Please join Dr. Ferenc Borondics to hear about how cutting edge IR spectroscopic tools, such as the breakthrough technique of Optical Photothermal Infrared (O-PTIR) is being used by users from a wide range of application areas, many of these based on recent publications in high impact factor journals
Additionally, Dr Mustafa Kansiz, Director of Product Management and Marketing at Photothermal Spectroscopy Corp, will provide a brief technique overview together a range of application examples in this two part event.
A summary of the webinar includes;
Introduction to submicron O-PTIR and simultaneous Raman (IR+Raman) microscopy with application examples
Submicron IR (O-PTIR) applied to;
– Cultural heritage – View publication here
– Neuroscience – View publication here
– Kidney stones
– Hair analysis
– Polymer orientation
[Applause] Hi everybody and welcome to today’s webinar it’s been a while since we’ve been back i think we did the last one in late december we’re happy to be back and today we’ll be talking about submicron simultaneously threatened ramen and how it’s being used at multi-user facilities like synchrotrons
From applications from life sciences to cultural heritage all the way across to polymers uh and much much more today’s guest speaker will be dr frank veronics he’s the principal of the inline scientist at the sims beam line of the solay synchrotron just outside of paris a lovely place to be
A couple of casper’s housekeeping uh points in fact they’re really only one housekeeping point and that is uh any questions that you come up or have they come to mind throughout the webinar please type them into the chat box on your screen uh and when we get to the
End when we have time and we will have some time if we want to get through all of them of course anything we don’t get through we’ll get back to you via email so really without further ado since there’s so much to talk about i’m going to jump straight into it
So the outline today will be i’ll take you through uh the or some of the challenges and issues um that that people doing ftr and rama microscopy are facing today i’ll take you through how opti overcomes virtually all of the issues that you’re having some pain with i’ll take you through
Then a range of applications uh that people have published with in fact most of my examples are based on actual applications or rather i’m based in publications and that’ll be about the first half in fact a little bit less than half in fact and then the majority will be then
Um we’ve got frank burondix uh going through his experiences using this rptr technique at the survey synchrotron and what his users uh that cover a wide range of replication spaces i’ve been doing what they’ve been publishing on so uh i’m going to assume most of you are familiar with vibrational um
Spectroscopies of us to say that it’s all about functional groups and each peak of course is representative of a particular functional group and it’s been it’s been used for decades across a huge and wide range of application areas however it has been around for a while it is
Quite a mature technology uh and it is really at the limits its fundamental limits and primarily it’s all about spatial resolution because we’re using long wavelengths in the infra in in the infrared those traditional techniques such as the ftirs and some of these emerging q cells that employ long wavelengths are
Limited sort of 10 to 20 micron region other issues are that the best spectra are obtained in transmission rate but for that you need to cut them quite thin 5 to 20 microns at most but cutting things thinly is sometimes difficult or often even impossible
In those cases you may be using a micro atr such as the one pictured here but that requires contacts difficult to position there’s contamination concerns the crystal and or the sample can be damaged uh so there are some challenges there and that here’s an example of how
The sample surface can be damaged with an atr uh these systems often nearly always require liquid nitrogen cooling which is a bit of a hassle to handle uh but one of them you know what i consider the underappreciated issues with these sort of traditional fdir and qcl sort of direct microscopes
Is these dispersive and scatter art effects so imagine you have a thin film of a polymer that’s a pma thin film you measure that in transmission you get a lovely looking spectrum nice flat baseline symmetric peaks all looking good but if you take the exact same
Material and make them into a bead 15 b this example measure that in transmission well the spectrum now looks very different baseline offset you’ve got weird bass lines peaks are shifted and they’re split change the shape keep the material the same the space would look different yet
Again so really go to show how spectra can be very very much particle shape size and even surface roughness dependent uh in addition to any chemical differences so you’ve got to be very mindful of that when it comes to raman by far the biggest biggest issue is autofluorescence and that can often
Swamp the signal you can mitigate some of that by going to longer wavelengths but then you take a massive massive hit on sensitivity and that’s on on top of an already limited sensitivity that’s just inherent and fundamental to raman spectroscopy uh you can’t collect single frequency you must collect
Hyperspectrals which i’ll talk more about singular frequency imaging in the coming slides but you know full hyperspectral imaging can be relatively slow uh there’s power sensitive and power issues because it is a low sensitivity technique you’re typically wanting to put in as much power as possible and it
Ends up being a fine balance between as much power for best sensitivity versus minimizing or id having absolutely no sample damage and then there’s the issue of the raman spectrum themselves can be dependent on the excitation wavelengths this could be sort of sample related with particular resonances visible resonances that may be excited
In the sample for example dyes or inks for example can can have some strong resonances it could also be the substrate glass for example doesn’t work very well with 785 right um so all of those issues i’m guessing you know you probably have experienced some or most of those all of
Those have pretty much gone away completely when when dealing with optical photothermal infrared as a technique iptr is a pump probe optical spectroscopy technique where the pump is an infrared laser that’s the one that excites the sample the probe is the short wavelength typically a green 532
Or an infrared laser and it’s the probe and it’s through the pro beam that would detect uh the the effect of the absorbances input absorbances uh so with opti we’re delivering raman like spatial resolution but with the richness of of the infrared spectral region we we operate primarily in reflection mode
Although certain applications do require transmission but even in in reflection mode we end up generating transmission ftir transmission like or hdi or atr like spectra in reflection mode so none of it the typical distortions artifacts or fringes uh that you might get with ftio we’re not creating reflection mode it’s
Not contact and we’ve already mentioned that and the spatial resolution uh is actually independent of the wavelength so it’s constant across the entire wavelength how might this uh technique work i’ve got a little video to take you through it so it all starts with the uh microscope objective it’s a reflective cassegrain style
Through that we shine a pulsed infrared laser beam and being infrared it’s fairly abroad maybe around 10 microns at the same time we’re shining in our probe being the green that’s about 500 nanometers the infrared generates thermal expansion as it’s as it’s expanding and that thermal expansion
Changes the way the green light is reflected or scattered back right so as we’re tuning our infrared laser from one wavelength to the other we monitor the intensity of the green light reflected back and it’s from that that we calculate out what is essentially a pure infrared spectrum collected in reflection mode
So speaking of modes uh one of the simple modes is where you just point and shoot if that’s your field of view you can mark spots and you can collect spectra and each one of these spectra will come from about a half a micron spot size you can work in a rain
Mode where you can draw a line you can collect a line of spectra with a minimum step size of 15 nanometers or you can work in a traditional mapping mode where you draw a grid and you end up with a with a three-dimensional data cube i call this discovery mode because
That’s great for when you really don’t know what might be changing but if you do know what you change what might be changing i call this targeted uh targeted mode imaging you can employ for single frequency reading so rather than collecting the entire stack of images you can collect only certain
Discrete frequencies that’s of course much faster in this slide here i’m going to attempt to compare and contrast traditional ir and i’m going to lump uh ftir and some of these emerging emergency microscopy techniques into the one bucket when you compare that with rahman against a whole host of key microscope
Characteristics or attributes first one being spatial resolution and if you care about that typically raman is going to be your your choice in fred as we know traditional defense says poor special resolution if you weren’t worried about fluorescence of course rahman is poor with that and ir has notes and fluorescence issues
Spectral sensitivity ir will win that race and that kind of ties into speed of measurement as well uh when it comes to the extensive missile libraries or the spectral interpretability it’s important to to remember or if you don’t know to be made aware the fact that commercial libraries out there
Have uh about 10 times as many infrared libraries or infrared spectra in their libraries as they do rather so there’s a huge wealth and depth of databases out there in the infrared so i’m giving that a green and raman is much much less uh if you want to work in
Reflection right which is the easy mode of operating it’s kind of coin and shoe silver prep isn’t really so much of an issue ramen’s great for that ir is really poor when it comes to any reflection work water vapor can be an issue with ir not with ramen water
Solvents if you’re working with live cells for example traditional ir is a real pain for that ramen is great glass mostly works well with garment does not at all with infrared uh and this idea of having a spatial resolution being way back independent well traditional ir it’s not
At all but with ramen it is so if you look at that it’s no wonder it’s no surprise that most labs will have one of each instruments depending on the needs of the experiment you may go to one you may go to the other or you may even take the
Same sample and measure them on both instruments and then of course is the challenge of finding the exact same measurement spot well what we do with opt-ir is we take all of those and literally put them into one instrument so when i say you combine the best of both
Of traditional iron ramen into a single platform i actually mean that quite literally now at the heart i think i’ve mentioned the heart of this at the beginning of all of this is is the infrared light source and that’s the qc on this case so uh there are there
Have been some developments in those over the last year or so uh out of the traditional or our standard qsl it covers an 1800 to 950 and that’s done with three chips these three sort of mini lasers within the one box um if you’re interested in some of the
Higher wave number um areas you can you can opt for an additional opio laser that’s 3600 to 2700 uh my favorite uh most interesting things for me are these sort of more custom cuban technicians operations called custom anymore quite mainstream uh the it’s the ch chip in particular
I’m quite excited about the thousand and twenty seven hundred and you can add that as your fourth um chip because you can house up to four chips in these in these boxes and that gives you this range and that so that really covers the key functional groups uh in the midfield
Spectrum if you’re interested in the silent region you can perhaps replace the ch with the solid region chip or if you’re interested in the long long wave in the low wave number into the spectrum uh you can opt for your fourth chip to be that one in which case you
Can go down to 1800 uh your in terms of probe and the probe of course doubles as your ramen excitation laser you can opt for a 532 or a 785 uh it’s important to compare i think just to really appreciate that uh opti spectra are indeed very much compatible and comparable to
The decades of ftir history and libraries out there so in this slide i’m going to compare reflection sorry transmission are data that have been collected in transmission mode of thin films these are these are library spectra and i’m going to compare them to the same material that collected in reflection
Mode of a thick block probably some millimeters without ptire so this so first example is polypropylene the reds the fti are thin transmission reference and opti is our thick reflection measurement as you can see that’s a spot-on match same thing with polyethylene p-e-t nylon and polystyrene
Right and then the spectra here are in no way um treated transformed these aren’t literally raw spectra that you’ll get on the screen spatial resolution is something we talk a lot about but understand that’s some basic fundamentals here uh spatial resolution can be approximated by the rayleigh criteria
Uh so 0.6 times the wavelength in microns divided by the numerical aperture of the objective and for traditional ir and qcl type measurements this wavelength is actually variable hence if you plot this equation you get a curve out here in the low wave number into the spectrum which is the long wavelength and
Your spatial resolution is much worse and it gets better as you get to the short way they think uh but with opt-ir which is very similar to raman we have this we have a fixed um v probe wavelength in this case 532 so plug that into here that’s where there was a numerical
Aperture you end up with 416 nanometers and that’s constant throughout the entire wavelength i mean that’s where your up to 36 30 x improvement comes from okay but one of our most exciting features is the fact that we can really fit and run together so these have been two complements
Complementary techniques that have been rounded together often operating side by side but never together and certainly never simultaneously so now we can truly truly claimed same spot same sub micron resolution all that at the same time so it’s pretty useful to go through a very simple schematic of the instrument
So it all starts as i always say with the qcl later that is shown through our objective and focus down at the same time we introduce that green um propane co-lingually that’s also focused down to a tighter spot so then this is where that sort of photo thermal magic happens the reflected light
Comes back and goes onto a visible detector where our infrared signals are extracted uh but the magic or the really novelty here the simple novelty as well is the fact that we use a raman grade visible probing a protein laser so that means we’ve got raman scattered photons here all the
Time whether we like or not whether we end up even using them or not they’re there so we’ve taken advantage of the fact that rather than scattered photons are there by putting in a dichroic filter in here so that’s separating out those wavelengths shifted and only those wavelengths shifted photons and that’s
In the master arm spectrometer where we get out around the spectrum and those unshifted photons continue to the visual detector where we get our infrared beam and thread and rather and so in this in this fashion we end up and that’s how we get that simultaneity right so this really takes full
Advantage of the complementarity of ira rather it’s confirmatory as well as here i can confirm the raman uh and the realm converts io and vice versa okay so that’s really kind of the first half of my uh presentation in the sort of second half i’m going to take you
Through a quick road show i’m going to be pretty quick here because i’m pretty over over already but i want to show you some examples of our publications i think that’s what drives many people out there is all of the publications being normally first and speaking of publication they’re
Certainly very much on the rise and this is probably not the latest screenshot from our website we’ve had probably about five publications that have come out already in the last four weeks uh and perhaps the biggest publication was late last year and that was um in in science nanotechnology that has a
Massive impact back to 39 and this was actually a really really thorough study this is where they looked at how baby bottle teats or nipples depending on which country you call them how the steam sterilization which was always thought to be really safe actually isn’t it actually sheds a lot of silicon micro
Plastics micro particles uh you end up etching the surface uh and through the unique capabilities of opt they were already able to able to dig in to that and and look at the chemistry of of the etch surfaces versus the unetched surfaces uh they were even able to you know pull
Out small particles that were as little as i think these are sort of 600 nanometers that’s actually a really really good study i recommend anyone interested in couldn’t read it that one uh we also had a new one come out a few weeks ago um out of out of frank’s
Lab in fact or at least the users came by his lab uh this was a cultural heritage example looking at um metal and glass objects from a cultural heritage perspective and this is a novel result because doing this with a regular ir wouldn’t work you you can’t take uh
Such a rough material and then point an infrared beam at that and get interpretable spectra back but well you can with and so this is the specs you get out of it you see glass piece you see four main carbonates and in this case they’ve done a small two-dimensional hyperspherical mapping exercise
They’ve generated some maps single point spectra from these points you can see and the spectral here are pretty much uncorrected this is what you see you don’t get these sort of dispersive scattering artifacts another hot off the press paper um is one from roth readers group and university houston where he couples uh
Some polarization rotation to look at collagen orientation within tissues as an added channel of inflammation in addition to the chemical distribution it’s also uh you can get some regardless of information from the orientation and really up until now uh ftir has been used a lot in some of these emerging qcl techniques
But they’re always limited in their resolution you can see between a between a and b here there’s a huge difference in resolution between fdir and obtainer and if i can zoom in we can actually look at these individual one micron collagen fibers which fdi cannot see but you can see with opt-ir right
And then on this far right column here they’ve looked at the amy2 to a mid-1 ratios as a function of different polarization angles and this is what the spectral look like as well so we’ve got glass and this is actually on glass and that’s another real benefit of this technique is that you
Can use glass as a substrate and when you’re interested in examine one enamel two ratios it’s really quite simple through it’s all software controls polarization ratios ratio changes um one from probably 2020 now another high impact factor journal looking at neurons i’m looking at protein secondary structure differences
In neurons um this is like i like this figure because it’s really showing the incredible resolution even down sort of 282 nanometers and how between those you actually get real chemical differences indicating in this case more beta sheet structures in one at one of those points uh very quickly this was a polymer
Example looking at a biodegradable polymer laminate pla and a pha layer laminated interested at what’s happening at the surface at the interface rather so we can do a simple uh one-dimensional line line array or a linear binary of that collecting opt-ir and ram spectra simultaneously if we just focus on the
Opt-ir data uh across that interface all of these spectra here are separated by only a hundred nanometers even with a hundred nanometer steps you see strong chemical differences um taking the single frequencies of those at 17 25 and 1760 we get a an image that you’d expect
But taking uh a line profile across here uh we can see how sharp that edge is around 327 and that one don’t show it here through some of the 2d correlation data analysis have worked out that the reason why these two layers are actually quite compatible when they’re
Expected not to be is the fact that the pha at this interface when in contact with dna is actually a lot more amorphous immense and thus it’s a lot more compatible with the already quite amorphous dla layer this is a really good example to show of where when ramen fails opti will not
Cannot so this is a forensic application looking at a paint chip cross section from a sort of vehicle accident i think it was uh so single point specs were taken in these red blue and green dots at these three different layers and the ramen this is the simultaneously
Collected ramen uh so if there’s fluorescence of course even the the raman channel on our instrument will still start through fluorescence as it did in this case with the red and the green the blue uh works but even when the even the red and green spots the optics vector are completely unaffected
By fluorescence fibers are an interesting example because they’re actually quite difficult with traditional ir and gcl measurements in this case we’ve been able to collect high quality spectra again without any data processing this is these are raw spectra as you can see evolving on the screen and along the length of this
Fiber we can see that there are additives that are changing along the length but whether it’s a 20 micron fiber or an 800 nanometer fiber the spectra still look like interpretable recognizable spectra very quickly this is a phase dispersion i like this one because you can zoom in
And do a really high resolution step image you can see some incredibly small features um and spectra on and off these hotspots look like as you might expect and you roll this this is a ratio which by the way it’s 1759 and 17 33 and we can see where the ratio
Where the contrast in these images are originated from if we zoom through that one so even some of these small ones can be in the sort of a couple hundred odd nanometer region um cells in water are actually relatively easy with this technique this is a cheek cell in water example that
Cheek cell uh placed into the calcium fluoride slide the calcium the right coverslip on top uh when i collected this is actually one of the one that i did myself when i clicked some single point spectra from around the cell i saw that there were some lipid features
There was some nucleic acid feature of course protein is quite ubiquitous and when i collect a single frequency image on those three wavelengths alone and combine them together in this rgb overlay low in the hole it actually looks like stunning almost fluorescent-like image where you can see very small lipid droplets and nucleic
Acid of course concentrated in the center some of these are sort of half a micron in size um in terms of particular examples especially in the context of microplastics this i think is a really powerful example showing of how 500 nanometer polystyrene beads are measured in seconds simultaneously within reading ramen all
Right so here this is this is in red we have the opti spectrum in green you should label these in green we have these we have this is the ramen so again half a micron bead measured in seconds but whether it’s a half a micron bead or a cluster
Of of two micron beads the specs look the same there are no dispersive scattering artifacts so opti generates repeatable spectra that are independent of particle shape and size or certain samples okay study out of the the andy old group in michigan looking at atmospheric particles where he used both infrared and raman
Kathy gough out of the university of manitoba in canada looked at collagen orientation again taking advantage of the inherent polarized nature of two cells and rotating the polarization um i’m going to quickly go through these these these are the half micron collagen fibrils um where the amount will name me two ratios that
Change quite effectively depending on the polarization orientation through those uh these were um cancerous and normal cells on glass that were able to differentiate um based solely on their open tire spectra again on glass so this is very clinically translatable and just to give you a sense of the
Quality of the spectrum they obtained so these these are single scans about one second each you you see some glass supports because they’re on glass the rest of the spectrum is super high quality half micron spot no processing whatsoever and you can do lipid chain length images with take
These ratios or the proteins uh and obtain some pretty stunning images i think um live cell imaging this is a paper out of kindergarten group in manchester where they compared fixed and live cells uh doing simultaneous ir and ramen and of course this sort of work is quite difficult with regular fti because
They’re going to use the seven micron path themselves incredibly difficult to work with but here they use this sort of upside down cell configuration where the cell is actually sitting upside down so the ir light hits it first doesn’t have to go through all that water uh and then we
Measure the probe being actually in transmission mode so that was a study that worked out quite well i think it’s my final example looking at back single bacteria up until now the only chance of doing single bacteria would have been with around the microscope but now in the
Infrared you can do single bacteria as well and they have actually now upgraded to raman so they can do infrared and raman obviously the bacteria but in this example they had uh well they they fed the bacteria in different isotopes of carbon and nitrogen uh that greatly shifts down one one and
A by two ratios uh and threw some citizens together the mouse of that a lot can be gleaned about the metabolic pathways of these of these cells uh it’s an example of a e coli cell that’s been measured imaged at 50 nanometer step sizes just a simple a1 image and even an intracellular
Spectra from a single bacterium uh shown here as well and this was a starman region chip so we’ve actually got some cd absorbances to these e coli were grown in in heavy water partially heavy water and i think my final example here is of a simultaneous iron raman spectrum of a
Single e coli bacteria so you can see the collection time here is about a minute ir spectra really high quality ramen decent quality but of course ramen is always will always lag behind when it comes to seedling noise but for the first time now we can collect infrared and raman from single bacteria
Which i think is pretty exciting i’m going to quickly move on to my little takeaways my conclusions by combining infrared with opti we can get sub micron inflation so you see a lot more detail it’s non contact it’s reflection mode so there’s no cross combination the preparation is easy
No dispersive scattering artifacts so the spectra are incentive insensitive to sample shape and size and only sensitive to the chemistry which is really of course what we care about at the end of the day a little to our subway prep new new sale options uh expanding the range of applications
And we’ve also recently added the ability to have a fluorescence module on top so you can actually do co-located by ptir and raman based off fluorescence images and i’ll finish off by saying ir pass raman it’s all about the same spot at the same time saying resolution
All right everybody thank you and i’ve just come to the end of my first half of this talk without any further ado i’ll pass over to farink for as our main guest for the second half let’s talk to see what he’s been doing at the solaire synchrotron with this tool
Very over to you thank you very much mustafa and thanks for the very nice talk and the introductions on the on the technique and everything and so today i’m going to talk about opt-ir in multi-user facilities and as you can see here i have two logos one is the
Institute where i work is the sole synchrotron in france and the other one is the logo of my beamline which is the smith beamline which basically works with infrared spectrum microscopy so one thing i want to point out here is that if you want to look at the
Slides that i’m presenting today that they are available under this link so don’t make any notes just just go to this bitly slash smith opt-ir 22 link and then you can see what you are going to see right now so multi-user facilities uh in our case
This is a synchrotron and i prepared one or two slides about what actually a synchrotron is so synchrotrons are large-scale facilities last case science facilities which are extremely expensive so we operate 24 7 and we operate in a proposal based access mode so basically you have to write a peer-reviewed proposal and then
Depending on the peer review process you either get measurement time or not and then you can come and do your experiments at a synchrotron so the synchrotrons are particle accelerators that are producing light and they are very much tuned for x-rays but as you will see they also have infrared beam lines
So just a few words here synchrotrons they are made up of different subunits one is a linear accelerator which produces electrons typically and they accelerate them to a certain energy and then these electrons are injected into a booster synchrotron which increases the energy to to some value
And then after the booster we inject them into a storage ring where they are circulating and circulating and in each turn each red or orange structure here which are magnets they produce light and these are typically as i said x-rays and where they produce the light we couple them into a specific
Let’s say spectrometer which we call a beam line where you can do different types of experiments so soleil is a 2.75 giga electron volt energy third generation synchrotron we are very close to paris about 30 kilometers and we are on the paris acclai campus um which is one of the biggest
Universities now in uh in france currently we are operating 27 x-ray beam lines and two infrared beam lines at the same time so you can imagine that we are doing 29 experiments at any given time and the two infrared beam lines are called l and there’s miss and i said
Smith is a my beam line so i’m going to show you just a map about infrared beam lines around the world so here on this map you can see the little markers these are the synchrotrons currently operating all around the world and the red ones are the ones which have
Infrared beam lines in in the facility so you can see that synchrotron infrared is an important uh contributor to the synchrotron world although again synchrotrons are tuned for x-rays and and we are contributing with the with the worldwide presence we have a very nice infrared user community
And if we uh come to my beamline which is uh again the smith beamline in the solar synchrotron we have uh been collecting and building our instrument park for for uh quite some time now and so here you can see an overview of our capabilities in terms of
Instrumentation so we can go basically from a few tens of nanometers through a few hundreds of nanometers to microns to even centimeters uh infrared spectrum microscopy and of course each one of these instruments have their own particularities and their own capabilities but today we are going to talk about the
Photo thermal infrared and its combinations with all kinds of all kinds of instruments but first i want to point to one important aspect that in microscopy light cannot be really focused into an infinitely small point but rather into a let’s say a pattern presented here on
The right and you can see this in black and white but if i invert the figure then you see that there are basically concentric circles uh around the center point and we call this pattern the area function and with this area function we can define the really diffraction limit which i’m
Going to show you on the next slide but basically one important aspect here is that there is a very strong source limitation because thermal sources that are found in any laboratory-based infrared microscope or synchrotron and laser sources behave different differently and the point here is that with synchrotron and laser sources we can
Reach diffraction limited resolution so what this means is that if we take this area function here you can see it in two dimension and on these figures you can see it in one dimension so basically just a slice of the image we can define a specific limit which is the rayleigh limit
Where the maximum of the of one area function coincides with the with the position of the first ring of the of the secondary function and we call this the rayleigh limit now if we are our area functions or objects for that matter are further away from each other than this real limit
Then we can call this resolved and if they are closer then we can call this not resolved so diffraction below the diffraction limit and uh in typical absorption spectroscopy this is pretty much what we can do if the objects are too close to each other then we cannot distinguish them they
Have to be at a certain distance and in the in the infrared this is typically several microns or several let’s say tens of microns or below 10 micrometer in the in the mid infrared now the the opt-ir technique uh applies a very nice trick to overcome this diffraction limit
And as you have heard from mustafa the the system basically uses two lasers to to do the measurements there is one laser which is an ir laser this we can consider it as a pump laser and a visible laser which we can which we can use for probing the effect of the
Infrared illumination so in principle actually this is a diffraction limited system but since our excitation is in the infrared and our detection is indivisible in our case it’s a green laser you can see that the the infrared diffraction limit is bypassed by several times so instead of having let’s say a 10
Micrometer spot we have a 500 nanometer spot because that’s typically the wavelength of the green laser so opti our spatial resolution goes from let’s say 4 to 10 micron to 0.5 micron which is a huge improvement and there is an additional benefit that typically in absorption spectroscopy we are limited uh
By the wavelength which also means that at 4 micron wavelength we have 4 micron resolution and at 10 micron wavelength we have 10 micron resolution but in opti this 505 nanometer resolution doesn’t change throughout the whole spectral range so that’s a very nice aspect of the technique um
As you’ve seen uh on my one of my previous slides where i was talking about the instrumentation of the beam line you can combine uh with quite a few instruments and i’m going to show you a few examples of combinations so typically what we would want to achieve is high spatial resolution
Large spectral bandwidth and lots of information content and of course everybody would like to have everything and if we can find the the cross section of the middle then then the study that that is in progress is is probably benefiting the most of all kinds of instrumentation
So i’m going to talk about how to combine mirage with the synchrotron ir with fdir imaging roman which you’ve already heard about from mustafa with x-ray fluorescence from the synchrotron x-rays confocal fluorescence scanning afm techniques and even microct so let’s uh dive in and start with the with biology and first i’m
Going to talk about alzheimer’s research which the first example is going to be with synchrotron and and the opt-ir combination this is the work from oksana clementieva from the lund university in sweden who we are collaborating with for many years now and so the point here is that dementia has a huge social
Impact and if you want to imagine the numbers every three seconds there’s a new diagnosis of dementia in the world and the the projection is that by 2030 this is going to be two trillion u.s dollars cost for the treatments so if we can mitigate these these cases or we can
Understand better the the processes underlying the the disease it’s going to be a huge economic impact and a positive one and so here we have used opt-ir and syncroton ir to image neurons which were grown to have expression of beta amyloid plaques which are responsible for the alzheimer
The development of the isomer so here on the left you see the optical image of a neuron and on the right you can see two single wavelength images one at 16 30 and then at 1650 wave numbers and one of them is as the alpha helix and the other one is the the
The beta amyloid signature so we can see that there are clear differences between the two images and the ratio of the two images can really describe where the beta amyloid is accumulating in the in these neurons so this is the the data that opitaria provided and then we have been verifying this
With the synchrotron infrared which is not as high resolution but provides extremely nice uh um signal to noise ratio at even even at the at the single cell limit and the databases are really well known and the the description of the the beta amyloids and alzheimer has been well established
So what we could see that indeed uh our opt-ir data can show the beta amyloids and we could we could really nicely verify this with the with the synchrotron uh infrared data and we published this in advanced science in 2020 now after this point we were trying to
Dive in even more to this to this problematic of alzheimer and we tried to combine other synchrotron techniques with the opt-ir and oxana got beam time at the at the beam line called nanoscopium in soleil which is an x-ray fluorescence uh mapping beam line with very high resolution so typically about few
Hundreds of nanometers but at some cases even higher so the the length scales are very nicely compatible with opti ir so what we what we basically did we took uh opti our data i mean optical images first we took synchrotron x-ray fluorescence data then the infrared data and then we combine these
To really understand the chemistry of the samples from two sides and the the two sides are that infrared can really provide information about the beta amyloid and the and the vibrational signatures of different molecules and the x-rays they can really nicely follow heavy metals so and not not only the the location of
These heavy metals but also the oxidation states of different different metals so therefore we can we can infer let’s say oxidative stress and the accumulation of amyloid data so if we plot all these together put all these data together then we can very nicely show the the protein signatures
Inside these neurons here is an optical image here is the the ir image and then we can mask this and match the same neuron with the with the x-ray beam line and then see where the iron for example is accumulating or where the iron and oxidized lipids are co-localized
And or even iron and beta sheet structures so this is my my point here that this is an extremely powerful approach where you can understand the elemental speciation and the chemical uh constitution of neurons then uh another combination of uh of opt-ir with with another infrared technique was uh was to
See if we can see even uh higher details within these neurons so you already know about opt-ir here we are using an ir excitation and visible sampling and the resolution is typically on the order of 500 nanometer however if we uh exchange the green laser in our case to an afm tip
Then we can still use ir beam to probe to to excite our sample and we can get the the response from this afm tip which is extremely sharp so now instead of 500 nanometers we can go to 20 nanometer resolution and we can be extremely surface sensitive as well so
These two techniques are different have the different probing depths and this work was uh again in collaboration with oxana clementiev and raul freitas from the from the brazilian synchrotron and i’m just going to give you a highlight here about the results so again on the on the left
This is what we have previously also seen in neurons we can very nicely record spectra of the of the protein structures and the image where the the amyloid beta is accumulating in or not accumulating in cases of wild type neurons and then with the transgenic neurons we can see the increase of
Amyloid beta and then we could put all these uh cells these that express amyloid beta structures into the scanning microscope the afn microscope that combines infrared and afm and you could you could see again the same type of uh of data here this is a larger picture here you
Can see that the scale bar is 500 nanometers and we have extremely high resolution data where we can also record spectra and again see the increase and the change of the of the amyloid beta and the alpha helix uh structures so so this was a very nice because both
Techniques are below the infrared diffraction limit they have both high resolution high spatial resolution but we could even discover heterogeneity at the 20 nanometer length so that was a that was very nice study if we consider other biological systems i have a couple of other examples one is uh on
Biomineralization which basically occurs all around the human body and it’s usually not a favorable process for example if we think about kidney stones many many people are suffering from this kind of disease and very often the chemistry of these biomineralized stones is very important and it can
Determine the treatment that has to be followed so the problem of course is that these these diseases are hard to measure for multiple issues in ftir we can have light scattering i mean regular absorption spectroscopy we can have light scattering and in roman spectroscopy which is higher resolution
The samples can be extremely light sensitive so so it’s a tricky type of sample to measure but opti-ir can also address this question and in this case what we did we use the mouse model which expresses an abcc6 mutation which basically leads to deficient deficiency in pyrophosphate that is a
Key inhibitor for calcium phosphate crystallization so what this results in is that if we have this mutation then we can have some some calcium deposits but if we add vitamin d then this calcium deposits even further increase and so the the colleagues of dominic bazaar wanted to understand this kind of
Processes and see really the the chemical spectroscopy aspect of this kind of images that they could they could do with the just stained sections so here they combined the scanning electron microscopy energy dispersion dispersive x-rays and and mirage and they could really nicely see from after locating on the optical image they
Could nicely image the deposits in the scm they could really measure the elemental composition so they can verify that indeed these deposits are largely made of calcium and then after taking it out from the asean they could drill in further with the with them with the mirage
And see how the calcium the phosphate basically accumulates in these these deposits so that was also a very interesting study on biomineralization and they keep pursuing this uh this line and they are working on other kidney stones and mineralization processes um for finishing the biology part i have
Basically just one slide for you here on on cosmetology or cosmetics and this is the work of one of my colleagues here at the smith beamline christophe sonde who is uh who’s studying hair for many years now and so he has uh recently discovered that that hair is although it’s quite well studied
Already can still present some kind of surprises and so he combined the two-dimensional hyperspectral ftir imaging synchrotron ftir and the opt-ir and so what he discovered that in the medullas which is basically the center part of the of the hair uh sometimes we can see unexpected uh chemistry and so what he saw
Is uh basically steroids can accumulate inside the medullas so you can see that here the blue spectrum is a typical metal spectrum and the purple one or magenta one is a calcium steroid from a database and moreover he could also match different type of steroids so different
Chain length uh or organic chain length steroids within the within the hair and these two actually all these spectra that you see here they are recorded in the synchrotron uh what uh what the opti reveals is even higher heterogeneity at the submicron level and i’m not going to show any of
The figures here because uh this has not been published yet so so we cannot uh cannot show the actual maps but uh stay tuned and then you will see how this is unfolding even further at the higher resolution limit uh if we shift gears a little bit i i
Would like to show you uh by highlighting uh other fields how versatile the opti technique is and so we have some colleagues who’ve been working on cultural heritage and art conservation so what i’m going to show here is the work of victoria beltran from the university of antwerp and in this study
They were measuring uh samples from a van gogh painting this which you see on the right here so the peculiarity of these paintings is that uh sometimes the the pigments that van gogh used and actually some of his contemporaries as well were not so stable so what i mean by
This is that if you for example look at the side of this painting and you can see that the pink is much pinker than on the front and the reason is that the the side was in the frame and obviously the front was exposed to light and the environment
So the protected part retained much more of the color that than the exposed part and this is a this is a problem if you have a van gogh that you probably bought for a few hundred million euros and it’s simply just fading away so it’s a very important thing to
Understand the chemistry and the the processes behind this so what is happening it’s very clear that it’s a photo damage of the pigments and the result is that the colors are disappearing so what you see here is a is a the sample so we didn’t have the actual
Painting under the microscope we just had some chips which were on the order of 100 micrometers and then we we section these to make a nice surface and again we we used two techniques synchrotron-based ftir and opt-ir to understand the chemistry inside the inside these pigments so first i’m going to show the
Synchrotron ir mapping here um what you can see is that in this section we could very nicely identify all the constituents basically so we could see epoxy which we used for the embedding proteins inside the structure so it’s most likely some kind of uh carrier that they disperse the pigments
In we could see cellulose from the canvas the oil for the paint some minerals and lead white as well which was uh which was also a pigment for for white color and we could see we could record the the spectra of all these um constituents the paper is uh is now out
It was published last year and it was deemed as one of these hot papers in the anger and academy and after doing the synchrotron mapping we of course turned into the higher resolution opti-ir technique and now we have been concentrating on a much much smaller area at a much higher resolution
And again we can see still see the optical image of these sections the sections basically and then by by mapping out uh the different uh infrared absorption peaks we can identify with the infrared where these components are located so this was very nice and and confirming the the synchrotron ir results but very
Importantly um so we could also record the the spectra with the op tire very importantly we could match some of the spectra from the from the high resolution maps with the geranium lake reference so these are the pink pigments that are basically light sensitive and and this is a very important Step towards understanding the the photochemistry of these these pigments and of course eventually protecting the paintings from fading away between between the creation and the and as time passes so another yet another field is a chemistry the application of opi there in chemistry and here i’m going to talk about uh high resolution
Three-dimensional molecular orientation and this is the work of thomas robel from the solaris synchrotron who we are collaborating with and so just a few words to to better present this about the light polarization so if we have non-polarized light and we put a molecule into this this light beam if
The molecule orientation is like the the red arrow then it’s going to absorb mostly from the polarization that falls into this direction if it’s in the orientation of this blue arrow then it will prefer absorbing the the blue part of the of the beam the blue polarization
And if we translate this into uh a polarized case which is actually the opt-ir then what we we can use this for if we have a polarized laser then we can put molecules in different orientations and if the molecules are in the red orientation then they will not
Absorb if they are in the blue then they absorb maximum and if they are in between then they will absorb some so we can very easily get two dimensional orientation information from just just a simple measurement but we can go one step further and so if we use uh instead of just one
Uh dipole moment that that i was showing you in the in the previous slide we use several bonds let’s say two bonds that are mostly perpendicular to each other and we use four angles basically four measurements then instead of just a two-dimensional orientation map we can calculate the three-dimensional orientation of
These molecules and here’s a pretty complex scheme of how to do this i think it was yes it was this paper that that introduced this method but the bottom line is that we need to make four measurements at four different polarizations and then we can calculate the orientation maps of our polymers
So let’s see this is how the the results look like i’m not going to go into very many details but i want to point this out that it’s a it’s a new approach for chemists to describe uh polymers and here in our study we actually combined again opt-ir ftir imaging with a
Two-dimensional detector and roman microscopy as well so here on the right you can see that we have the two dipole or transition moments because in in the case of roman we are talking about polarizability but the the technique is equally applicable to this as well and we could very very nicely
Calculate from the fdir from the opt-ir the orientation vectors of these these molecules and you can you can see this it is published already on the chem archive and hopefully it will be soon uh fully published in germany so i came to my conclusion which is pretty simple in this case i wanted
To show you that opti is an extremely powerful technique and i think especially in combination with other modalities like synchrotron ir ft ir imaging raman synchrotron x-rays and there are many advantages for uh for opt-ir the sample preparation is quite simple and this is this is often um uh
Factor that that we need to consider so i said in the beginning that you can you can see my presentation but you can also go to this other link where you can see all the papers that are published from the beamline and you know dig further
And and enjoy the science that we are producing i just have two more slides to add here if you are interested to work with us either with the with our mirage or even the combination of the synchrotron techniques then as i mentioned in the beginning soleil has a proposal cycle which is two
Times a year typically february and september and i think the next deadline is going to be around september 15th so please visit the solei website and then send in a proposal it’s it’s quite easy and yet another teaser for you uh here there is a very nice study that that you can see
This is not opti but it is infrared so you can see what we are working on typically on many other groups and this in this case it’s uh it’s space samples so this is all i i would want to say about opti and if you have any questions
I will be happy to answer them thank you ferry for that really exciting and interesting and very broad talk as well i suppose that was the point of this webinar now i’ve been sitting here in the background watching lots of questions flying in i’m also conscious of time so
We’ll ask as many as we can and we’ll see how we how we go for time the very end first one i would say i’m going to point your way says could you comment on the summer preparation requirements especially for the combination of different techniques please
Yeah so um as i said in the in my my last uh conclusion slide slide um for opt-ir sample preparation is quite simple i we rarely have to uh do uh you know suffer uh so basically we if we have just any kind of thin sections for biological samples or cells
Then we can directly use it for measurements typically for combination techniques the requirement the stronger requirement is from the other side so let’s say if we are doing ftir the samples have to be thin enough for trans to to be able to transmit the light or they have to be
Reflective to to reflect the ir or often for x-rays they have the specific requirements for special substrates and and so on but the opti itself is quite quite an easy technique for sample preparation okay uh next one i’ll send your way as well theory so it says how comparable
Are the ftir and opti spectra when working on biological samples themselves so i will probably refer back to the beta amyloid studies so i think i don’t know if if you are very familiar with the with the bio biospectroscopy but if you just look at this figure
I mean it looks very nice and and it’s uh it’s quite comparable to normal ftir it happens sometimes if especially in the in the case of the hair study that we we could see that uh because of the averaging of of normal ftir we can have some
Surprises but this is not coming from the actual um uh technique differences it’s coming it is coming from from the heterogeneity difference so let’s say the fdir averages over a large spot and then it looks like a normal biospectrum and when we look at it with the with opt-ir then we
Can have a surprise and that just because because for example there is a tiny calcium steroid grain right where we are measuring and then it of course looks very different from a normal bio spectrum actually if i may even add a little bit to that very if i may uh it’s important
To also remember that um with opti we’re typically measuring most biologicals in reflection mode so there’s a certain uh simplicity and ease of sampling that comes with it and even though we measure opti and reflection mode the spectra that we acquire are completely comparable to those that have been collected with
Fti are saying transmission mode or with atr mode um next one i think i’ll take on my own actually this one says how thick or thin can samples be are there any limits and make me a fairy feel free to jump in as well uh but you know i know in our
Applications lab we’ve gone as thin as something like 50 60 nanometers perhaps and as for thickness well there is really no limit uh so that again one of the the nice aspects of working in this uh reflection mode uh we thickness isn’t a concern so whether you’re working with say 100 nanometer thin
Section or a or a centimeter thick section it really doesn’t matter it’s only the top uh few microns that we’ll be sampling from so from that sense you know whether it’s a thin slice or a thick slice really doesn’t matter um yeah anything to add from your side of the theory
No no i think you covered it pretty well the the samples that we have measured were the dried uh neurons and where they are really thin then they are a couple of hundred or even just a hundred nanometers thick so yeah so it’s quite accurate all right um this one i’ll throw
Your wafering um is it possible to measure in liquids and live cells uh so i yeah so the answer is yes uh we have uh tried liquids already and i think you have tried the live cells right yes i have and there’s some exciting stuff that’ll be coming out um in the coming
Couple of months so i may tease the audience with a little bit of that and i’ll just suffice to say that live cells uh will be extremely easy to measure with the water dipping objective i’ll say that much but they’ll be coming out in a couple of months
Um okay and this one i’ll take as well this may be the last one i think yeah this will be the last one uh what is the total laser power and actually i know there you’ve done a lot of work on this as well so of course it depends there’s there’s two
Lasers of course there’s the infrared and the visible um probe laser on the infrared side depending on where you are in the spectrum it could be as much as say quite a few milliwatts to some tens of milliwatts of power and of course all the power is very controllable in the software
And like with any sort of laser-based system you do need to be mindful of power and you will just adjust it accordingly for particular sample needs on the probe side this is something that i don’t think i covered actually i always forget to do this for those samples that are particularly
Sensitive to green light the 532 which by the way is probably outputting by the time it reached the sample you’re probably looking at many tens of milliwatts maybe several tens of many watts but for those samples that are very sensitive to probe green light we can run that down to few tens of
Microwatts of power and we can do that without really sacrificing sensitivity which is in which is in contrast to raman where if you had to lower the laser power down you also suffer correspondingly or proportionally a drop in signal noise and with opti we actually switch detectors we go from a standard silicon
Photodiode we switched to an avalanche photodiode detector an apd which is a great detector but it only operates well in very low light conditions and is ultra ultra sensitive so when we switch to that low level low light level detector we get signal noise as good as uh standard detector though we operate
You know assuming that you can operate as low or tens of of microwatts and and and i know fairy you guys use the apg a lot in fact probably more than i do yeah i think we really prefer this uh this sensitive detector because it allows us to really decrease the
The light intensity okay all right i think um we’ll have to wrap this up now so uh ferry thanks again for your time i know um you’ve been amazing with with her you know giving various users access to the lab and that’s now being reflected in the number
Of publications coming out of your lab that are based on i know ptire so it’s good for you it’s good for us um it’s good for staying in the community and so again thank you for your time uh well it says for the audience members of
Course thank you guys for logging in and listening in this webinar is uh being recorded and will be available on our website we’ll send the link out within some days and feel free to send that link on to friends and colleagues and with that i’ll say goodbye everybody thank you [Applause] You