SpectraMax i3x assay optimization guide

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My name is Caroline Cardenal. I am an application scientist for Molecular Devices,

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and in today's video, we're going to review how to optimize an assay in

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fluorescence or luminescence using the SpectraMax i3x.

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First of all, let's review the three optimizations you can do with the i3x.

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First of all, the microplate optimization.

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This optimization is recommended to realign the read head with the center of the

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well where the signal is the highest, and you will need a fluorescence or

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luminescence solution in the four corners of the microplate.

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This optimization is recommended for 96 well plate, and it is really a necessity

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for 384 well plate if you want to increase the sensitivity for your assay.

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The second optimization is a Z-height optimization.

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This is for the PrID only, and in that case, it will be a live in focus.

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Exactly like a camera, the lens will go at different height

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in order to measure the signal, and you will report the height at which the signal

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is the strongest.

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The last optimization is the wavelengths optimization, and this is basically to

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find the best excitation and emission couple for your assay.

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Let's go into SoftMax Pro to have a look how to set up those optimization.

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You can select the instrument setting from the main menu or by selecting the second

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icon of the plate menu.

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You will have the optical configuration, and you can choose either monochromator or

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fluorescence or luminescence cartridge.

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In term of read mode, this optimization is only for fluorescence and luminescence.

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In the wavelengths window, the default option selected is known.

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But here we're going to select unknown in order to start the optimization result

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when we click on Read.

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Then we're going to select a plate, and here we're going to select a 384 well

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plate,

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and we're going to select standard opaque for fluorescence intensity.

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In TNT objects, you're going to have the set height value that is

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set to one millimeter at default and will be updated when this is done.

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And in more settings, the pre-read optimization option should be selected.

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Click OK,

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and then we need to click on Read.

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The spectral optimization wizard will now appear, and you will have to adjust the

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wavelengths for the excitation and the emission scan.

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As guideline, we will take the literature value and add plus or minus 25 nanometer.

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Here we're working with fluorescein, so we're going to choose start for the

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excitation scan at 460 and the end of the excitation scan at 510.

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For the emission scan, the start is going to be at 500 and the end is going to

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be at 550. You need at least 10 points for the excitation and 10

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points for the emission for the scan to be able to work.

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Then you have to select a blank well, which will be your buffer,

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and click on Next, and the sample well, which can be any well of the microplate,

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and select Next.

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The optimization wizard will start, and it can take seven to eight minutes or

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longer, depending on the number of points you have in your scan.

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Once the optimization is finished, you will have a heat map that will appear, and

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it will show you a cursor showing the best excitation and emission couple for your

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fluorophore. This is the excitation versus the emission, and we have three zone,

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a light gray zone that is not allowed, a dark gray zone where you can have some

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excitation like in the emission spectrum, and the last zone

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where you have no excitation in the emission spectrum.

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The cursor will show you the best excitation emission peak, but you can move that

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cursor to any region of the scan. The value next to the cursor

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is showing the signal-to-background ratio,

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and have a look at that value as it might be in units, in tens, hundreds, or

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thousands.

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You can restart the visit if you don't like the values you're getting, or you

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can reset to the optimized peak if you have moved that cursor.

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You can then click on Read to use the optimized wavelengths.

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A proofing window will appear, and you'll be able to select microplate

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optimization and Z-height optimization.

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Click Run Optimization.

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For the microplate optimization, this is to remind you that you need to put some

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fluorescence or luminescence material in the four corners.

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The optimization will start, and it can take between six to seven minutes.

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When the optimization is finished, we'll have a shooting target-like picture that

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will appear, and you have to realign the cursor with the center of the microplate

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where the signal is the highest. You have to do this for the four corners, one

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after the other. And here you can see that with a 384 well plate, the read

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heads and the center of the microplate are not properly aligned to start with.

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So by adjusting the cursor to the center of the microplate, we are readjusting

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the read head. When this is finished, a window will appear showing the new name for

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the microplate and the offset updated dimensions. Click Save.

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The next window is for the Z-height optimization, and you will be able to choose

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any well of the microplate.

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Click on Next. The optimization wizard will start and will take only 30

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seconds. This will optimize onto the volume of liquid you've got into the

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microplate. So you need to do that every time you change volume, or you can do that

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before every read if you can spare those 30 seconds.

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You will then have the optimized read height that has been found, and

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you can click on Save.

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Then you have the option to read the plate, and you will carry on reading the plate

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with all the optimization. Or if you click Cancel, the optimization will be saved.

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And you can see here in the right of the plate that you got the

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optimized wavelengths, the new plate, and the optimized height.

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You can then save this as a protocol file, so you can open this with all the

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optimization when you read your assay.

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Thank you for your attention.