 As I mentioned briefly in the last lecture, if you are doing the high throughput experiments what becomes really crucial that what are the quality control checks you have done. While it is very easy to generate big data now with the advent of latest technologies especially in the field of proteomics and genomics. But what becomes very crucial now how close the tension you are paying in terms of your reagents available, they are good quality reagents, your assay performance, the quality control checks for that and then your various control spots or control features which should guide you whether experiment works or it has lot of you know non-specific values. In this light micro experiment becomes very crucial when you have thousands of spots printed on a given chip and you have to now perform your you know clinical sample applications or various type of protein which you want to test out on the chip, you need to ensure that you have the good guiding controls for the entire experiments. You have good positive controls and negative controls printed on the chip. But then what also becomes very crucial if you are printing thousands of features on the arrays how reproducible your printing is, how close your spot morphologies are from one to other. Have you paid enough attention to ensure that there is no batch to batch variation between the slides because if for the biomarker discovery program if you are using large number of patient samples let say 100 samples you want to use for a biomarker discovery program so you need 100 slides one for each patient. If there is a variability from slide to slide and batch to batch you already have so much variability from the individuals that is the biological variability which we cannot avoid. Each one of us are very unique very different and the patient sample will have many things which will be not so reproducible across hundreds of samples. If your features itself are printed on the chip are also non reproducible then you cannot make sense of the data. Therefore making a good chip with the proper quality control checks becomes very crucial. So in continuation to the last lecture Dr. Saloni Sonawalla the application scientist from Array Jet Technology will demonstrate you how to perform reproducible and high quality printing for microarray based experiments. You will also learn about iris which is a camera and advanced technology that is used for this type of printing of microarray slides. So let us have Dr. Sonawalla to give her talk. I'll go back and I'm going to open the command center for you. That's your command center. That's the software where the magic happens. This is where all the development of arrays like Hubpro and other industrially supported arrays have happened. So all these people use the command center. The command center is something that you can use. It's like your paint software. You can design which area you want, what arrays, what plates you want. You can pick and choose how many samples you want. You can customize the whole experiment sitting on a computer and designing it from these particular features that I will take you through. Obviously this is the first tab that gets open. You've got an option. So what should we do? Let's do one example of something that you want to develop. Just anybody has any idea what kind of array you want or what design you're looking for. I'll leave it to you. Just give me an example. Maybe you have 34 antibodies. You have 12 antibodies. Just give me a number. Anyone? Any number? 22. So that's a good number because it can do 12 times 12. So what will happen is your two of that will be blocked. So we can design a 22 sample run very easily and I'll show you how. So obviously most of us use 34 384 sample plate. I'm going to reduce this to one. This is your 384 samples. Now you set 22. Now look what happens when I type 22. The most minimum sample that you can print or you can have is 12 because I showed you the jet spider. The lowest ability is 12. The highest ability is 32. So now it's a good one that you've given me 22 because it's we're working around it. So 12. If I want to increase this number it's 24 36 48. These are your samples. This is where your sample goes in the plate and that's how you can fill the plate. And it tells you exactly where you want to put your sample. So everything is automated. You design your run and the software will tell you how to prepare your sample plate. So it'll give you a print out that okay if you want to print 22 samples I'll show you where your 22 samples will go so that your experiment will look like how you wanted to look. So the software will tell you exactly how to design all that. So I'm just extending this but I don't want that many. I only want 22 but I can't get 22 I can get 24. So I'll leave it here and I'll go to the next step. These are the nozzles of the printhead. This is sample one is distributed across four nozzles. Like that there are 12 samples. Now again I wanted to tell me how I can get 22 samples instead of 24. And I'll tell you this I'm able to block some of these dots. I'm able to block them so I don't use them at all. So any any idea? What I will do is I'm going to use 11 out of these 12 from the jet spider. I'm going to block this one. Why? Because it's 22. So what I want to do is I can do 7. If I want to only have 7 I'll block these. So basically the jet spider will go inside your printhead but those that I've blocked will not use your sample. You can put a blank or a buffer or something there. So I will only use 7 and they get printed across 7. So let me try and block them and let me show you what happens. That's your blocked. So now I've got 11. If I move back I'll have a set of 22 because it's blocked too. Do you see that? So now I can print 22 samples because I was able to flexibly just go here block one go back 22 samples. I could block if I wanted to just do 7. I didn't I had only 6 samples. I'll block all these and I've got now I've got only 6 samples that I'm working with. So now you've got 12 because your time's 2. So it's 12. So this is where you can modify how much sample you're printing and what do you want the jet spider to pick up. So it's not that it'll go inside the plate and pick up all your sample and bring it back. It'll go inside the plate and only 1.3 micro litre will be picked up each time and that is more than enough for your entire assay. It's a very good question. There are two options you can do. The filling of the samples in the plate can happen vertically. So it goes a set of 12 goes first corner of the plate picks up the 12 then it goes down and it picks up but you also can do horizontally. Now I've moved this to horizontal. Let me go back. So these are 12 minus so there are 6. So what happens? It has to fill up because the jet spider is 12. It goes alternately. So alternate well. So it needs to complete that first top corner before it moves to the next section. Now look what happens. I'm increasing the samples left to right. Yes. So the printing happens in left to right motion on the fly because that is how the spots get printed from that edge. So the first reference edge is your top right corner and then it gets printed off like this. So set of 12 set of 12 set of 12 set of 12 but because the way the plate is designed that is why the software will tell you where to put the sample. So you know you can decide where how you want the array to look. So if you tell me that this is how I want my samples to look we will we will feed it into there is another option in the software where you can feed your requirements and it will generate a plate map for you on how you want. So you can generate your plate map. You can generate your data sheet. So you can generate these looking at how you want it to fill. So if your plate right now has certain samples in certain specific locations then the software can also go and pick up that sample from that well. So it's so flexible because there are so many different applications it's actually a very interesting question and I can I'll spend another two hours telling you how we are doing this mechanism of generating the well plate. So the jet spider needs to fill up one corner of 12 because it's in multiples of 12 it has to finish the first 12 go to the next 12 wells go to the bottom 12 wells go to the next bottom 12 wells it has to finish one section before it moves printing and takes another set of 12 samples. So that way you are able to save the time it takes to move around it'll just pick up 12 at a time finish go back 12 at a time and done. So that is the so when we are doing assay transfers we show you a full demonstration of how your assay manually can move horizontally but this is this is only to prepare your source plate this is not how it will look when you print it. This is your 384 well plate so this is something like this plate let me show you this one so this is like any plate where you put 20 microliters of sample. Okay now this this feature of the slide shows you where you have to put your sample so so that the array can look how you want it to look the so an aliza can look how you want it to look this will only this is only for the reasons of where to put your sample once you know where to put your sample you move to the next stages this is a fun part this I really enjoy doing because it's fun now somebody give me a normal number how many pads or let's take an aliza let's take 96 I'm going to take it to making so what I'm going to do is divide this full slide into 96 squares with the software so right now we're doing let's I'll just go to I'm showing it this because I want to show you the difference in the pitches so let me give you a six by six example and increase the space for the purposes of explaining think that will be better so with the help of a software I'm changing how I want my asset to look this is where the slide properties happen the next feature when I move is the spots so what I'm going to do now is this is the distance from one spot to the other spot it is called the spot pitch the pitch is the distance from the center of one spot to the center of another spot and that has to be consistent if you want to give consistent data it has to be a consistent spot so let me increase let me decrease this when I decrease it what is that tell me it tells me that now there'll be some space from one spot to the other spot and right now because we have many we have many samples and we have such a small area it cannot fit all the sample let me go back to the basic you said 22 so let's stick to 22 so that's my 11 and now I can so from one slide let that be a blank slide but from my blank slide I have made many identical assays many each assay contains 22 samples so my 22 samples are distributed across the entire slide in the way I wanted because what I'm going to do after this is I'm going to post-process it I'm going to put my secondary antibody and I'm going to make that as one reaction and that'll be my one assay done so you can multiplex it so right now I have given it right now I have given it several squares and I can duplicate it so most of the work in protein obviously you want to get high reproducibility so you duplicate you triplicate it so the hue pro is of a duplication so you've got you've got 19,000 times two features I can triplicate so this is how it looks this is the final tab how my arrays are going to look and if I want to make them centered I can make them centered and I zoom in these are my spots and if I zoom out you can you can see that each square is its own little Eliza if that's how you want it to be so instead of doing separately at the end of the day you will have a complete data sheet which will show you that my first spot for example my first spot I'll increase the space because right now they are a bit clustered so you can't appreciate it I hope you're seeing this because it's not letting me increase it and there is a reason because it's me reach its maximum limit so let's see how it looks now so this before you start your entire experiment you know that there is something which is not suitable because it's either it's going outside the dimensions or it's inside the dimensions so you can alter your entire print run based on how you want it to look so now let me go back I'll make this slightly smaller and I'll make these slightly bigger and now I will give them some space to grow are you seeing any difference in how it's looking that's better isn't it so this is your one square and now I've designed it just while talking to design it in such a way that it looks pretty it's good morphology and differentiated spots so now you can tell that yes this is something that I can work quick I can develop my assay there could be many other biological samples that you can get in this square so this is just one so let me go out of it that's your first row that's your second row that is your one slide like this the same time it takes for one slide is the same time it takes for 25 slides so really doesn't matter if you are doing one slide five slide 10 slide 25 slide your entire assay can be replicated identically with all these squares which is why it gives you the the fun of designing something and transferring your immuno assay or your chemical or any other biological component assay on to the inkjet platform because many other people can use it because of the software because it's so flexible you can design your assay and when I press start I can go home there is no need to sit with the computer wait which is why it's 100% automated so finally I'll go back to Iris the camera which has changed the world of proteomics why most of the people prefer this camera is because it remembers it recognizes and it reprints and these are the three hours that change the life of any protocol or any assay because you don't want to waste your experiment if things don't work because of a platform that is in your lab that's not your fault but you don't want to then have to go back and repeat anything just because it didn't work so we have decided to put in a secure quality system itself on the platform that it'll remember if there is anything that you've missed if there's anything that you've forgotten mistakes happen it's okay so what we are doing is we'll remember it that yes that is the place where she's forgotten these areas but don't worry we'll go back we'll print it and that's why what comes back at the end of it as a solution is 100% healed these are some of the case studies that I have put on the table this is the benefits of reprinting it's a technological advancement and no other sort of proteomics solutions provider has a camera attached to the printer where the camera knows what sample where whether it's there it's not there whether it's missing it'll tell you a full report and that is completely automated so what is this camera that I'm talking about it's called the iris camera iris it has two twin cameras on both the sides of the printhead and it flashes when it moves from left to right right to left it flashes so if you are sitting and looking at an area you can tell that the camera is working because it's flashing it's taking real-time images every time a spot is getting dropped it takes a picture takes a picture and all these pictures get accumulated in its database in the cloud and then it'll tell you oh by the way this slide has a dirt or a sample is gone or maybe something has been brushed off or you know you're missing an antibody because you forgot to fill the well because you know so many things so it detects missing spots artifacts merged misaligned all these problems that come with micro ring as traditional micro ring are eliminated that's how it looks so these two are the cameras so the camera is actually here so when it moves the camera flashes on the slide and it takes a printer it takes a captures an image real time of every spot that gets printed every section of spots that's 12 that's 32 in a row it'll take pictures of all 12 at a time so it's a real-time imaging capabilities it's automated defect detection that means it's missing artifact merging misalignment all that is it's completely quality controlled so you don't feel that you have to go back and repeat something just because in the first time you miss something you always have the option that you switch on the camera it'll tell you remember it'll go back and print it and that's it so look at this experiment this is an overview slide that I created and this is only one small square section this is a this is I think more than thousand in one square thousand samples this is just a one small part of it but this is what's one one of our customer observed because he was having was a lab with a lot of dust particles and things and he wanted to characterize this so he selected his threshold that I want my spots to have a certain threshold of quality he chose how much percentage of missing spots he wants so many of the high throughput proteomic research institutes have criteria so if they want a successful assay they have criteria it my signal should be not above not below my my spot has to be certain percentage I have to have certain number of assays so you have criteria for every assay to make them successful we can set these criteria for the camera itself so when it starts printing it is able to detect upon these threshold that you have set and it'll give you a positive accuracy this is something that was developed in China they've used the iris for their array production and capital bio it's a very big microarray provider in China and they use our system to develop some of the high quality assays high quality immune assays but the reason that the iris works is because they need to make sure it's it's including all their criteria that they have in an assay or your your supervisor or the head has these criterias for making an assay work you transfer all of those criterias onto the platform what's a pro license pro license is something that has the unique ability to remember recognize reprint three hours and that is an advanced feature of the software that comes along with the camera so think about it this way you need a camera to detect your issues but if you want the camera to remember and reprint you need an advanced software so your current software which doesn't have the camera is not going to be able to print something if it is missed without the camera so the software that is a standard software does not include the options of a camera if the camera is being used it has to come with a special software that can remember and reprint your spots that is why you have something called the pro license so pro license why is it extremely useful is it can automatically refill your spots or reprint or it can manually do it as well you've got advanced data recording you can store all your content all your data in a cloud monitoring system so it's all up and no issues with backup and data safety etc so it's it's all there and you have improved visual parameters so you know exactly what is going on and you don't need to be so if it's a long assay if it's going to take maybe four hours and you have other things to do you can press start you can go back come back after four hours you'll have few files waiting for you in your cloud right this is my assay these are this is how successful it was how many missing features were there whether it was able to detect all my antibodies what is my data it'll give you a couple of reports so you just take those reports and then you can post process them automatically with your secondary antibody or whichever method RPPA whichever method you're using to characterize this is how the pro looks now I've shown you the the work that I was showing you before if you have noticed it was on the pro it showed advanced pro in the previous feature that I was showing you it was a rigid pro that's the software where all the camera and reprinting happens so it's an artificial it's actually a part of artificial intelligence software which evaluates your slides real time it's automatic or manual spot refill and it provides hundred percent yield no missing data sets so there is no need to repeat your experiments for a whole month so it remembers that there is something that is missing here and what it does the next time you have a spot here so here it's blank it remember so remember these are the pictures that you get automatically from the camera this is the picture that it showed without the spot then it recognized remembered reprint and it printed the spot so you have an antibody there instead of wasting four or five slides without the antibody it is able to track it is tracking the antibody and printing it again so there are spot refill feature now spot refill means you can reprint that spot wherever it is a missing place it'll go and put your sample so it evaluates the likely cause of the missing sample sometimes what happens is air goes inside or you've forgotten to fill a well because these are 384 well plates if you fill all the wells sometimes the human eye cannot really see if all the wells are full or not I have had my own experience where with pipetting so many different samples in the 384 style if you miss one well and if you put that plate to a reader what's going to happen to that one antibody it's not going to print it's not going to get picked up it's not going to get detected so you've missed the whole set you have to go back you have to fill it again and start so it evaluates what's the issue whether it's an air it's a thread it's a fiber it's any other problem that you've either missed it you've forgotten it it evaluates it and it will show you before it starts the printing it'll show you yes I'm able to successfully detect it and I'm able to print it but there are methods where you can do a manual printing as well where it evaluates the cause of the missing sample or the empty well where you've forgotten to put a sample in it locates it from the whole slide deck of hundreds of slides of many arrays it'll be able to detect where exactly it has been missed and it replaces it with the sample that is already in the printhead I showed you in the the second of the first slide that it has some capacity the printhead has capacity to store your samples so from that capacity it will use some of the samples for this reprinting if it doesn't have enough sample in its in its nozzles in its capacity then it will go back to your plate it'll pick up some more sample 1.3 micro-eaters or 2.5 micro-eaters and start again and it re-checks before it prints it re-checks so this is your plate map this is what I showed you this is your plate now remember you've forgotten to fill this well and that's okay so what happens is when you're doing the manual method it'll go back to your print run your print design and it'll show you this is the place where your sample is missing it'll be able to show you that this is where you've missed it so you can take the plate you can have a look yes it's missing it's right it's missing I've genuinely forgotten which is fine put it back you put a buffer in or you say okay it's not too late if it's if I missed it I can put a sample again so go to your lab quickly prepare a sample put it back and put the plate inside the reader and it starts where it has left off and then when I start this overview run the green area is where it'll show me to confirm to double check to verify that yes my antibody that I just went and refilled is definitely going to get printed it's not that I'm missing my entire experiment and the whole thing has gone to waste it'll remember it and it'll show you just to double check are you happy with this can I start can I press yes are you happy with this you said yes and then that's it so you've got this is your missing so this is your manual method where you've got the spot which is missing you see this and then this is your image this is your array this is where you've missed it and without having to do everything again you just go and fill this one sample and it'll show you that yes your antibodies as a now you have complete data set you have a complete array with all data sets to be able to characterize it so you are not wasting enough time to go back and do it this is the gal file reading this is what happens when you have the pro license which is able to show you where you've missed your spot or where things have gone wrong and it has corrected it for you and this is where your data file goes and merges on top of your sample file to give you your data set so if you've done some work with gal files probably before you probably have an idea or I can take you in detail offline with how you can do this but it'll tell you exactly this is my spot zero samples at one this is my diameter this is my pixel this is my advanced diameter this is the distance this is the circularity 98% which is almost 99% so the morphology is extremely round so you've got a nice good protein content in that one spot so it gives you complete details to be able to verify or plot a standard curve or get a t-pad squared test etc so to conclude it's multi-purpose it's scalable it's obviously next generation of printing it's the fastest technology in the market and it has a large customer base which is why it's important to address issues in proteomics in R&D more than established industries because here we are at a stage where we are trying to develop something and we need technology that can help us develop it faster and more accurate if the technologies itself are not accurate and how are you going to develop something and make it work after two years so that is why we support them through grants through any of the collaborative studies we support R&D work because we want to develop that assay from a non-standard platform to a standardized assay which will work and if we have got an experience in so totally I think from all of us in the team we have about 75 years worth of microarraying, RPPA, NAPA, Hubro experience which is why we're able to support all these industries that are here today who say talk about printing arrays are printed what did you use to print them so if they are having any issues with printing arrays then we're not only promising improved results but we're also partnering and it's a joint effort to make sure that the assays little more sensitive highly sensitive than what you were previously getting but it's also highly accurate so you're saving a lot of time if you've got any assay that you want to discuss with myself or my team that you feel we can totally transfer and it'll save me time money and energy let me know and I'll be more than happy to talk. I'm sure by now you are convinced that while it seems it is very easy to do microarray based experiments and one could screen thousands of proteins just with the you know small volume of the patient sample or clinical sample or even small drug what you is available to you you can just have a few micro liters of droplets and put on the micro reslites and get data for thousands of proteins however to get reproducible data and which meaningful data which could make sense is not so easy and that's where lot of technical expertise is required I hope you are now convinced that microarray printing plays a really important role and technologies like bio printing provides innovative solutions to the researchers for the biomarker discovery based programs or drug development programs or to even various diagnostic based companies so microarray technologies microarray printing platforms especially from the array jet which we discussed today provides unique non-contact inkjet printing to offer throughput precision and consistency and it could deliver high quality reproducible microarray based printing which is really required if the goal was to perform experiments on large number of samples if you are doing experiment where you need only 3 slides probably even with you know very minimal variability among the slides you can still do corrections but when you have large number of samples a large number of microarray slides to perform then a technology which can deliver high reproducibility with the high throughput manner that becomes very very crucial in the coming lectures you will be exposed to more such advanced technologies and their applications in high throughput areas for various type of life science applications including clinical applications thank you.