Hello everybody. Welcome to today's webinar. My name is Simon Moe, Associate Product Marketing Manager here at Permega, and I'm excited to be your moderator. Today's webinar is titled Advancing Cellular Imaging with Enhanced Tagging Technologies. Before we get started, I want to cover some housekeeping items on your screen. There are multiple windows, all of which are movable and resizable, and I would encourage you to move them around to get the most out of your desktop space. We also have many ways that you can interact with this webinar during it. Feel free to submit a question anytime during the webinar. We'll be answering those during the live Q&A session at the end of the webinar. There's also a resource library on your screen that contains a bunch of helpful material, including copies of today's presentation. So I would encourage you to explore those both during the webinar and afterwards. If you're interested. After the presentation, there will be a survey. And so I would encourage you also to take a moment to answer those questions. We really appreciate any feedback you have on this experience. And we'd also encourage you to share the webinar with any of your colleagues who might not be able to attend today. So hopefully they can learn a little bit more about what we're discussing. And then I will now introduce our speakers quickly. So this is an agenda for today. We have two different speakers. We have Doctor Lisa Vu and Doctor Mark Willett. And so we'll be breaking this webinar down into two sections. The first will be led by Lisa. The second part will be led by Mark. And so a little bit of a description about Lisa. She's a senior scientist within the Advanced Technology Group at Promega, covering some of the early research division, and is responsible for assessing and investigating external technologies in the field of life science in collaboration with both academic and governmental partnerships. She received her PhD at the University of Wisconsin, Madison and has worked in the biotechnology field on various products from concept to commercialization, such as micro fluidic cellular assay devices, biochemical assays, instrumentation, and cellular imaging tools. And so now I will hand it over to Lisa. Thank you for that introduction. My name is Lisa Vu and today I'm going to be discussing advancing cellular imaging with enhanced tagging technologies. So what is Halo Tag? Halo Tag is a 33K Dalton monomeric protein that binds irreversibly and covalently to chloralcane groups. Because of that, you can fuse Halo tag to your protein of interest and use these chloralcane Halo Tag ligands to attach any functional group of interest you want to their protein. So what are the applications our users have been using Halo Tag for? Well, our most common application is fluorescent labeling of your protein of interest. We have a series of fluorescent Halo Tag dyes which allows for cellular imaging, functional imaging and super resolution. You can also take your Halo Tag construct and use it as a reporter system through CRISPR insertion. You can then sort for your positive clones using these Halo Tag dyes. Also, if you have a Halo Protac, you can take your protein fusion, attach a Halo Tag protein and then look for specific degradation of your Halo Tag protein via recruitment of an E3 VHL ligase. Also, you can use Halo Tag in conjunction with Nano Luck to look at protein protein interactions. If you have a Nano lock fusion protein with a Halo Tag fusion protein, when the two proteins interact you will see a Brett signal. Finally, you can use Halo Tag to capture and isolate your Halo Tag protein as well as any complex that are associated with it. Today we're going to be focusing on using Halo Tag for image based applications. So taking a look again at how a Halo tag fluorescent dye is generated. Our chemists can take that chloralkane binding Halo tag group, attach it to a dye of interest in different colors, and also tune these dyes to maximize photo stability, brightness and fluorogenicity. So what is fluorogenicity? Fluorogenicity is an increase in fluorescence when the Halo tag is bound or there's a particular chemical environment. So in this case, in the context of a Halo tag, when a fluorescent dye is not bound to a Halo tag, it's going to be dimmer, whereas it when it's bound to Halo tag, there's going to be an increase in fluorescence allowing for lower background. So why is this brightness and photo stability so important for imaging? Well, the brightness isn't just for creating a prettier picture. Actually, with an increased brightness, what it allows you to do is do more enhanced life imaging. By having a really bright die, you can actually tune your laser power lower to be less photo bleaching and less phototoxic to cells. Also, brightness is essential for endogenous imaging where your protein may be expressed at low levels. Brightness and photo stability are essential for super resolution microscopy, which will be discussed further in this talk in order to be stable in the presence of the stronger lasers that are used for super resolution microscopy. Also, since Halo Tay combined to many different colors including far red dyes, far red dyes are going to have innately lower background and also a far red laser is going to be less toxic to your cells. Also, the ability for Halo tag to bind to many different colors allows for more advanced imaging sauce such as pulse chase experiments as well as functional assays. So taking a look at some of these applications that researchers have used Halo tag for, here is an example of which researchers have used Halo tag as an endogenous reporter for their protein of interest via CRISPR insertion. This allows users to interrogate their protein at physiologically relevant expression levels. So here are just three different examples. Grab 2 NRAS and HTAC Halo tags and which have been CRISPR inserted with Halo tag at in different cell lines and showing localization to different areas of the cell. Here's an example where researchers have used Halo tag for pulse chase experiments. In this case, researchers had tag Halo tag tagged ribosomal protein RPS 9 with Halo tag. They stained this ribosomal protein with a red Halo tag dye, washed out the excess dye, and then three hours later stained with the newly synthesized Halo tag protein RPS 9. As you can see here, initially in the red stain, you can see Halo tag is localized to both the cytoplasm and the nucleolus. However, the newly synthesized ribosomal protein is only or mainly seen in the nucleolus and over time, you could see both the red and green protein are distributed primarily into the cytoplasm. Similarly, using Pulse Chase, users have also used Halo take for something called chloralcane penetration assay. What users use this for is screening the solubility of molecules through pulse chase. By attaching their peptide or lugonucleotide of interest or molecule of interest with the chloralcane group, they can assess if their peptide or the lugonucleotide localizes to their organelle of interest. So here's just an example where the researchers had taken a Halo tag GFP construct and localize it to the nucleus. With GFP, they can use it as a control to ensure under all conditions their protein is being localized to the nucleus. In the absence of the red Tamara Halo tag dye on the top panel, you can see there's no Halo tag staining while there's GFP staining. In the absence of their molecule of interest, you can see a Tamra stain of their Halo tag protein lights up beautifully. However, now when they pre incubate their cells with their peptide or a little nucleotide of interest, they're able to block the staining of this Halo tag dye showing, showing that their molecule of interest is indeed localizing to the nucleus. The users went on to do dose response curves of their molecules to show that they can generate IC 50S using this method. We also have a set of Halo tag and permeable dyes. So these dyes are not permeable through the cell membrane and are going to be best for all your Halo tag proteins that are localized to the cell membrane. Researchers have also used this for functional assays to look at protein recycling. So in this case they have a protein that is localized to the cell membrane and initially when they stain the dye or the same the protein with the Halo tag dye, they see a low level amount of fluorescence. However, over time that protein becomes endocytose increasing fluorescence also as new protein becomes placed on the membrane. And then after a longer amount of time, the proteins are saturated with staining and we see a maximal amount of fluorescence. We have two new impermeable dyes available now and Early Access 1 is a far red more fluorogenic Halo tag ligand JF 635 as well as we've added a new color to our impermeable dyes, a red impermeable dye JF4549I. Also, I'll touch a little bit on super resolution imaging as Mark Willett, our next speaker is going to be going into more detail about this later. But I just want to share some examples of Halo tag being used in different forms of super resolution imaging. In the top left panel, our researchers are using it for stead imaging where they have clathrin coated vesicles that have been stained with Halo tag. And you can see a comparison in resolution with the confocal versus the Super resolution stead imaging. Down below we have storm imaging which can get resolution down to 10 nanometers of Halo Tag histones and in the far right panel users have used Halo Tag to do single molecule tracking of a transcription factor in the nuclei of yeast. Some of our collaborators have actually gotten to be more creative with the Halo Tag protein itself and created a Chemi genetic calcium biosensor based off of Halo Tag. In this case, they've used a circular permuted Halo Tag one end or the C terminal and being bound to a calcium binding calmodulin and the amino terminal and bound to an M13 peptide which only binds the calmodulin in the presence of calcium. So in this case, in the absence of calcium, there is very low fluorescence using our fluorogenic JF 635 dye. However, in the presence of calcium, it generates a micro environment for the dye in which we see an increase in fluorescence. And below is an example where the researchers have transduced their neurons with this calcium halotic biosensor Halo camp and in the presence of voltage see an increase in fluorescence. They've also done this to detect voltage by attaching Halo tag. To a. Rhodopsin and then membrane localization sequences. When a membrane is hyperpolarized in the absence of voltage, we see fluorescence of this biosensor and in the presence of voltage with depolarization you get actually a FRET quenching of the Halo tag ligand. So we've launched in this past year a series of Genelia floor Halo tag dyes, 1 green, 3 red and three far red, all with their own strengths. For example, our JF 549 has a, our red JF 549 has a nice balance between brightness and background, whereas our red JF 585 dye is has a lower brightness but is also very fluorogenic. And for this case, it would be useful to use this dye for addition only type of experimental protocols, oftentimes useful for cells that don't bind to glass or bind very poorly to glass. And also we have our JFX dyes which are going to be our brightest dyes and our most photo stable dyes. We've taken these dyes and reformulated them into a new user friendly format. In our original Halo tag dye format, what we had was we provided the customer with a solid, a tube of dye containing solid dye and often times this was provided in excess of being allowed to stain 2 to 396 well plates or 8 cell slides at a time. However, users were telling us that oftentimes they waste the dye as it's not recommended to freeze, thaw the dye and oftentimes do not even use this these many samples in their experiment. Further, the dye had to be re suspended in DMSO and then diluted in media before it could be added to cells and our newest lyophilized format. You can clearly see your lyophilized dye and it comes in five one nanomolar aliquots which allows for less waste of the dye. Further, there is no DMSO required. You can simply add your cell media or your buffer of choice to the dye and then add it to your cells. So looking a little bit further at Workflow and our first original TMR Halo tag dye, what it required was a dilution of this dye into media, adding it to cells, incubating, going through one wash, then another wash and then a final wash, incubating that final wash for 30 minutes and then replacing media. With our newer Genelia 549 dyes, we had the solid format in which there was a resuspension in DMSO. However, we got rid of those wash steps and you only needed to replace the media in the end. With our new lyophilized 5 pack of this version of a dye, you can direct directly dilute this dye and sell media and dispose of the dye when you're done. So with that, I'd like to just summarize with the few takeaways that the Halo Tag dyes has unique features to improve and expand these imaging capabilities. The brightness and photo stability and fluorogenicity of these dyes are going to enhance your cell live imaging capabilities as well as your endogenous imaging. The variability and colors that are available for Halo Tag is going to allow for pulse chase studies. Photo stability and brightness is also going to be essential for super resolution. The newest Halo tag guy dyes come in allopholized 1 nanomolar flat format, which is going to be clearly visible in the vial for users, allows for aqueous dilution with your cell media or your buffers of choice and allows for a faster workflow and comes in these 5 pack of single aliquots that aids for less waste of your dye. So with that, I'd like to thank you and take any questions. All right. Thank you, Lisa. Now we will switch over to the second-half of today's webinar. This one will be presented by Doctor Mark Willett. He's the head of the Imaging and Microscopy Center at the University of Southampton, UK, and he got his PhD in biochemistry at the University of Sussex. So now I will turn it over to Mark. Hi there, my name is Mark Willett are in the imaging facility at the University of Southampton. I'm here to talk to you today about super resolution imaging and how Genelia floors can be used to enhance users super resolution imaging experience. Before we discuss super resolution imaging, I'd like to talk a little about the technique that genelia flaws could potentially replace. Green fluorescent protein is a gene derived from jellyfish and its discovery was a major breakthrough in fluorescence imaging. This is because it allowed users to express their protein of interest mutant or chimera visibly in live cells and organisms or cells and organisms that have been post fixed. To do this, our protein of interest was cloned onto a plasmid that contained the sequence for fluorescent protein. This is then transfected into the cells that we want to express it in and is then expressed in a fluorescent manner. However, using fluorescent proteins is not without problems. If we want to do a pulse chase experiment, we need to switch the fluorescent protein on at the beginning of the experiment. For that we need specific photo switchable variants. Present. Proteins are sensitive to photo bleaching and they don't last very long in live imaging experiments. Also they don't provide as much signal as synthetic flaws like genelia flaw. So your signal to noise ratio might be quite poor. Genelia flaw works in a similar way. We clone our protein of interest and then we add that sequence onto a plasmid that already contains sequence for the Halo tag. This is then expressed in cells as before, but this time the fluorophore is added in afterwards. This means that we can use a much stronger, brighter, higher signal to noise ratio synthetic fluorophore rather than the quite weak naturally occurring fluorophore like GFP. Just one thing before we go into super resolution microscopy. If for example, you're on a help desk or you're trying to sell Genelia Floors and users want to know how the Genelia Floors will work with their specific microscope filters, you should direct them to use an online Spectra viewer. Many of them are available and some of these will provide details of Genelia Floors. For example, Flora Finder. If we then click on the floor in Fluora Finder. We can see the excitation and emission Spectra of that fluorophore, which laser to use, and common emission filter sets that will work with that fluorophore. Now I want to talk about super resolution microscopy and how we define it as breaking the diffraction limit. So you can see on top of this figure here a diffraction limited image. This is a standard microscopy image. Below that we see a storm image. This is a super resolution image and you can see clearly there's much more detail available. Before we talk about super resolution, we have to define what ordinary resolution is and why we are limited to that type of resolution. Ordinary resolution is caused by a process called diffraction, and you can see it here. It works with any medium that is comprised of a waveform. So in this example we're looking at waves in the ocean. As waves pass through an aperture, they are bent. They are actually slowed down on the edges as they pass through the aperture, and we get a curved wavefront. But you might notice the wavelength of the waves doesn't actually change. This process is called diffraction. Also, we need to consider another physical phenomenon which is destructive interference and constructive interference caused by waveforms that interact with each other but are either out of phase or in phase with each other. For example, if we have two waveforms that interact that are out of phase with each other, they will cancel each other out. This is destructive interference. If we have two waveforms that are in phase with each other that interact, they will amplify each other. This is construct of interference. The same thing that happened to the water waves passing through an aperture happens to light waves when they pass through an aperture. Also, as light travels at different angles, phase differences occur, resulting in parts of destructive and constructive interference. Also, if the light is passing through a small aperture, for example a low NA objective, we get more of the light diffracted, so we get a large pattern. If the light passes through a large aperture, less of the light is diffracted, so we get a small pattern. This is what gives us the fundamental unit of resolution in microscopy, and it's this diffraction pattern is called the point spread function. Each point spread function represents one single fluorophore that has been used as a label in the specimen. The size is related to the wavelength of the light and the objective lens aperture, not the size of the fluorophore. This means we can't measure anything smaller than the point spread function. His size is 200 to 300 nanometers. Because this is caused by diffraction, This is why we call this range of resolvable sizes the diffraction limit. However, a single fluorophore that we've labeled a specimen with is about one to five nanometers much smaller. Each diffraction limited image that we take is actually a mosaic of overlapping point spread functions, one for each fluorophore, and these can severely limit the amount of information that we can see on the image. Super resolution microscopy therefore, is the process that we use to break the diffraction limit. However, it's not one single technique. There are multiple different super resolution microscopy techniques. They use completely different principles and I'm going to talk about the three main ones that are used, structured illumination, also known as SIM, single molecule localization microscopy known as SMLM. And because we're talking about Genelia floors, I'm going to talk about stochastical optical reconstruction microscopy, which is a type of SMLM and that is called STORM. The third type of microscopy I'm going to discuss is stimulated emission depletion microscopy, also known as STEAD structured illumination microscopy or SIM. To understand how this works, we have to look at moire patterns, and you might have seen these if you've taken a picture of somebody with a stripy shirt. You might see some interference patterns. Also, if you have a polarizing filter over your computer monitor, you might see some interesting moire patterns appear too. And that's interesting because the pixel is too small to see, but the pattern from the pixel isn't when we put a polarizing filter over it. So we can derive from that that Moiri pans can be bigger than the structures that made them, as you can see on the panel on the right. So we might not be able to see structures that are diffraction limited. They might be too small, but we can see the Moiri patterns that they make when a grid is passed over them. We need to move the grid around the specimen and rotate it 120° three times to ensure that we get all the information we need and an image of the MOIRI pattern is acquired at each position. Typically structured illumination uses around 15 separate images with different grid positions and rotations to reconstruct 1 SIM image. So there's about 15 times more photon dose to the specimen than standard epiflorescent imaging. We then use a reconstruction algorithm and we reconstruct super resolution image in a typographical manner. Final image resolution is around 100 nanometers laterally, which is A2 fold improvement over normal wide field microscopy. However, due to improvements in actual that's Z or Z resolution, the final volumetric improvement is actually 8 fold. This can yield images such as this one. But Dappy, the fluorophore that binds to double stranded DNA was used and also a thymidine analogue Edu was used and that was incorporated in a pulse chase experiment into newly synthesized DNA. On the right you can see the SIM image and you can see it's much clearer. We have a better signal to noise ratio and particularly on the actual image at the bottom right, you can see there's much higher resolution in Z or Z. Here's another structured illumination image. This one is quite interesting. In this case they expressed GFP tagged Ebola VP protein, and this made virus like particles that were then extruded from the membrane of cells. So what we're looking here is actually the surface of the cells and you can see these sort of filamentous structures and these are these are actually analogous to the shape of Ebola viruses. You can see there's a significant increase in clarity and signal to noise ratio and in detail compared with the turf image on the left, which is a wide field technique. So what are the fluorophore requirements of structured illumination microscopy? Well, structured illumination is compatible with live imaging, but fluorescent proteins are not so good. We need to take about 15 structured illumination images to make one reconstructed super resolution image. So there's lots of photon dose. Photon dose is toxic to cells. Halo tag, the nearly A floor system, is more efficient than fluorescent protein, so lower laser intensity is required and that equates to less photon dose on the cells. GFP isn't very resistant to photo bleachers. That means that you're restricted in how many frames you can take, how long your time course could be, or how close together your time lapses could be when you were taking a movie. Being a synthetic fluorophore, Halo tag has a much higher resistance to photo bleaching versus fluorescent proteins, so many more imaging cycles are possible in super resolution. A high signal to noise ratio is a requirement for image reconstruction. Otherwise remember this is a computed image. The algorithm might try to reconstruct the background and we get some strange looking images. Halo tags in alia floor has a higher signal and therefore superior signal to noise versus fluorescent proteins and this helps alleviate this issue. So which Genelia floors would you recommend to users? Well, all Genelia floors will work with SIM, so long as a user's microscope has the appropriate filters and lasers fitted. However, it's strongly recommended to choose a pair of fluorophores that can be separated by microscope filters, but that are spectrally close enough together to minimize chromatic aberration, which can interfere with image reconstruction. And by that I mean I would use a red and a far red or a green and a red or a blue and a green. But I wouldn't use a blue and a red because they're spectrally separated. And the chances are that the user will find that one of the channels will reconstruct well, the other channel will not because they're too far away from each other on the spectrum. Using halotactonellia floor exogenous expression of mutants is possible where fluorescent proteins alone might not have yielded the requisite high quality conditions for SIM in both fixed and life specimens. So it's a good choice. Next, I'd like to talk about single molecule localization microscopy SMLM. And as I stated previously, the example we're going to use because it's relevant for Genelia floors is STORM. Imaging principle of SMLM is simple. You need to take a movie in which only a subset of fluorophores are active on each frame so that you can see the centre of each individual point spread function without any overlap. We then plot the centre of each point spread function on each frame of the movie, so each frame now has a single pixel rather than the large concentric circle point spread function pattern that we had previously. We then overlay the plotted centres from all of the frames in the movie to make a super resolution image storm SMLM exploits the photo switching properties of some fluorophores. In correct conditions, fluorophores are transitioned to a temporary dark state by a high-powered laser. So they're switched off. They know no longer for us. They then transition to and from the dark state into a activated state and back in the range of milliseconds to minutes. This means that fluorophores start blinking randomly and in an asynchronous matter manner. Apologies. Which allows us to have only a subset of point spread functions on each frame as our move of our movie as we acquire it. So here's a case study. If you want to know more about Storm, I would sincerely recommend reading Christophe Litteriot's papers. His research is into the periodic actin spectrum scaffolding neurons which is diffraction limited. You can't see it in normal microscope images because the periodicity of the scaffold is about 170 nanometers, while as discussed earlier, the point spread function is around 200 nanometers up. So resolution in storm is about 10 nanometers, so 10 times better than structured illumination and 20 times better than wide field STORM is not compatible with live imaging. If you're interested in that sea palm, another SMLM technique. And for perspective, single proteins can be in the region of five to 10 nanometers in size. So we're approaching single Mac macro molecular imaging here. So what sort of fluorophore should we discuss when we're looking at STORM? Firstly, STORM is not compatible with live imaging. It takes too long to acquire the images. But fluorescent proteins don't work for STORM in fixed specimens either, because they don't fluctuate. So your fluorophores must be able to blink, in other words, enter the dark state. Genelia floors enter a dark state and blink under reducing conditions and are therefore suitable for STORM, while standard fluorescent proteins don't. A high signal to noise ratio is a requirement for image reconstruction, otherwise the algorithm tries to reconstruct the background, same as in SIM. Halo tag denilia floor has a high signal and therefore superior SNR versus fluorescent proteins. Halo tag denilia floor enables the user to investigate exogenous mutants. Conventional STORM usually uses antibodies or nano bodies, so it can only probe endogenous targets or antibodies without tags, sorry antibodies against tags which lower resolution. Combining Halo tag with immuno methods would allow the visualization of mutant activity against an endogenous background. And by that I mean using immuno methods in another channel. For example, we could use Halo tag JF549 in the red channel, but in the near infrared channel we could use an Alexa 647 tag nanobody so we can mix and match our techniques. Or alternatively, we can do 2 channel storm just using Genelia floor. In this case, I'd recommend in the red channel, Genelia floor JF549 and the near infrared channel JF646 Stimulated emission depletion microscopy or STEAD. This is the final super resolution technique that I want to talk to you about. So instead what we have is a laser which is doughnut shaped. That laser is very, very powerful and the outside of the doughnut has the role of pushing fluorophores into a dark state, much that we saw in storm. However, the inside of the doughnut we have a separate laser whose job is to reactivate fluorophores that have been pushed into a dark state. So the outer doughnut shaped laser is called a depletion laser because it's pushing the floors into the dark state and the central laser that's stimulating the emission of the fluorophore is called a stimulated emission laser. These two lasers are then scanned together across the specimen and the size of the hole in the doughnut shaped laser is dependent on laser power. Higher power yields higher resolutions, the more photon dose as higher laser powers cause us to have a smaller hole in the middle of the doughnut shaped laser. So this small hole in the middle of the laser is what gives us our resolution. So this diagram is just explaining what happens here. So our electron in our fluorophore starts off in its down in its ground state. Our depletion laser then pushes it up into a dark state where the fluorophore can no longer fluoresce. Then our central stimulated emission laser causes the electron to drop down back down to its ground state and therefore emitting fluorescence. So this happens on multiple steps as the laser is scanned across the specimen. Resolution is dependent on laser power and can be as high as 5 nanometers in inorganic scenarios. However, in cells expect a resolution of about 40 to 50 nanometers, so five times better than wide field, but potentially about five times worse than storm. Storm is somewhat compatible with live imaging, however only somewhat due to its high photon dose. It's the most expensive method too. However, it's very fast and accessible and doesn't require extensive imagery construction like SIM and STORM because the resolution is already defined by the hole in the middle of the doughnut. So what kind of fluorophores do we need? Well, similarly to STORM, we need a fluorophore that has the ability to enter the dark state and to be resistant to float photo bleaching. So the same ones that work for storm should generally work well for stead. Genelia floors could make limited super resolve live imaging much easier because trying to do stead on GFP for example, you're just going to photo bleach your GFP straight away before you get an image. So this could be a sea change. In stead, imaging using Genelia floors would enable us for example to see mutants against an endogenous background because they're much brighter and more bleach resistant than fluorescent proteins. However, due to the powerful depletion laser, the number of time points will still be somewhat limited compared with wide field techniques. So we shouldn't raise the hopes of the user too much that they're going to get days worth of imaging trying to take a live self dead image. As mentioned, Halo tag Denilia floor enables the user to investigate exogenous mutants. Our conventional STEAD usually uses antibodies and nano bodies with synthetic floors attached, which means that normally using stead you're just probing endogenous targets. The floors used tend to be red or near infrared because of the wavelength requirements of the depletion laser. Therefore, I'd recommend the following fluorophores for the near infrared channel JF635 or 646 and for the red channel JF549. Or 585. Thanks for listening to my talk. If you've got any questions I've added my e-mail. Please feel free to e-mail me with them. All right. Thank you so much. All right, so now we are going to transition into the live Q&A session. And so I'll be moderating this. We have Lisa here. Mark unfortunately was unable to attend. But if you do have questions related to his portion of the talk, feel free to submit them in the Q&A as we're going through the questions that have already been submitted and we'll do our best to to answer them or send them along to him. And so with that, I'll start with some of the questions that have been submitted. So someone asked about recommendations for buffer conditions for the Genelia 4 dyes, such as D Storm. Yeah, I'm we don't have a standard buffer recommendation that we think is going to work for everyone's experimental setup, but we can definitely send out a reference to what people have done in the past and hopefully that would provide a good starting point. Great. And then I also another question was about cytotoxicity. So the question was, do the dyes show any cytotoxicity to the cells? Maybe one specific type or if different cell types have been tried? Yeah, we've just tried it on one cell type and but we've done 72 hour studies of just leaving the dye at at a saturating amount of concentration for three days and have shown no cytotoxic effects of the dye on the cells. Great. And then this question just came in, it says hi, sorry if this is a simple question. Is it possible to use multiple Halo tags at once? Can the Halo tag be fluorophore specific? For example, if I used 2 CRISPR insertions together, would it be possible for one protein to be tagged with blue and the other red? Yeah, that's a good question. I mean, they're both going to be Halo tag protein. So if you stain with your red and green dye or blue dye at the same time, they'll they'll probably both light up red and green. So one dye doesn't prefer one target versus the other. They would both stain with both dyes. Unless you can temporarily control when your other target is being expressed. Possibly you can stain one protein and maybe turn on gene expression of the other protein and then look at it that way with a different color. Great. And then this question kind of relates to that. So it was asked, the ratio is a dye to Halo tag labeling at a ratio of 1 to one. Is a dye to Halo tag label racheling? I mean, theoretically, yes, But when we when we stain cells or stain cells with dye, I always add a saturating amount because you're going to lose some, not all of it is going to get into the cell and not all of it and some of it may stick to cell cell organelles and whatnot. But yes, theoretically it should be a one to one ratio and that there should be only one chloralkane binding site on your halotype protein. Someone else asked how long does the Halo tag protein say stay stained by the dyes? So that's also a good question. The the issue is more so as what is the turnover of your protein in your cell. So you can stain it with the dye, but the question is does your protein get turned over and get chewed up and in which case it would no longer be labeled and new protein that doesn't have dye in it would be generated when you don't have the dye. However, I will say that we've had users use these dyes and organoids which obviously lasts for weeks. And after they stain their organized, they can still see Halo tag staining clearly remaining after three days. And I myself have used them in or not three days, 30 days rather. And I myself have used them in and neurons as well on like a A2D2 day 2D surface and seen same level of intensity after day four of four days post staining. But again, this is so the dye can be still live and active in cells afterwards. But it's more so a question of does your protein get degraded? All right. And then another question about the the specific applications someone asked, do you see any nonspecific accumulation of the dyes in cells? Yeah, there is going to be. It's, it's not a perfect dye. You're going to see. Yeah, you're, you're gonna see some nonspecific staining of your dyes. And but I will say and if you're looking at very low expression levels, if you have a very robust protein that's stable expressed on like say ACMV promoter that's or overexpressed, your diet is going to be so bright that it any non specific staining is or residual dye that's leftover is going to be really low. And if background is an issue for you, I would recommend using our most fluorogenic dyes, which would be JF 635 and JF 585. Those are most fluorogenic. So when they're not bound to Halo tag, even then may they may be still not washed out or or present within your cell, you're going to see very little background with those dyes. Great. And then a question about storage after diluting, how long can the dye be stored? Yeah. So with the JF dyes, the solid versions we don't recommend after re suspending and DMSO, we don't recommend storage afterwards. They they will degrade after freeze thaw. With our latest versions, they're in small alequat. So we're hoping you're going to use most of it after we give you these small alequats, these these one animal alequats. I will say that we don't do stability tests on these because what could because they can be re suspended in media or any buffer of choice there that it's too variable what a user may want to re suspend it in, but I will. So we haven't done a clear chemical analysis on it. But I will say I've personally used it probably a week if you re suspended. I re suspended my media, used it a week later after storage and 4° and it was still lighting up my cells just the same. But I would say it's a use at your own risk type of deal. But like I said, I haven't had a problem with it a week after after being stored at 4C. And just be sure to use the dye in access. You know, if you're right on the cusp of, you know, it just barely stains your cell at this concentration, you might have a little bit of breakdown in which case you're going to see a decrease in staining. Just make sure you use it in Access then. All right. And then we just had a question come in asking for in your experience a comparison with snap and clip tag compared to Halo tag in terms of signal strength background. Any other factors you think might be important to consider? I I have not personally done a side by side comparison with those tags. I will say that I've just heard people say that they prefer Halo tag in terms of signal strength and as well in terms of stability. Some people have talked about having issues with losing signal on the snap tag, but I personally have not done a side by side comparison so. OK. And then a couple more questions specifically, I know Mark isn't here, but he was talking about photo stability for the techniques he was talking about. Do you have any specific amongst the Genelia floor products that you would recommend as like the most photo stable if that's an important consideration for the application someone's using? Photo stable JF the JFX dyes are going to be your most photo stable dyes. So JFX 554 for the red and JFX 650 for the far red. Nice. And then I did also want to ask. So we do, we do have, you know, non Genelia Fluor fluorescent Halo tag proteins. Yeah, I guess. Is there anything else you'd want to speak on comparing the Genelia Fluor dyes to maybe the the more traditional fluorescent dyes that someone might be familiar with from Promega's portfolio? Yeah, I, yeah, as as I pointed out, the the Genelia floor dyes is going to have a better workflow. They do offer the next level of brightness, JF XS being our brightest dyes and but even our JF at 64 and 549 are red and are are far red and are red dyes are going to be brighter than our current red dye. And we currently compared to the old portfolio, we did not have a far red Halo tag dye. So the JF 646 and JFX six 50s and 6:35 is isn't a different series of far red dye. So now you can image in that far red region compared to our our older dyes. We we didn't, we had a far red dye but it was only an impermeable dye, so these would all be sell permeable. OK. That's I think super helpful to consider when thinking about like the breath of of Promega's portfolio with Halo tag as we think about comparing it to kind of all the different applications. All right. So looking back down to the Q&A, we have a question about specificity. Do you know anything about, yeah, I guess the, the antibody for Western blot for the Halo tag antibody? I mean Halo I would assume it's a very specific protein. I mean Halo tag is not antibody it, it's not expressed in other any other mammalian cell types and and so there's not going to be a lot of cross reactivity with other at least in a mammalian cell of its presence there. OK. That's, that's helpful to think about. And yeah, so I did want to mention be just because Mark isn't here, if there are any additional questions relating to specific super resolution applications or really fine detailed points, we'll do our best to pass those along to him or someone affiliated with him that we can that we can do our best to answer those. But I did want to move into kind of talking about what's coming next. And so there is an upcoming webinar happening December 4th that is a follow up to the webinar that you just finished watching. It's titled Using Halo tag and Genelia Floor dyes for live single live cell, single molecule imaging. It's going to be presented by Young Schmidt, who's a professor at Michigan State University. And so I do think that what we just heard and what we listened to does a really good job of establishing the baseline for thinking about super resolution applications as well as how Genelia flora ligands really fit into these applications. And so as, as we kind of lead into December 4th, I would highly encourage people who are attending this one to consider attending this next webinar that's coming up. And so if you're interested in registering for that, there is a registration link that is on your on your screen right now. And so feel free to click that. Otherwise, be on the lookout for an e-mail promoting this as we, as we get closer to December. And so with that, I will move to thanking everyone for attending. I really appreciate you all for your time and listening to us. And again, if your questions were not answered, we'll do our best to follow up with them after. So thank you very much. _1734847117561