personalities of Fluorophores (no one is perfect) - However, I started this blog with not all fluors are created equally, even when they share a family name and a dominant physical characteristic like polarity. For this example we can stick to the Alexa Fluors. The smallest Alexa Fluor is Alexa Fluor 350 which is a sulfonated coumarin derivative. It has an EC around 30K. Even if it had a 100% QY, it inherently can’t absorb enough energy to be independently bright. Another variable in our application of blue dyes (that I’m not going to go deep into in this blog) is that eyes, PMTs and cameras are also inefficient at detecting such a short high energy wavelength. Listening to a talk at the AQLM course in Woods Hole one year, the instructor stated that blue dyes are optimal for super-resolution imaging because their short wavelength confers a tighter diameter of airy disc, which in theory means that two individual points of light can be closer to one another than green or red-emitting dyes and still detect them as two individual sources of light https://images.squarespace-cdn.com/content/v1/5efa95517826cc473e77a4b4/1594613770371-GA94BQ188KYBL9QGJELG/BV421+brightness.png Blog - The different Personalities of Fluorophores (no one is perfect) - Sadly, blue dyes like Alexa Fluor 350 are so dim and can’t be amplified sufficiently to be individually useful for super-res in application. Their dimness makes their photobleaching rate even more exacerbated. Also, in another blog I covered sources of autofluorescence which also is prohibitive to getting a “bright” good S:N in the blue emission range. This is one of the reason the Brilliant Violet organic polymers were so important. Being able to multimerize an individual violet/blue fluor increased the total signal strength. https://images.squarespace-cdn.com/content/v1/5efa95517826cc473e77a4b4/1594613883707-G5LRVI14OAKSCI9IKK2E/structure+differences.png Blog - The different Personalities of Fluorophores (no one is perfect) https://images.squarespace-cdn.com/content/v1/5efa95517826cc473e77a4b4/1594613958282-1GRZFPK0H2RDGZCOU9PG/Bodipy%2BFL%2BLDL.jpg Blog - The different Personalities of Fluorophores (no one is perfect) - So, back to the physical characteristics. As an example, let’s compare and contrast two branded families of fluorophores, the Bodipy and Alexa Fluor families, both from Molecular Probes/ Life Technologies. Bodipy dyes are characterized by neutral polarity and smaller size. That may seem simple but it translates to use in distinct applications. Bodipy dyes would be the preferred dye to label lipids due to its neutral polarity. For example, if the application is to label a phospholipid or lipid raft to look at membrane dynamics, Bodipy dyes would be at their highest quantum efficiency when nestled into the membrane and would least interfere with molecular function in such a surface. An odd side-effect, though, is also that if the compartment is heavily concentrating the probe, it will increase the total hydrophobic force of the compartment than the unlabeled probe alone. https://images.squarespace-cdn.com/content/v1/5efa95517826cc473e77a4b4/1594612882086-26V4I9LXXE7U2TS0UW4K/Screen+Shot+2020-07-12+at+9.00.48+PM.png Blog - The different Personalities of Fluorophores (no one is perfect) https://www.cytometry.me/blog/electrophiles-and-nucleophiles-basic-bioconjugation-strategies monthly 0.5 2020-08-14 https://images.squarespace-cdn.com/content/v1/5efa95517826cc473e77a4b4/1597179100321-M89YYTMZ38WP0OL6Z3JN/Screen+Shot+2020-08-11+at+1.26.16+PM.png Blog - Electrophiles and Nucleophiles: Basic Bioconjugation Strategies - The most difficult condition to control is protection of the NHS ester from oxygen and temperature. This is why the reagent is resuspended in DMSO or DMF that is >99% anhydrous. We’d routinely have people call tech services that had resuspended their reagent in DMSO that was left on the lab bench intended for freezing down cells. https://images.squarespace-cdn.com/content/v1/5efa95517826cc473e77a4b4/1597178535736-1A1OJ9STFE0OQCWC3TMG/image-asset.png Blog - Electrophiles and Nucleophiles: Basic Bioconjugation Strategies - When I began working at Molecular Probes in technical services, my experience was cell biology-centric. The only chemistry knowledge I had back then came from basic courses in university. But, Molecular Probes was at its core a chemical company, albeit fluorescence chemistry. The reagents might be used on cells, but the chemistry was our business. In tech services, our bible was called Bioconjugate Techniques by Greg T. Hermanson. I still have my torn up, rabbit eared copy sitting next to me on my desk at home. It’s like a safety blanket. https://images.squarespace-cdn.com/content/v1/5efa95517826cc473e77a4b4/1597179910928-ZYP52FQ535HJTVE575SN/Screen+Shot+2020-08-11+at+1.35.04+PM.png Blog - Electrophiles and Nucleophiles: Basic Bioconjugation Strategies - To bring all three of these common conjugation methods together, the most difficult of all the bioconjugations you might do is a large protein to another large protein, for example a phycoerythrin fluorescent protein that is 240kD to an antibody that is 150kD. Because of the size of the phycoerythrin, you may want to localize the fluor to the hinge region of the antibody (thiol-mediated) to reduce the risk of losing binding affinity of the antibody. In this instance the goal is to activate the primary amines on the PE molecule with an SMCC cross linker. https://images.squarespace-cdn.com/content/v1/5efa95517826cc473e77a4b4/1597179503739-GZQL44GC2XG01I570ELI/Screen+Shot+2020-08-11+at+9.08.23+AM.png Blog - Electrophiles and Nucleophiles: Basic Bioconjugation Strategies https://images.squarespace-cdn.com/content/v1/5efa95517826cc473e77a4b4/1597179741490-KTSRCRRBDRJ2X3P6J4F5/Screen+Shot+2020-08-11+at+1.32.26+PM.png Blog - Electrophiles and Nucleophiles: Basic Bioconjugation Strategies - When you have a fluorophore that is REALLY big, like the KIRAVIA or the Horizon Brilliant polymerized fluors, the randomness of conjugating primary amines can be undesirable. The risk of conjugating too close to the antigen recognition site or changing the folding and function of a protein can be too much. Instead, a more reliable, predictable target can be a cysteine, thiol-containing side chain or the disulfide bond of the antibody hinge region. 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She is an industry-wide recognized subject matter expert in the applications of fluorescence chemistry particularly flow cytometry and microscopy multiplexed cell analysis. She has spent her career being the bridge between advanced instrumentation platforms/ novel technology with biological applications, reagents and bioassay methods first with Molecular Probes/Invitrogen and later at Biolegend. Her experience is focused on cell biology, neuroscience and particularly immunology/ Immuno-oncology research application areas. 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