 laser always sounds very exciting how are you going to apply it ready we're going to learn in this talk now Dr. Matthias Koch is a biological physicist and on the side he also was interested in building gardens whereas PhD he was applying lots of different technologies for laser spectroscopy he's also interested in compilers biology he also dances Wendy hub please welcome welcome to this Congress and here the stage is free for Dr. Matthias Koch hello hi Matthias Koch I would like to first give a quick introduction have a PhD in biophysics I was using this technology for my PhD thesis while I was studying I was thinking what is happening today and I was thinking and it would be better if I investigated this myself and here I am with this topic what happens when we're shining this in we have a lamp here and first we're going to look for an overview of what is happening here sometimes it happens that the molecule after the excitation drops back to another band and so maybe and maybe there's a difference between the absorbed photon and the radiated photon so maybe if we shine a red blue laser into into a solution we also get a green and red light out so and so the first excited state if we sometimes the photon then gets absorbed and there's an interaction between the molecule and the light and this is called the the Raymond Stokes radiation and and sometimes it is lifted to another vibrational state and and it and it goes back without radiation into the low state of the first excited state and then there are many possible state changes for the lower band for the electronic base state and this means that the radiation is rather broadband and because of the first first state that doesn't radiate and so the state the frequency of the radiated photon doesn't really depend on the laser that you shine into it so now we look at the spectrum and the wavelength scale shows the rainbow from violet to red and that's a wavelength of 400 to around 700 nanometers and the sensitivity of eyes at the edges is relatively low and the photon energy is between 1.8 electron volts to 3 electron volts at the violet and so this setup we have a glass and there are algae in it and so it's liquid in the liquid and it's a special algae and and it can be used for creating dyes and in this glass there's a plexip on a perspex plate and there are lamps in it that are used for calibrating the spectrometer and below the perspex plate is a mixer and the culture to to mix it so that each individual molecule will only come into contact with the laser very briefly so doesn't isn't changed by that and and there's also a fiber bundle and to shine the laser into into the solution and there are different other fibers which pick up the light and send it to the spectrometer and we have a blue laser with 473 nanometers wavelength and 50 milliwatts and this laser is a it's a single mode laser and has a very narrow band of only 12 picometers and then we have a spectrometer which also has a CCD camera and we see the broad back from the spectrometer in this case without the low pass filter and this very strong signal is the Rayleigh diff Rayleigh diffraction of the and in red we can also see some artifacts that also across by missing the low pass edge filter and there's something between 550 and 600 nanometers it's also an artifact and the inner surfaces are not perfect in the spectrometer and part of the light is going to other directions but in a very strong signal there also can be relatively strong artifacts so we have this low pass edge filter and the Rayleigh peak and the little artifact peak are now gone and the strong signal that we see now between 650 and 750 nanometers is the chlorophyll fluorescence chlorophyll fluorescence is from the green in the leaves and absorbs blue and red light and and also the low red and here's the same spectrum with a logarithmic intensity scale and the chlorophyll peak is in green and we see in between 550 and 600 nanometers there's a little bump this is the spectral line of water and we can see that the line is much smaller than the chlorophyll fluorescence and I marked this one little peak and it's a permanent line of the carotoid and this is the place where the filter becomes transmissive the calibration for the calibration we have these little neon lamps and it lights up in orange but it has also lines in the green blue range and they can use them for calibration if there are many there are many ways to calibrate the spectrometer but the little neon lamp is a very simple way to do that neon has very very many lines so we can so we can use them to calibrate it and here we see that again and now the chlorophyll fluorescence has been cut off so you can see the Raymond lines much better and the strong signal here from the water at 560 nanometers and we can also see the neon lamps so the position of the spectral lines can be compared to the published values and we see that it doesn't fit completely but it's quite close and with a correction of 5.25 nanometers then we have the exact position of the neon of the neon lines the small range around 500 nanometers has another neon spectral lines and it shows which range the spectrometer can normally record and this is the very wide range that the spectrometer can and this is to show the total spectrum and to create that it took about half an hour to record this total spectrum and here are the Raymond lines and there's a shift compared to the excitation and in the Raymond spectroscopy we can sometimes use a scale of the so called relative wave number and it's a very strange unit but it is proportional to the and it is created from the reciprocal reciprocal from the two wave lengths and here we have cut off the Raymond line and is the Raymond spectrum of the current noids and this is created by matter that is only present in small quantities and there are several algorithms and I have invented one of those and published it in a scientific paper and to remove the baseline when we move the baseline we are left with the Raymond spectrum alone and who wants to know more about this and has to do some detective work with the literature and so the Raymond lines above 2,000 wave pairs and the lower Raymond lines who can read anymore in the literature can find these but this is outside the scope of this of this talk to show this I have shown the pure better keratin so only carrots and excited it with two different wave lengths in the blue line we have 500 nanometers and then we have the green line to compare this with the spectrum line you see that it's a little bit steeper it's a little bit higher so you see that it's a little bit stronger this line the frequency is dependent so that as it's a growing stronger you see the blue line the here we see the keratin and ethanol resonance card resonance map this small part of the spectrum it shows as it goes up that the frequency and these things are relative to each other there's something to watch here we have to count relative waves we have 500 nanometers it's better keratin like in the map together we have it with the spectrum resonance and absorption it's a maximum it's the first response and see how it goes up high then it goes down again so there's a response from the chemicals the the status goes high and then back the proton is responding to the more energy it's very very strong here so this is an excitation wave lengths a different and through the logarithmic scale we can also separate the anti-stokes lines the special of the anti-stokes lines is they cannot be covered by fluorescence and they they have a dependency on the absolute temperature because the vibrational states correspond to the Boltzmann distribution and there you can so you can measure the absolute temperature with a laser with a laser beam for after this small overview of the effects it's now we are talking about how we can use that at first the selection of the laser we usually have to use a blue or violet laser that's a very simple reason for that if you know where it is even if you use the laser laser protection goggles other lasers may be much more extensive more expensive but with a red laser you might be tempted to remove your goggles but you should never do that so you can see the light so a blue laser is good so you can use a piece of paper with a sharpie and then you can hold it into the beam and see where it is and the skin will also fluoresce a little in yellow and so you can see with your finger where the laser beam is for here's a small do-it-yourself for a spectrometer and the spectral range is important and you if you have the whole visible range that's good but if you want to do the rayman spectroscopy you need a very high resolution and you need to be able to filter parts of the spectrum and the sensitivity is a bit like the aperture in photography and so you have a grid and the camera and if the slit is very low then there's only very little light and there's only very little light on the camera but so good resolution and so it depends on how the optics are built if maybe there are other apertures or small small parts and you have to look what is better here so for rayman spectroscopy it's very important to be very precise there and you have to experiment a bit another important part is the dynamic range a normal camera has maybe 10 to 12 bits dynamic range whereas the camera which I used to record it has 16 bits of dynamic range and you have to experiment a bit with that as well and but with a webcam which has only eight bits per channel isn't very helpful if you don't want to look at the fluorescence but also some some other more interesting things and another part is that if you exceed the maximum contrast from from from the camera of the camera then you are looking only at so if you have a very strong a very strong beam then it may be diffracted inside the camera and then you if you have optical low pass filters they are very expensive but the spectrometer if you build it yourself you can disassemble it and and put a little a little blind there and so you can easily find that and block it out if there's and maybe you already have something like that in your do-it-yourself box and LEDs can can be used as sensors for example and I have measured a few LEDs and what their receptive spectrum is and I have published it the most easily accessible range is is dependent on the material of the sensor for example silicon can use thousand hundred to a thousand and and you have to improvise maybe if you need another range other semiconductor materials have other ranges and the silicon carbide goes of 200 to 400 indium gallium arsenide goes from 800 to 1700 and germanium is from 800 to 1800 nanometers and and some some tubes also create ultraviolet light and you have just have to experiment a bit maybe it's also possible to you open up a germanium power transistor and use that as a sensor but the sensitivity will probably be very low and for measuring rayman lines that's a really technical challenge and the absorption can be measured relatively easy so I want to show you some examples how such measures can be broken down such measurements for classical measurements where you use a lamp and a monochromator and then you have use a photodiode as a sensor and then it goes through a beam splitter and through a photodiode and then through the through the liquid that you want to measure into a photodiode and then you can find out the specific wavelength and the absorption is dependent on the thickness of the material and and an absorption coefficient that is also dependent on several parameters besides the lamp and a monochromator and and there may be variations in the intensity of the lamp and to compensate for that if we can have something where the very LEDs have a very very stable intensity and so the intensity can be measured with a photodiode and the intensity of the LED itself can be measured that way for example there's a probe both of those can be shined through the finger and a photodiode can can be measured with a photodiode and the blood oxymeters work like that and shine through this through the finger and that way you can measure how much oxygen is in the blood and you can use a spectrometer to look at the light of the sun and with several LEDs with different wavelengths you can also experiment with ours a good example is the infrared hygrometer it used the sun as the light source and shines them through through as a sample and use LEDs as the sensor for 880 or 940 nanometers and the water vapor has an absorption line near 900 nanometers so you can figure out the water vapor concentration in the air and can build a hygrometer that way and here's another example if you want to examine chlorophyll fluorescence if you look at the leaf with a shine on it with a blue or green lamp and with a blue LED you can shine at the plant and use a dark red filter but it's not easy to record that on video but we'll see you can still see it in this photo and you have perhaps seen these spectacular infrared photography photos and you can use a dark red filter for that and you can see that the sky becomes blue because the blue is absorbed by the filter and the leaves become very bright and on this photo you can see on the right it is very bright and the tree on the left side is much less bright and that is because the chlorophyll fluorescence is a reaction to get rid of superfluous additional light and so that the leaves don't get overloaded with light you can also take photos like this from satellites or airplanes and can figure out where it is dry or if there's a danger of forest fire and if you measure the chlorophyll fluorescence you need three LEDs a blue one for excite the fluorescence and two red ones for detecting the fluorescence and the emission spectrum of this LED is at 700 nanometers and you can see that they overlap and the reason is that we have a semiconductor with a band gap between and so red and yellow LEDs have similar band gaps and so they can see their own light and the green blue ultraviolet LEDs have an indirect band gap semiconductors and emission and sensitivity are separate so they cannot see their own light but only in different bandwidths and the sensitivity is either it starts at the emission spectrum or is and here is a bright red light red LED and if you look at the sensitivity of the two LEDs together with the chlorophyll fluorescence you can see that the light red LED cannot see the chlorophyll fluorescence whereas the dark red LED can see the chlorophyll fluorescence and so we have everything that we need and we can excite it with a blue LED use the light red LED as a reference and use the dark red LED for measuring the data and you can see the result of the chlorophyll fluorescence from a leaf which I have created with these three LEDs and for the blue for this measurement the blue LED will flash and then I use the two red LEDs with an operational amplifier and you can see the red curve is the measurement and the blue curve is the reference and you flash the blue light into the leaf and sometimes the LED will light between the measurements and and they fluoresce very strongly for a short period of time and so continue the time adding so this curve is known as the Kautzky effect at the end I give you some literature tips for people who are still curious and some areas that can be very interesting for example lighting the light emission of the sea and you can use a microscopy for example and so and the main component analysis can be done and maybe you can use hyperspectral cameras which can record a very large spectrum and they use a diode and two very shortly very short excitations so and see you again in the Q&A so what a complex matter my head is really rotating I learned a lot thank you very very much for this huge impact I feel like I'm fried by a laser and and saturated with information thanks thanks a lot due to touch with the laser the probe one moment the laser only has 50 milliwatts for the probe and the probe is touched and there's only a part it it only is together for a current moment a fast moment that means you tickle the probe with 50 milliwatts yes it's a clear answer yes sondern mit lebendigen organismen setzt du diese proben nicht unter stress is not stress for the measurement does it influence it matthias has replied that yeah there may be surprises and for example that maybe foaming or abiding over