SpectraMax iD3s and iD5e overview

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Good morning and good afternoon to everybody, and welcome to today's

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webinar. My name is Michael Mendelson, and I'm a

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senior field application scientist in molecular devices.

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Today, we're going to be discussing the next generation of molecular devices plate

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readers, the SpectraMax ID3-S and ID5-E.

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These plate readers are an evolution of our ID series instruments, which have been

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positioned globally as one of our flagship readers.

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Today, we're going to be discussing the ID series instruments and their basic

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features. We'll also give you the brief differences between the ID3-S and the

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ID5-E.

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After going over the basic features of the ID series instruments, we are going to

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go into talking about what's new on the ID3-S and ID5-E.

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This is where we'll go over our new innovations to improve the ID series

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instruments.

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After that, we will go into SoftMax Pro and give you all a brief introduction on

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our industry-leading software, SoftMax Pro.

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This is a software that's able to do both acquisition and analysis all in one basic

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package.

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We will now move on in the webinar by providing you with an introduction to the ID

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series instruments, the ID3-S and the ID5-E.

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In this portion of the webinar, we will talk about what makes the ID3-S and ID5-E

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both unique and their shared features.

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To begin this discussion, we will start by talking about the ID3-S.

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The three in the name of the ID3-S stands for the read modes that it's able to read

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within. The ID3-S comes with three read modes, including UV-Vis

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absorbance with a spectral range of 230 to 1,000 nanometers.

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The ID3-S can also do top and bottom reading fluorescence, as well as

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luminescent-based applications.

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The ID3-S by design is a monochromator-based system.

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Monochromators provide you with wavelength tunability within all of the read modes,

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where you can tune your wavelength within one nanometer increments.

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Within each of the three read modes on the ID3-S, there are four read types.

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An endpoint read will take the point of detection at the center of the well at a

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single time point. This will include applications like your BCA assays for

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protein quantitation or your DNA quant applications.

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The kinetic read type will allow you to take multiple reads across a defined

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runtime.

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The well scan read type is very beneficial for any assays with adherent cells at

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the bottom of the well.

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Well scan allows you to take multiple points of detection across the area of the

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well. And the last read type is spectrum scanning, which allows you to

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scan across the spectral range within each of the three read modes.

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The ID3-S also has the ability to be upgraded with a dual injector module that has

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an ultra-low dead volume. We will talk about this more in later slides.

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The ID3-S also comes with SoftMax Pro, which provides it with ease of use

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and a package that provides data acquisition and analysis.

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The ID5-E is going to come with a few extra read modes that we call our

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advanced read modes, bringing it up to five total read modes able to read within

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the instrument.

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The ID5-E will come with the same three read modes the ID3-S has with absorbance,

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fluorescence, and luminescence, but the ID5-E will also have the ability to do

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advanced read modes such as time resolve fluorescence, fluorescence polarization,

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and fluorescence resonance energy transfer or FRET.

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To do these assays, the ID5-E employs a filter set to provide you with

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the increased sensitivity required for these advanced read modes.

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Just like the ID3-S, the ID5-E also has the upgradable option of a

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dual injector module.

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Using our ScanLater Western Blot detection technology, you are also able to read

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Western Blots on the ID5-E.

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The biggest difference between the ID5-E and the ID3-S is that the

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ID5-E is a hybrid optical system.

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It still has the tunability and flexibility provided by the monochromators,

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but the ID5-E can also include six excitation or six emission

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filters in the unit.

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This provides you with the ability to increase your sensitivity for assays which

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you are developing and also provide you with a wider dynamic range.

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And the ID5-E is also going to have similar features to the ID3-S, such

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as the ultra-cooled PMT and our auto PMT features, which we will discuss in later

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slides.

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Both ID series instruments come equipped with a high-efficiency four-monochromator

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design. This provides you with two monochromators on the excitation side of your

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light path and two monochromators on the emission side of your light path,

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providing you with more wavelength tunability while also conserving the photons

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needed for your assay and removing any background light from entering your light

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path.

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Both instruments also use a xenon flash lamp as their light source.

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The xenon flash lamp in the instrument only powers on when it's being used during

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an assay, so it's reliable to over a billion flashes with easy

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validation. One of the most important features on the ID series instruments is the

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ultra-cooled PMT. We will talk a little bit more about the ultra-cooled PMT in

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later slides. This is a patented technology meant to reduce background noise from

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your sample, providing you with a more sensitive signal and a wider dynamic range.

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The biggest functional difference between the ID3-S and the

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ID5-E is that the ID5-E has a hybrid optical system.

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This means that the ID5-E can use either monochromators or filters or a combination

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of the two to get the best results for every assay.

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Having flexibility between your wavelength selection really gives you the ability

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to optimize your assays and perform assay development at your own speed for the

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best sensitivity. The ID5-E can accommodate six filters on the excitation

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slider and six filters on the emission slider.

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Filters are easily swapped out and read automatically within the instrument and

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input into the software, where you can access them through the dropdown menu.

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We have a list of many filters for all different fluorophores and read modes and

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are able to custom build filters if that's what's required.

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This slide displays some features that are on both of the instruments.

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Both the ID5-E and the ID3-S can accommodate temperatures with temperature

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control up to 65 degrees Celsius.

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Both of the instruments in their standard configuration have three modes of

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shaking: linear, which would shake the plate side to side, orbital, which would

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shake the plate in an orbital or circular fashion, and double orbital, which would

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shake the plate in a figure eight or an infinity sign fashion.

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One of the newer features on the instruments is the option to have advanced shaking

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specific for microbial growth applications.

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We will discuss this in further detail when we talk about the new features on the

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ID series instruments. Both the ID5-E and the ID3-S can

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accommodate plate types down to six wells and all the way up to 384-well

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plates, and everything in between, including 12, 24, 48, and

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96-well plates. Both instruments have the option to add an optional

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cuvette adapter if cuvette research is something that you're interested in.

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Our cuvette adapter can accommodate three working cuvettes as well as one blank.

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And again, we cannot stress how important the patented ultra-cooled PMT is

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for better background noise reduction for a more sensitive signal in fluorescence

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and luminescence. We will go in a little bit more detail on the principles and the

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function of the ultra-cooled PMT in the next slide.

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You've heard me mention it a few times, but now I want to go in a little bit more

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detail on one of our unique patented features, the ultra-cooled PMT.

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If you're not familiar with what the acronym PMT stands for, it stands for

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photomultiplier tube. This is your platform of detection for any fluorescence or

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luminescent-based applications.

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When an instrument is operating, you can imagine that it's going to produce some

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residual heat within the system. A standard PMT will take that heat,

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convert it into signal, contributing to background noise in your sample.

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What we do on our ultra-cooled PMT is we cool the PMT down to negative five degrees

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Celsius, thereby removing any heat contribution from the signal, which will provide

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you with a wider dynamic range, lower background, and a better lower limit of

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detection. This can sometimes be the difference between a false negative

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and actually reading signal on your plate reader.

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The ultra-cooled PMT will provide you with more accurate distinction between weak

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signal and no signal, and can be the difference between an assay working and not

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working. If you look at the graph to the right, we will display this graphically.

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The grayer bar on the graph displays your dynamic range with a standard PMT,

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whereas the lighter shaded graph is going to show you your dynamic range with a

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cooled PMT.

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Another way that I like to explain this is if you're looking at a mountain and the

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top of the mountain is covered with clouds, that is what your system is like with a

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non-cooled PMT. With a cooled PMT, when you look at the mountain,

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there's no more clouds covering the peak, and now you can resolve the smallest

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portion on the visible eye of the mountain, which is the peak of the mountain.

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The ultra-cooled PMT will be on both of the instruments, the iD3s and the

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iD5e.

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The next patented feature that's going to be available on both instruments is our

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auto PMT feature.

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Auto PMT allows you to read assays with an extremely wide dynamic range.

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What the auto PMT does is it automatically adjusts the PMT gain according to signal

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intensity, reading each well at the highest PMT gain, where if any of those wells

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saturate, it will read them again under the medium PMT gain, and if any of those

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wells saturate, it'll read it under the low PMT gain.

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After this, it will automatically normalize your data based on the lowest PMT gain

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used, providing you with a dynamic range that extends up to seven logs.

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What you can see to the right is the settings in SoftMax Pro, where you have the

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fixed settings of high, medium, and low, and at the top, you have the automatic PMT

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gain setting.

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The graph to the left shows you the reads at the three different fixed PMT gain

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settings, where the high is in green, the medium is in blue, and the low is in

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purple. What you can see from this graph is that in the high PMT gain read, you

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have a lot of saturation at the higher end of your curve.

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When you read under medium, you're able to resolve a few more points on the curve,

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but you're still saturating at the top end of your curve.

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When you read it again at the low PMT gain, you're able to resolve the full curve,

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but you see that you start to lose linearity at the lower end of the curve.

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What we do to combat this is the auto PMT normalization, where we now normalize

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based off of the three points so that now you maintain linearity across the whole

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entire dynamic range of the assay.

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Just to conclude, this auto PMT feature is a very powerful feature if you have

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very, very bright and very, very dim samples on the same plate, where now you're

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able to maintain linearity across that dynamic range.

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With the use of SoftMax Pro, both instruments will also come with a few

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optimization features to help you in your research.

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The first optimization feature is going to be microplate optimization.

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The second optimization feature is going to be read height adjustment, adjusting

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the Z-axis of the PMT.

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And the last is going to be wavelength optimization, or our Spectral Optimization

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Wizard. We will go in a bit more detail in each of these three optimization

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features in the next few slides.

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To do that, we're going to start with the Spectral Optimization Wizard.

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The Spectral Optimization Wizard is an optimization feature that comes with

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SoftMax Pro that is available within the fluorescence read

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mode. The Spectral Optimization Wizard requires you

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to have a well with whatever your sample fluorophore is, as well as a well with

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whatever your blank or your buffer is.

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The Spectral Optimization Wizard will then perform a spectrum scan on both the

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excitation and emission wavelength pair for both of those wells.

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Once it's completed with that scan, it will provide you with the optimal excitation

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and emission wavelength pair for that fluorophore, given the signal-to-noise ratio

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between the fluorophore and the buffer.

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It will produce for you a graph such as you see on the screen here, where you can

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move the hairpin trigger to the most red spot, which would tell you what the best

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working wavelength set is for that fluorophore.

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This will also allow you to see any secondary peaks that you might have missed to

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show you how you can optimize your wavelength for that fluorophore even further.

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The next two optimization features that we'll discuss are both available through

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SoftMax Pro and are pre-read optimization options.

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Option one is microplate optimization, requiring you to have sample in the four

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corner wells of your microplate. This optimization wizard will then scan those four

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corner wells and spit out the optimal dimensions for the microplate and

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automatically input them into the software.

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You will also be able to visualize and choose the exact center of the well using a

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hairpin trigger to more accurately read your data and ensure that you're reading

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from the center of the well for that specific plate type.

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The Microplate Optimization Wizard is especially beneficial for six to 48 or

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384-well plates, where the dimensions vary from vendor to vendor.

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It can also be very beneficial if you're running 96-well half-area plates.

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This optimization wizard prevents you from having to surf the internet to find the

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specific dimensions for a lot and manually input them into the software.

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The next optimization feature we'll discuss is the read height adjustment

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optimization feature. This is going to be available in any of the read modes that

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use a PMT. The Read Height Adjustment Wizard requires you to have

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wells with your sample in them, where the optimization wizard will then optimize

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the height of the detector above the well.

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This optimization wizard is sensitive to volumetric changes.

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So if you ran the optimization wizard for one specific assay and then either

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reduced or increased the sample volume, you will need to run this optimization

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wizard again.

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The next shared feature between both instruments is the ability to add on a dual

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injector system at any point in which you have the instrument

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The dual injector module is housed in the right side of the instrument, as shown in

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the images to the right.

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It's a peristaltic pump that can move both forward and reverse.

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The dual injectors are enabled for all read modes and have a few unique features

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associated with them. One feature is overflow detection, where there's a sensor at

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the tip of the injector, where if a liquid or a fluid touches the tip of the

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injector, it will stop the assay from progressing to avoid overflowing the well and

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damaging the instrument and the assay.

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You also have a feature called Smart Inject, which will shake the plate while

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you're injecting into the well to ensure homogenous distribution of your substrate

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or your reagent. The dual injectors can dispense in volumes as low as one

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microliter and as high as the maximal allowable volume of your plate type, and

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within one microliter increments in between.

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It has a low dead volume of 10 microliters, and it achieves this by having the

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reverse peristaltic flow, returning any unused reagent back to its

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tubing infrastructure at the conclusion of the assay.

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The injectors are also going to dispense at a speed of 100 microliters per second

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and come with great tutorials for washing, priming, calibration, and tube

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replacement that will be available either on the touchscreen of the ID5e or within

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SoftMax Pro for the ID3S.

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These injectors are specifically tailored for flash luminescence assays such as

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dual luciferase reactions.

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We'll now move on to discuss new features that come on the ID Series instruments,

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which include an advanced shaking component and environmental control.

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We'll start by discussing the advanced shaking option.

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Both of these features are available on both of the instruments.

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The advanced shaking option is a feature that came to our plate readers through

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customer feedback from those wanting a more robust system for their microbial

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growth assays.

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The advanced shaking option will improve the robustness of the shaft that holds

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and shakes the plate within the plate reader.

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It also allows you to be a little bit more specific in terms of your speed,

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diameter, and shake mode within the instrument.

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This advanced shaking option is meant to maximize your microbial growth assays by

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enhancing the robustness of the reader.

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This is specifically for people who are doing continuous growth assays with

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continuous shaking over long periods of time, such as 24, 48, or 72

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hours.

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The shake option with advanced shaking allows you to adjust the shake diameter, has

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a configurable RPM speed, which is dependent on plate type, shake diameter, and

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shake mode, and allows you to be more specific in the total shaking cycle to

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better meet customer needs.

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When you have the advanced shaking option, you also still have the standard shake

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option. To initiate the advanced shake option, all you need to do is check a box

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within SoftMax Pro.

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The next new feature that comes on the ID Series units is the ability

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to have environmental control within your plate chamber.

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This is enabled by the SpectraMax AER or air exchange regulator, used as the

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gas mixer on the system. This should provide you with a walkaway solution for

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long-term lifestyle assays that maximize your lab performance.

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This is going to be compatible with both the ID3S and ID5e and can be

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added to the instrument at any time.

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The control of oxygen and CO2 is going to be software-driven, allowing you to

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change your oxygen within the plate chamber from ambient down

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to 1% within 0.1% increments, and the CO2

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from ambient to 15%, again in 0.1% increments.

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These values are traceable in the software and allow you to put

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them into the audit trail if you're in a GxP or compliant environment.

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In this slide, we just wanted to show you what the SoftMax Pro control interface

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looks like for environmental control.

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You will have in your instrument settings the ability to see what the set point is

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for your CO2 and O2 settings.

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When you click the gas control button, it will open up a window that allows you to

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turn the environmental control or the gas control on and dictate the

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percentage of either CO2, O2, or both that you want in the plate chamber.

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You're also able to use the gas control feature within our workflow editor to do

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more advanced protocol development within SoftMax Pro.

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You will also notice that in the settings information, it

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will give you the set points to the right with the rest of your acquisition

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settings for that specific assay.

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We'll use this slide to show you some data collected using the environmental

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control for three-day live-cell assays using U2OS cells.

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What this graph below is showing you that the performance of the environmental

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control is very similar to that of the incubator.

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The experimental info for this was the plate was using an IB seal to help with

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humidity control, where the plate reader was set at 37 degrees and CO2 set at

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5% in the SpectraMax AER.

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What you can see is the blue and the red are going to be the incubator and the

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environmental control, whereas the green is the use of no environmental control at

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all. The conclusion from this assay is that ATP levels measured with

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CellTiter-Glo demonstrate comparable levels for the incubator and the

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ID gas mixer, the ID Series gas mixer.

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The next piece of data we wanted to show you is just to show you successful

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real-time viability monitoring using the RealTime-Glo assay kit.

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What you're seeing here is cells plated at three different densities and tracked

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over 72 hours. This was done in the SpectraMax ID

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Series with an AER gas mixer to avoid the need of transferring the plate to an

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incubator after each read.

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This data shows successful viability assessment or successful growth across the

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72-hour period.

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We'll now move our conversation forward by talking about SoftMax Pro.

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SoftMax Pro is an industry-leading software that has both data acquisition and

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analysis all in one easy-to-use and refined package.

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When you purchase an ID3S or an ID5e, you will receive four perpetual

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licenses that you can apply to a PC to control the instrument with

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SoftMax Pro.

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What you're looking at now is the general interface for SoftMax Pro.

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Built within the Windows operating system and user interface, this SoftMax Pro

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interface is very easy to use with many one-click or one-touch

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accessibilities.

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Some benefits that we provide to our customers in SoftMax Pro include the protocol

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manager.

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The protocol manager has a protocol library of over 200 pre-populated protocols.

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These protocols are there to help you when you're ready to run an assay on our

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plate readers. You can use these protocols as a scaffolding or a foundation to

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build your own working protocols for future use.

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All of these protocols are going to be available to you upon installation of

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SoftMax Pro.

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One of the big benefits to SoftMax Pro is the fact that you can do data acquisition

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and analysis all within one easy workflow.

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What I want to show you now is how we can take an already created protocol and get

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to analyze data using that protocol.

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To do that, I'm going to open a protocol from my own personal protocol library.

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What you can see here is a template already applied to the protocol, having the

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different groups in different colors and duplicate columns.

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You can also see that we have our data layout already set in tables to give us some

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important pieces of information,

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and we have a standard curve that is waiting to be plotted.

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What I'm going to do is just copy and paste this data here as I'm in a simulated

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interface.

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And now you'll see that once we've collected the data, our tables will be filled.

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We'll have a lot of analyzed data and information.

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And when we go to our curve, you'll see that the curve is already plotted with a

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fit applied to all four of the graphs.

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That is to show you one easy way of using SoftMax Pro, by having a pre-written or

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pre-canned protocol, so that when you're ready, all you need to do is press play to

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collect data and have already analyzed data once the read is completed.

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The next thing that we'd like to show you within SoftMax Pro is how you can take

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collected raw data and easily get to an analyzed data set in just a few clicks and

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a few minutes. To do this, we'll access some pre-collected data.

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You're seeing two wavelengths collected here, one at 450 nanometers and one at 630

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nanometers. One of the things I can do to this data is subtract the two wavelengths

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with each other using the Reduction tab.

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This is where you can perform mathematical calculations to your raw data within the

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software.

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I'm going to go ahead and subtract the wavelengths from each other and then choose

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the Display tab to display my reduced value in bold below my raw

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wavelengths.

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You're also able to view your data in a color map

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and with the reduced number below the color map.

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Beyond data visualization, to plot into these tables and into the graph, you need

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to apply a template within SoftMax Pro.

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So to do this, we'll take our standards and quickly plot a standard curve.

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We'll go ahead and quickly plot our

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unknowns.

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And we'll quickly plot a plate blank.

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When you look at this and see what I'm doing, I just would like you guys to know

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that this is something that you'll be trained on how to do upon purchase of the

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reader. Once I click OK, you'll notice that my data changes a little bit to

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represent the background subtraction from our plate blank.

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When we scroll through the generated tables, you'll notice that there's a column

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for back calculation that has empty wells associated with it.

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To populate data in this column, we will need to apply a statistical fit to our

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standard curve. So let's go ahead and jump down to the standard curve and see what

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we observe.

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What you're seeing here is our data points plotted along the standard curve.

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If I want to apply a statistical fit, I can apply one of 22 different statistical

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fits into the data set.

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Once you apply your fit, if you scroll down, you'll get the logistical equation as

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well as the parameters that fit into the equation, your R squared, and in the case

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of a four-parameter logistic, the IC or EC50.

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These graphs are exportable. You can also save them, and you can add

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annotation as either text, image, or shape onto the graph.

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Another cool feature is you can right-click a data point and highlight the source

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for where that data is coming from.

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This is very beneficial to outlining or identifying why an error bar

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might be larger than the others.

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When we scroll through the tables now, you'll notice that the tables are fully

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populated

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and that now we have data that we can look at and observe.

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There is so much more to learn below the surface of SoftMax Pro, giving you the

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option to do many different forms of statistical or data analysis and customize how

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your data is output once you've collected it.

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That concludes the content portion of today's webinar.

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On this slide, you will see my name, title, and information, as well as information

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from Molecular Devices and our support team.

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Feel free to reach out should you have any questions or require any additional

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information about what we discussed today.