[Title fragment 1.1] [Title fragment 1.2] [Title fragment 1.3]
[Title fragment 2.1] [Title fragment 2.2] [Title fragment 2.3]
[Title fragment 3.1] [Title fragment 3.2] [Title fragment 3.3]



Tardigrades, carrot carotinoids and the DIY Raman spectrometer (V)

The scenario is remaining tough for our readers. On one hand our magazine is primarily intended to be entertaining - and sometimes sentimental, of a rather shallow intellectual depth. But on the other hand we want to help those among you who would like to reach out a little bit further and to become active themselves.

In the most recent issue of our magazine we illustrated in a generic manner how to integrate a - merely thumb-sized - laser into a microscope in order to provide the irradiation basis for Raman spectroscopy. Examples of the resulting experimental Raman spectra have been shown in older issues. Newcomers are encouraged to start reading at our February and March magazine issues.

For a rough understanding of Raman spectroscopy it is essential to focus on the bizarre combination of a very strong, single-wavelength stimulating laser light on one hand and the need for a tremendous amplification and protection of the resulting, very weak Raman bands against their "laser father" on the other. Basically and practically speaking we merely have to solve an ambitious filtering problem. Once this is understood and practically solved, natural physics will come in and take over, coming up with thrilling results.



[ 532 nm green laser signal as seen in the VIS spectroscopy ]

Fig. 1: The thumb-sized, solid state green laser (30mW or 50mW) for our Raman experiment was already depicted in our recent May issue. It is ideally suited, due to its intense green light, its well-defined output wavelength (532 nm) in combination with a reasonable bandwith and wavelength stability over time. No need for active cooling, control or recalibration. It is simply doing its job, without boasting, reliably. Exactly like some politicians, isn't it?

The output of this laser (as shown above) can be visualized by the same spectrometer as the one used for our Raman setup. But for this particular measurement the - tremendously intense - laser radiation had to be dimmed down by two greyscale filters. Furthermore the measurement time had to be reduced to 100 ms, whereas typical Raman analysis measurement times will span over a range between 10 seconds and several minutes. Otherwise the laser signal height in our display would have reached an impressive magnitude of ca. 100 meters! No need to say that most monitors are too small to show the magnitudes, even when turned to a vertical orientation  ;-).

[ 532 nm green laser bandwidth ]

Fig. 2: The output of the same laser signal as in fig. 1, but this time with a much better resolution on the x-axis. In this zoom view it is becoming apparent that our green laser (similar as virtually all green 532 nm lasers, so-called DPSS solid state lasers) is delivering a very fine signal at a position close to 532,0 nm. The bandwidth FWHM ("Full Width at Half Maximum") appears to be in the range of ca. 1 nanometer - perfectly sufficient for our needs.

[ Same as before but with Raman shift scale ]

Fig. 3: In the case of Raman spectroscopy the position of the Raman bands has to be translated to so-called Raman shift values. Those shift values are indicating the distance from the laser signal, with the laser signal arbitrarily set to zero. Furthermore the wavelength shift is being converted to its reciprocal value, thus ending up with a [1/cm] unit. At first sight all this might sound terribly confusing - but it is just a math trick in order to get identical Raman shift values even when using different laser wavelengths. As a consequence the Raman shift e.g. of a diamond will be always about 1332 [1/cm], no matter whether a green or a red laser are used. Those among you who are getting headache from this can simly use one of those fine online wavelength to Raman shift converters. In any case the wavelength of the green 532 nm laser light will then appear as a Raman shift of zero.

One might think now about simply switching on the laser, turn its beam towards the sample and hope to receive a perfect clean Raman spectrum in return (like the one shown in fig. 5). Sadly, no! First of all there is an excess of stimulating laser radiation which has to be filtered away - otherwise it would overwhelm and spoil the spectrum:


[ Carotin Raman Spektrum ohne Longpassfilter ]

Fig. 4: The Raman catastrophe - a hopeful spectrum of carotene, made without long pass filter, just for demonstration purposes. The spectrum is being overwhelmed by the laser radiation, drowning all the Raman signals in a terrible mess.

[ carotene Raman spectrum ]

Fig. 5: This is what we would like to reach instead: a perfectly usable Raman raw spectrum of ß-carotene (this time we used no tardigrade carotene, but intead carotene from carotene grocery pastilles for the hypochondriacs and artificial tanning addicts among us).
Strong carotene bands are observed at Raman shift values of ca. 1000, 1150 and 1510. When keeping in mind that the stimulating green laser is much more intense (by many magnitudes) it becomes apparent that most of its radiation had to be filtered out in order to generate the spectrum shown. In this case a highly sophisticated cleanly cutting ("long-pass") interference filter plus a laser line-filter (see below) were used in order to suppress all unwanted laser radiation, in particular the strong primary laser signal at Raman shift zero. As can be seen from the spectrum the long-pass filter is cutting off almost everything below a Raman shift value of about 350 cm-1. It is filtering out most of the primary laser light at Raman shift zero, leaving just a modest stub in place. Measurement time 2.000 ms. Averaged over ten runs, summing up to a total measurement time of 20 seconds.

[ carotene Raman spectrum without line filter ]

Fig. 6: But there are some more pitfalls to consider. In the spectrum above only a long-pass filter was used. This filter is well able to filter out the primary laser but it doesn't influence anything on the further right side of the spectrum, beyond 350 cm-1. As a consequence some very weak satellite signals of the triggering laser will become visible as well. Even though the laser is concentrating most of its radiation at 532 nm (Raman shift zero) it is still delivering some spectral impurities (satellite signals) which are becoming visible due to the enormous amplification needed for Raman spectroscopy. As a consequence additional signals, like the one at Raman shift 1900 are showing up in the spectrum. This is no problem for the whole-hearted spectroscopist who might be able to deal with the situation. But the beginner will be irritated by those ghost signals and - worst case scenario - interpret them as bands resulting from the sample under investigation. As a consequence a so-called laser line filter in front of the laser can be recommended. It is simply cleaning the laser signal, preserving the 532 nm signal and removing everything else. Nevertheless one should keep in mind that any laser cleaning filter will weaken the Raman bands as well. So, at least in some tricky cases or for the first experiments, one might be better off to work without the laser cleaning filter in order to make the Raman signals better visible.

[ Raman carotene spectrum - interference from an old-fashioned energy saving lamp ]

Fig. 7: Last but not least one should be aware of the individual measurement environment. As we are measuring in the wavelength region of visual light, fluorescent light, old-fashioned 'energy saving' lamps and also strong sunlight might interfere with our spectra. E.g. the band with a Raman shift of 2420 as shown above did appear only when an energy saving (fluorescent) lamp was switched on in the lab room during the time of the measurement.

[ Raman carotene spectrum - artifacts due to inhomogeneous CCD channel susceptibility ]

Fig 8: A further typical source of mistakes: in this example the sensor background noise was forgotten. As a consequence the resulting spectrum is an overlay of the actual spectrum and the (different) susceptibilities of the CCD sensor channels (pixels). Modern spectrometers are able to perform this kind of background correction automatically, whereas in the case of older spectrometers it might be necessary to make a measurement without the sample first and use it as a blank frame for background substraction afterwards.

Even though all this might sound tough for some of you it must be emphasized that experimental Raman spectroscopy can be tremendous fun and intellectually rewarding, in particular for those who cannot afford more expensive professional equipment at a price tag of, let's say 20,000 US$.
The pathway is open due to bargain green lasers, reasonably priced used CCD spectrometers and interference filters, all of which were not available a few decades ago. Lucky times, at least in comparison to the past ...



© Text, images and video clips by  Martin Mach  (webmaster@baertierchen.de).
The Water Bear web base is a licensed and revised version of the German language monthly magazine  Bärtierchen-Journal . Style and grammar amendments by native speakers are warmly welcomed.


Main Page