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hello this is a little tutorial a slight digression for me because this time we're talking about some biochemistry and molecular biology techniques and this is looking at some free software from the company like or which is called image studio light this is a Leica website if you do a search on google for image studio light you will get access to this site which allows you to download a free copy of image studio light image studio light is a fantastic piece of software that allows you to do densitometry analysis on western blots PCRs or other eminent blocks or gel electrophoresis experiments including silver staining and in Coomassie staining so this is what the interface looks like when you open up image studio light this is on my Mac so the PC version looks slightly differently but the great thing is it's available for both PC and Mac and the two versions appear to be almost identical in functionality it's got a windows look about it and it's designed to look a little bit like Microsoft Office in that if you click on little is logo at the top left here it brings down a very Microsoft s import/export save print acceptor box and this allows you to import images from image studio if you happen to have the full version of image Studio Odyssey in Perl which are two other image acquisition packages made by like or and the beauty is for the free version it also allows you to import Tiff's pngs JPEGs in other words images that you've captured using a desktop scanner or another densitometry imaging system and that's obviously the use that most of us will have for this software is importing images from other software once your image is imported it just sits on this little desktop window in the middle here and this is a Western blot in blue peter stylee that i prepared earlier and you can see here you can import various other westerns and I've got a little collection of westerns you can see that I've already done some analysis on on the bottom here and everything you import stays in this little bottom window you can expand this window and you can label various things it's quite a handy no but to be honest this is more for lab book generation I'm more interested in just doing densitometry so I tend to import pictures do the analysis and then close the pictures and delete them from this database just so I can carry on doing my work the menu is broken down into four different sections there's the image section which allows you to do various things like adjust your image change the channels you're seeing if your image in this case is black and white you just see the white Channel if your image is in color you'll also see the red green and blue channels and you can change various contrast and brightness of those different channels you can show your images or your lab book here in different modes either 1 or 2 or or more across so you can look at variations on your image you can do a slideshow the various images you've got which is fun if you like that kind of thing you can duplicate images rotate flip reduce noise assign channels and great one at the end here is you can crop so by clicking on the crop tool you can then just highlight the bit you want to crop down to under scale to make this one a little bit smaller there we go press ok and now it's cropped this image and saved it within the software your original image on your hard disk which might well be your full gel remains undeterred which is great reducing noise is a dodgy business especially Western blots it will essentially edit the document and as we well know we shouldn't be editing our scans by removing unnecessary noise because we may have to be removing some of our biological data so use that one with caution we can also change how we're going to be exporting or printing our image and we can copy parts of our image as well for pasting into other bits of software with the image loaded on the screen depending on which of these windows are open whether it's image analysis annotation a lab book you've also got the option of doing various things on the right-hand side and by default the display tab is shown which allows you to muck around with brightness and contrast and the saturation curves of your image for subtle changes and what you see down here you've got profiles concentration and a chart but at the moment we've not done any analysis so it won't show any data in those windows the analysis tool is where all the work happens and in the analysis tool you have a selection box which allows you to then select items that you've created on the screen and the rest of these boxes basically are to do with densitometry analysis of these various rectangles and various blobs you have on your western blots or your PC RS so there is two options you can either automatically add rectangles as depicted by add rectangle or you can draw your own rectangles and that basically allows you to draw a rectangle of a predefined shape or an ellipse of a predefined shape around a blob or Elaine on your band and allows you to do the densitometry from that I would always do draw rectangle this allows you to copy and paste a rectangle so that all of your gels have the same size rectangle for comparison it just makes life a lot easier for explaining later what the densitometry don't data mean so I would always do that but if you're feeling adventurous you could click on add rectangle which I'm going to do now press your mouse on one of the button on the one of their bands and you can see it's automatically drawn a blob around the band and I can draw it over all of these and you can see it's drawn a rectangle that encompasses what the software thinks is the band so I would undo all of that by pressing commands and or controls yet on a PC and I would draw my own rectangle so I'm going to draw a rectangle that encompasses my band and I'm going to use the select button to select the rectangle you can see it's selected when it's got a dotted line around it I'll make sure it's about the right size which it is then I'm going to press copy and paste paste again and in this style I am now adding these rectangles to my gel one might argue that you'd need to make your box a little wider for this last one to marry enabled you to encompass the entire band one would argue you could put all the others at the same width as well but for the moment I'll leave that where it is so now what is done is it's selected each of these bands and if you look over here now under profiles you'll see that it's created a peak profile that shows the band and if you click on each of these you can see the peaks change as our bands are slightly different densities which is good it's a good sign shows the software is clearly able to look at intensity but at the moment it's not subtracting any backgrounds and therefore we don't know how big this band is and you'll see underneath here it says n a.m. in other words it can't calculate the density of each of these bands so we'll move down down down down down over to here and this is where our background is set at the moment is set to user-defined that's my default option you've got a choice between none averaged where you can choose the top of your segment the top bottom or the right left so in other words it chooses a border around the outside of your square that you've drawn and you press save and you can see what it's done now is this little tiny line down here is our background line that's actually not a bad idea because of course if there's any variation in the long long to tune or section of your gel this would pick that up quite nicely you could of course go to average and choose top to bottom and you can see there it's taken an average of the density at the top and at the bottom and it will subtract that from the background and you see what it's done is has plotted a background and you can choose various other options the median for instance you can choose to choose the median or the average around all your gels or various parts and have a play with those settings and choose one you think works for you to be honest I think the average choosing average and choosing all is a fairly good option if your gel is fairly clean but if your gel is not very clean and there's a big gradient across your journal there's something strange you may want to choose user-defined and it'll tell you that the background method is user-defined but I need to assign a shape so I press ok and then I'm going to draw a rectangle for instance across the entire width of my gel and I'm going to right click on it and choose assign shape and now that is my background you can see here this is my background shape and it calculates all of my optical densities according to the subtraction of the mean of this large background blog you can experiment with this and you can select it make it different sizes move it around your membrane but you'll notice in this case it's affecting all of my vans equally and it's indiscriminate about how much it's taking off each band it's the same on all of them simply because the gel is fairly clean so you haven't got a big problem with that you can then unassign that shape if you choose not to use that shape and you can go back if you want to go to average and choose other options so it's a fairly flexible piece of software I'm just going to delete that background box there and we'll leave it on average for the time being so that's still into that one just by highlighting as you're pressing the backspace button so these are now my optical densities that it is determined from each of these subtracting the background and if you click on each of these lanes and choose the profiles you'll see it's done a pretty good job at taking the background noise away from each of the lanes if you're worried about it you can make your band taller and see how the numbers change to be honest it's not changing a huge amount so you could be fairly happy that what you're selecting is a good value the key to densitometry is also observation looking at these bands this is a fairly clean gel to look at you can see that that band is clearly darker than band number 2 band number 3 is clearly the darkest band on the gel 4 and 5 look much the same and maybe 6 is slightly denser than 5 and 4 and again slightly denser than 1 but not as dense as 3 and if you choose to look at these numbers here you'll see that those numbers correspond to the opinion that I've just made by looking at the gel and that's obviously a good sign of a densitometry program that actually agrees with what your eyes are telling you obviously it may bias your opinion but you have to be aware of that in all science obviously once you've looked at your profiles and you're happy with your background subtraction and you're happy with what you can see on the screen you can choose to show backgrounds show text show labels show the shapes hide the shapes or show them hide the quantification or show and hide the color bar so you've got lots of options to display things have a little play with that but down the bottom here you've got the chart option and you can see what it's doing is it's plotting a little bar chart to show you the relative densities of each of these you would of course do this in your graph program separately because you'd need to average data from many Westerns and many samples to get the decent data set but this just gives you a quick look-see to see what we're looking at and we can clearly see that we look at this gel here we've got six lanes and those six lanes are represented by different height bars you may get a bit annoyed and I certainly have on occasion where these boxes overlap our image and our image looks a bit small we can go back to the image window click on the zoom button and choose zoom to fit and it'll always fit the image into the left side window make our graphs smaller go to zoom zoom to fit and it'll fit again so the software itself is actually fairly self-explanatory it's not particularly complicated to understand the great thing is with the densitometry you've got here you can now look at what you want to print for instance you can choose the print area yep happy with that and then you can go to the window choose print and you can have a little look so I'm just going to go page setup make sure it's printing to my office printer and I can just choose print and it'll print this window with the densitometry the annotations and the numbers for your route book so all in all it's a very handy little bit of software not particularly complicated for those of you who are using much more complicated densitometry programs this it does appear to do the job I would certainly want to compare this with slightly higher end densitometric programs to make sure that the numbers are accurate but I think for a software that just needs to give you some densitometry numbers to be able to compare treatments during a biochemical experiment or cell culture experiment I think it's very capable so please go to the like or website there it is again just remember it's like allcom have a search for image studio light in there research tools and downloads and have a play because it's free