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+ Microalgae
+ Transmission electron microscopy (tem)
+ Nanoparticles
+ Sample preparation
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Martin Clarke

TEM microalgae samples preparation method ?

Donald Micheal Graham  Follow
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Amar Bashyal  Follow

Dear Maysan Nashashibi , thanks for sharing this interesting question. It is always a good idea to use the searching box into RG website, often you will get some very valuable informations such as those provided by Aarif Shah . Specially in the first link you can see a good TEM sample preparation protocol for bio-samples with nanoparticles.
That is a generic protocol, and it must be adapted to each sample and to what you want to characterize. For example, it will be different if these nanoparticles (NPs) are in the liquid media, on the microalgae´s surface, or dispersed into the microalgae´s tissues. Problems associated to NPs aggregation will probably happen in the two first scenarios, when the NPs are dispersed into the liquid media and by evaporation of it, they end up on the surface of the algae. Some NP´s could be attached to the algae surface and evaporation of the media could add more from the media. If you are interested about the NP´s already bonded to the microalgae surface, and there are NP´s dispersed into the media around, it would be useful to remove the algae from the media, wash them up with some clean media (bear in mind that this clean media should not remove the bonded NPs from your algae), then dry the algae as explained in the previous links or if it is available, use a TEM cell for liquid samples. Of course, the microalgae must be thin enough to work in transmission mode as pointed by Mohammed Amer Shaheed .
If the microalgae have the NPs inside their tissues, the chances of aggregation by drying are very few, the algae tissues will keep them apart, so that if you finally observe aggregation of NPs, it means -generally- that they were already aggregated before the drying process. Once more, Mohammed advice is key, you will need a very thin section of your algae to let the electrons go through the sample and forming a image. You can get such thin sections embedding the sample into a resin and cutting it with an ultramicrotome. Otherwise, you could use a FIB (Focused Ion Beam), a very focused and thin beam of ions to cut a lamela (a very thin section) of your sample, which is then placed on a TEM holder grid with the help of micromanipulators.
TEM works by transmission, transmission of electrons through the sample, so depending on the density of each part of your sample (the value of Z, the atomic number of the atoms on each part) you will see more or less contrast. For example, if your NPs are metallic, they will be denser than the organic tissue of your microalgae, and a good contrast will be expected. But if your NP´s are light, say carbon NP´s or Si NPs the contrast with the organic matter will be smaller.

It could be very useful, before to go to the TEM, to observe the samples with an optical microscope (may be a confocal, looking for some fluorescence from the NP´s), or a faster option, to use a SEM, specially if you are studying NP´s on the algae surface. Normally you would need to dry up your sample as with TEM, but here -with SEM- you don´t need to worry about sample´s thickness too much. SEM often requires conductive samples or coating your sample with a very thin conductive layer (gold, carbon, iridium...), but modern equipment also let us to use the sample uncoated, thanks to charge removal methods.

Hope this helps. Good luck with your research work and my best wishes.

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David Ford  Follow
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David Johns  Follow

For TEM, samples must be cut into very thin cross-sections. This is to allow electrons to pass right through the sample. After being fixed and dehydrated, samples are embedded in hard resin to make them easier to cut

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Barry Smith  Follow

For TEM, samples must be cut into very thin cross-sections. This is to allow electrons to pass right through the sample. After being fixed and dehydrated, samples are embedded in hard resin to make them easier to cut

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Connor Leech  Follow

Dear Maysan Nashashibi , thanks for sharing this interesting question. It is always a good idea to use the searching box into RG website, often you will get some very valuable informations such as those provided by Aarif Shah . Specially in the first link you can see a good TEM sample preparation protocol for bio-samples with nanoparticles.
That is a generic protocol, and it must be adapted to each sample and to what you want to characterize. For example, it will be different if these nanoparticles (NPs) are in the liquid media, on the microalgae´s surface, or dispersed into the microalgae´s tissues. Problems associated to NPs aggregation will probably happen in the two first scenarios, when the NPs are dispersed into the liquid media and by evaporation of it, they end up on the surface of the algae. Some NP´s could be attached to the algae surface and evaporation of the media could add more from the media. If you are interested about the NP´s already bonded to the microalgae surface, and there are NP´s dispersed into the media around, it would be useful to remove the algae from the media, wash them up with some clean media (bear in mind that this clean media should not remove the bonded NPs from your algae), then dry the algae as explained in the previous links or if it is available, use a TEM cell for liquid samples. Of course, the microalgae must be thin enough to work in transmission mode as pointed by Mohammed Amer Shaheed .
If the microalgae have the NPs inside their tissues, the chances of aggregation by drying are very few, the algae tissues will keep them apart, so that if you finally observe aggregation of NPs, it means -generally- that they were already aggregated before the drying process. Once more, Mohammed advice is key, you will need a very thin section of your algae to let the electrons go through the sample and forming a image. You can get such thin sections embedding the sample into a resin and cutting it with an ultramicrotome. Otherwise, you could use a FIB (Focused Ion Beam), a very focused and thin beam of ions to cut a lamela (a very thin section) of your sample, which is then placed on a TEM holder grid with the help of micromanipulators.
TEM works by transmission, transmission of electrons through the sample, so depending on the density of each part of your sample (the value of Z, the atomic number of the atoms on each part) you will see more or less contrast. For example, if your NPs are metallic, they will be denser than the organic tissue of your microalgae, and a good contrast will be expected. But if your NP´s are light, say carbon NP´s or Si NPs the contrast with the organic matter will be smaller.

It could be very useful, before to go to the TEM, to observe the samples with an optical microscope (may be a confocal, looking for some fluorescence from the NP´s), or a faster option, to use a SEM, specially if you are studying NP´s on the algae surface. Normally you would need to dry up your sample as with TEM, but here -with SEM- you don´t need to worry about sample´s thickness too much. SEM often requires conductive samples or coating your sample with a very thin conductive layer (gold, carbon, iridium...), but modern equipment also let us to use the sample uncoated, thanks to charge removal methods.

Hope this helps. Good luck with your research work and my best wishes.

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