I´m afraid you are not going to get any spectroscopic information with the laser lines you have.

You need to use the 1064 nm laser as excitation dource, as many of the papers in literature are showing.

For example, see Analyst, 2003,128, 82-87, where authors use Raman spectroscopy to characterize different species of trees, or Journal of Molecular Structure 920, 2009, Pages 128–133, for different systems such as lichens.

I'm afraid the problem is high power density (focusing through lens PD~10kW/cm^2) that burns your samples. To avoid this problem you can:

1) Use Retro-Raman configuration, where typical PD~0.1W\cm^2, but the studying area is very high.

2) Use LWD objectives with large-area spots (for example x10).

3) Use nitrogen box to cool your samples to avoid burning.

4) Also you can make low power 532nm by neutral density filters (0,001%) and high accumulation time.

We have found strong orientational dependence of fluorescent signall, i.e. the problem is better or worse depending on the angle between the polarisation and the wood grainl. We found transmission x-ray diffraction on an area detector (WAXS) is a much easier way to make orientational analysis of spiral angle of cellulose.

When decreasing the power, the laser doesn't burn the sample anymore, but there is still a very strong background, from which peaks are very difficult to see, even after hours of accumulation. Even with a 10 x objective, instead of 100 x. What I don't understand is how some authors explain that they used a 532 nm laser with a 5 mW power, and could get spectra without baseline correction ? These are the conditions I also used, but the results are totally different. Could the kind of wood be responsible of such effect ? With aged wood samples, the result is even worse, as the peaks are even much smaller whereas the background almost saturates the detector. In some cases, getting understandable spectra is impossible. What should I do ?

Alain

@ Chris. Interesting remark. I always did the studies by focusing the laser along fibres. I should check what happens in the perpendicular direction.

Alain

I´m afraid you are not going to get any spectroscopic information with the laser lines you have.

You need to use the 1064 nm laser as excitation dource, as many of the papers in literature are showing.

For example, see Analyst, 2003,128, 82-87, where authors use Raman spectroscopy to characterize different species of trees, or Journal of Molecular Structure 920, 2009, Pages 128–133, for different systems such as lichens.

Dear Alain,

I am writing to you in reference to the question you have made to Dr. Rafael Alvarez Nogal, related with the Raman microscopy and fluorescence.

I am Silvia González, a colleague of him, and I will try to help you with this issue.

At first, try to use the 780nm laser instead the 532nm in order to achieve photobleaching. Some equipment has a tool called “photobleach” that usually helps.

Varying the acquisition conditions also improves the situation. Try with very low laser powers (1mW or 0.5mW) and play with the exposure time and number of accumulations, in instance: 1s exposure and 40 accumulations, or otherwise, 10s or 60s and only 5 accumulations.

I hope this will be helpful.

Best regards,

Silvia ([email protected])

Dear Silvia,

Thank you for your help. However, I am afraid that I already tried this. Reducing the power indeed decreases the fluorescence background, but also the intensity of the peaks, so that the signal / background ratio is not higher.

Maybe the only way is indeed using a 1064 nm laser, but I definitely don't understand how some authors got nice spectra at 532 nm. Can it be explained by a different orientation of the laser with respect to wood fibres ? Or another experimental trick ?

Alain

Transfer the answer to Silvia.

Please write directly to she:[email protected]

I also experienced laser induced burning during mapping, but spending days with patience I managed to produce spectra and images. Since I studied extracted wood section (about 200 nm) mostly, the fluorescence effect was minimized. However I experienced fluorescence with 532 nm laser while mapping normal wood, but it was not so severe. Of course background was subtracted afterwards.

You can not avoid fluorescence with this laser you should change the laser wavelength source.

Another way which may help ton avoid fluorescence is rotating the sample by a motor which the sample is firmly fixed on its plate.

You have to switch to 1064 nm.

Thanks to all. @Sayyed, please explain why rotating the sample may help: do you mean a continuous rotation, or a rotation until finding a minimum of fluorescence and then stop moving the position ? Is there any influence of the sample's orientation (parallel vs perpendicular to wood fibres ?)

Alain

Yes some peaks have an intensity dependance on the rotation with respect to the laser polarisation. I've used this to determine the alignment of cellulose molecules with respect to the grain. The angle of the spiral around a fibre axis or microfibril angle.

@Chris: so the experiment should be done with a polarised laser ? Which kind of polarisation ? Should there be a polariser also on the detector, and of which kind ?

Thanks for your help.

Alain

Dear Alain, rotating does not allow the beam to focus on one point, therefore prevent the sample from heating up.

You can not take Raman spectrum of wood material. One way is preparing the samples as small pills and then mounted on a rotating disk (with about 2000 rpm) to avoid heating the sample. Be careful the lase spot not hit the center of sample. 

I just saw your question and may be, by now you have found a solution. In addition to the answers above the following information may be useful. By the way, we have worked with woods and Raman spectroscopy for a long time. You can check out a # of our pubs. in case more information is desired.

There are two classes of fluorescence-causing-entities in woods - lignin which is native to wood and extractives which vary from wood-species to wood-species.  In the case of the latter, if the wood of intertest is dark, it can produce overwhelming fluorescence and no matter what you do in terms of laser wavelength change or sample rotation or power reduction all will fail, more or less. The only solution is remove extractives as much as possible by solvent-extraction (that's why they are called extractives). However, it is posible that some extractives will remain, the ones difficult to remove by most solvents.

Once wood has significantly brightened, it is possible that the still-remaining fluorescence is dominated by lignin. The latter is not easily suppressed but the best way, without chemically treating lignin (in wood), is to use the 1064 nm laser (785 or shorter nm excitation may also suffice). There are two main ways to treat lignin to reduce fluorescence - NaBH4 reduction and alkaline H2O2 treatment. Both these retain lignin in wood for the most part but brighten the lignin, and therefore, wood. That helps a great deal (see some of our pubs). Further, depending upon the nature of the information desired from a wood spectrum, I mean if lignin information is not important,one can remove lignin completely and a big reduction in fluorescence occurs. In that case, one can use visible lasers.

Dear Umesh,

Thank you for your detailed reply.

I would be delighted if you can forward me your relevant publications on this topic.

Best regards,

Alain

Alain:

Most of our pubs can be found in the links below. Considering that there are too many to send you, I would rather ask you to check these first and in case you do not find what you need,  you can contact me again.

Umesh

https://www.researchgate.net/profile/Umesh_Agarwal/contributions?ev=prf_act