The Ra­man spec­tro­meter

Our Raman spectrometer is combined with an atomic force microscope, AFM. (© Max Planck Institute for Marine Microbiology/ K. Matthes)
Our Raman spectrometer is combined with an atomic force microscope, AFM. (© Max Planck Institute for Marine Microbiology, K. Matthes)

What is Ra­man spec­tro­metry?

Ra­man spec­tro­metry is named after the In­dian phys­i­cist, C. V. Ra­man. In 1928, Ra­man was the first sci­ent­ist to demon­strate that light is scattered in­elast­ic­ally by mo­lecules or solids. This means that part of the kin­etic en­ergy of the light is trans­ferred to the mo­lecule or solid, thereby ex­cit­ing it. This ef­fect is what Ra­man spec­tro­scopy is all about.

What is a Ra­man spec­tro­meter used for?

The Ra­man spec­tro­meter is used to in­vest­ig­ate ma­ter­ial prop­er­ties. Ma­ter­ial prop­er­ties refer to the com­pos­i­tion of the chem­ical com­pounds of an ob­ject or a sample as well as prop­er­ties such as the type of struc­ture in which the mo­lecules, atoms, or ions are ar­ranged (crys­tallin­ity) or the pres­ence of any for­eign atoms (dop­ing). RA­MAN spec­tro­scopy is used in many dif­fer­ent fields. For ex­ample, in min­er­alogy to identify min­er­als, in ar­chae­ology to ex­am­ine finds, and in food chem­istry to ex­am­ine car­bo­hydrates as a com­pon­ent of food. At our In­sti­tute, we use Ra­man spec­tro­scopy to ex­am­ine bio­lo­gical samples. We work with bac­terial cul­tures or mi­cro-or­gan­isms from dif­fer­ent hab­it­ats (e.g. oceans and fresh­wa­ter lakes).

The great ad­vant­age of the device is that it al­lows non-de­struct­ive and non-con­tact meas­ure­ment. Fur­ther­more, only very small amounts of a bio­lo­gical sample are needed. For ex­ample, RA­MAN spec­tro­scopy can help us de­term­ine whether bac­teria in a wa­ter sample con­tain cer­tain stor­age sub­stances. The stor­age sub­stances can be dif­fer­ent for each spe­cies.

How does the Ra­man spec­tro­meter work?

The laser of the Raman spectrometer. (© Max Planck Institute for Marine Microbiology)
The laser of the Raman spectrometer. (© Max Planck Institute for Marine Microbiology)

As the name sug­gests, the spec­tro­meter can be used to dis­play spec­tra. A spec­trum provides in­form­a­tion about the fre­quen­cies or in­tens­it­ies of ob­jects in a set that all have a com­mon prop­erty. The best known is cer­tainly the col­our spec­trum. Here, dif­fer­ent wavelengths of light be­come vis­ible as col­ours. At our In­sti­tute, we mostly work with mass spec­tra, which are non-op­tical spec­tra. The res­ult is a line spec­trum in which fre­quen­cies are dis­played as peaks.

Ra­man spec­tro­scopy makes use of the ro­ta­tion, os­cil­la­tion, and re­align­ment of the spin of a particle when it is ex­cited by the (mono­chro­matic) light of a laser. This means that when the laser is fo­cused on the sample, the mo­lecules start to os­cil­late, stretch, or bend and de­form. This move­ment or change can then be shown in a spec­trum.

For ex­ample, this hap­pens with bonds between car­bon and car­bon or sul­phur and sul­phur as well as with bonds between car­bon and many other atoms. The light scattered by the sample pro­duces a spec­trum and looks dif­fer­ent for each sample.

Lasers with dif­fer­ent wavelengths can be used. Mostly lasers with longer wavelengths are used. Our sys­tem has a red laser with a wavelength of 785 nm and a green laser with a wavelength of 532 nm.

Drawing of molecules excited by a laser and their possible directional movements during oscillation, spinning, and rotation. (© Max Planck Institute for Marine Microbiology/ D. Tienken)
Drawing of molecules excited by a laser and their possible directional movements during oscillation, spinning, and rotation. (© Max Planck Institute for Marine Microbiology, D. Tienken)

In con­focal Ra­man spec­tro­scopy, only a small part of the sample to be ex­amined is il­lu­min­ated with the laser light. The un­focused light is shiel­ded. This in­creases the depth of field and con­trast. This can also be ex­ten­ded to a three-di­men­sional scan. Non-de­struct­ive, non-con­tact meas­ure­ment is pos­sible with only small amounts of sample ma­ter­ial (bio­lo­gical samples). The lat­eral res­ol­u­tion is less than 200 nm, and the ex­pos­ure time is only seconds or mil­li­seconds.

The Ra­man spec­tro­meter in ac­tion

Ra­man spec­tro­scopy al­lows state­ments to be made about whether the ma­ter­ial in ques­tion has a di­morphic or poly­morphic struc­ture. This means that for com­pounds that have the same mo­lecu­lar for­mula – as is the case with the min­er­als pyr­ite and mar­cas­ite (both have the mo­lecu­lar for­mula FeS2) – the Ra­man meas­ure­ment can dis­tin­guish between the two min­er­als based on their dif­fer­ent struc­tures.

The dif­fer­ences be­come vis­ible in their spec­tra:

Pyrit: Rruff data base; Pyrite R050070, Rock Currier 	           Markasit: Rruff data base; Marcasite R060882,Bob Jenkins
Pyrit: Rruff data base; Pyrite R050070, Rock Currier Markasit: Rruff data base; Marcasite R060882,Bob Jenkins

Or as in the fol­low­ing ex­ample of calcite and aragonite. Both are cal­cium car­bon­ates with the mo­lecu­lar for­mula Ca­CO3.

Spektren von Calcit und Aragonit. Beide sind Calciumcarbonate mit der Summenformel CaCO3.
Rruff data base; Calcite R040070, University of Arizona Mineral Museum 6965 und Rruff data base; Aragonite R040078, University of Arizona Mineral Museum 3887

Ra­man spec­tro­scopy is also used in vari­ous aqueous sys­tems (bi­otic and abi­otic).

In this way, it was pos­sible to show where cer­tain bac­teria store their stor­age sub­stances and what was in the areas.

In the fol­low­ing ex­ample, bac­terial fil­a­ments of the spe­cies Beggiato spp. were ex­amined with Ra­man, and the spec­tra of dif­fer­ent areas of a sur­face were re­cor­ded us­ing a ras­ter scan:

Bacterial filament Beggiatoa spp. (Max Planck Institute for Marine Microbiology)
A) Light microscope image of the bacterial filament Beggiatoa spp.
B) The same section of the filament was recorded with the Raman during a raster scan. In yellow/red, you see the sulphur (main peak at 473 cm−1) and the autofluorescence of the cells in green.
C) A recorded spectrum on a yellow/red dot shows sulphur inclusions in the form of cyclooctasulphur and as polysulphide. Above is a spectrum of an upper bacterial mat. In the third spectrum from the top is a lower bacterial mat. At the bottom is the spectrum of polysulphides.

Publikation: Polysulfides as Intermediates in the Oxidation of Sulfide to Sulfate by Beggiatoa spp., Jasmine S. Berg et al, Applied and Environmental Microbiology, 2013

Im­age A is a light mi­cro­scope im­age and shows a fil­a­ment of the spe­cies Beggiato spp.

The ex­ample shown above is from a pub­lic­a­tion on poly­sulph­ides as in­ter­me­di­ates in the ox­id­a­tion of sulph­ides to sulph­ates by Beg­gia­toa spp.

Stand­ards were meas­ured for com­par­ison. This was ele­mental sul­phur, which has a ring form (cyc­loocta­sul­phur). This sul­phur ring shows three char­ac­ter­istic peaks (second po­s­i­tion in Im­age C). In the up­per mat of the Beggiato spp. colony, the three peaks in the Ra­man spec­trum were also found (see

first spec­trum in Im­age C). This means that cyc­loocta­sul­phur is also present here. The lower spec­trum in Im­age C shows a stand­ard of sul­phur in chain struc­ture (poly­sulph­ide). The spec­trum is dif­fer­ent; the first two peaks are smal­ler than in the cyc­loocta form. This struc­ture was also found in the lower mat of a Beggiato spp. colony.

An­other com­mon method is the ras­ter scan – also called Raman mapping. Here, many in­di­vidual point spec­tra are re­cor­ded. These are com­bined line by line over a cer­tain area to form one large im­age.

They help to find the areas of the cells where stor­age sub­stances are present as well as how much stor­age sub­stance is present in each area. The res­ult of such a ras­ter scan can be seen in Im­age B. It shows a Beg­giato spp. fil­a­ment with red and or­ange areas. Spec­tra of sul­phur are loc­ated in these areas. The auto­fluor­es­cence of the cell is shown in green.

Who uses the Ra­man spec­tro­meter?

Sci­ent­ists, PhD stu­dents, tech­ni­cians from the Department of Biogeochemistry, MarMic students dur­ing the lab ro­ta­tion, and vis­it­ing stu­dents.

Please dir­ect your quer­ies to:

Group Leader

Greenhouse Gases Research Group

Dr. Jana Milucka

MPI für Marine Mikrobiologie
Celsiusstr. 1
D-28359 Bremen
Deutschland

Room: 

3128

Phone: 

+49 421 2028-6340

Dr. Jana Milucka

Scientist

Biogeochemistry Group

Sten Littmann

MPI for Marine Microbiology
Celsiusstr. 1
D-28359 Bremen
Germany

Room: 

3136

Phone: 

+49 421 2028-6720

Sten Littmann

Technician

Biogeochemistry Group

Daniela Tienken

MPI for Marine Microbiology
Celsiusstr. 1
D-28359 Bremen
Germany

Room: 

3131

Phone: 

+49 421 2028-6402

Daniela Tienken
 
 
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