En­vir­on­mental scan­ning elec­tron mi­cro­scope

Our scanning electron microscope FEI Quanta 250 FEG ESEM (© Max-Planck-Institut für Marine Mikrobiologie, S. Littmann)
Our scanning electron microscope FEI Quanta 250 FEG ESEM (© Max-Planck-Institut für Marine Mikrobiologie, S. Littmann)

What is a en­vir­on­mental scan­ning elec­tron mi­cro­scope (SEM)?

When ex­amin­ing and ima­ging minute ob­jects such as mar­ine mi­cro-or­gan­isms, we quickly reach the lim­its of op­tical mi­cro­scopy. In or­der to be able to im­age these di­verse or­gan­isms, we must use scan­ning elec­tron mi­cro­scopy in­stead. In the Biogeo­chem­istry work­ing group, we have a scan­ning elec­tron mi­cro­scope (SEM) for this pur­pose. It is equipped with vari­ous de­tect­ors for im­age gen­er­a­tion and ana­lyses.

How does a scan­ning elec­tron mi­cro­scope work?

SE image of a dinoflagellate from the MPI pond (prepared with CPD) (©Max Planck Institute for Marine Microbiology, S. Littmann)
SE image of a dinoflagellate from the MPI pond (prepared with CPD). (© Max Planck Institute for Marine Microbiology, S. Littmann)

An SEM al­lows a higher res­ol­u­tion (down to 1 nm at 30 kV in high va­cuum) and depth of field than a light-op­tical mi­cro­scope. A field emis­sion cath­ode gen­er­ates an elec­tron beam that is fo­cused on the sample by elec­tro­mag­netic lenses. The sur­face of the sample is then scanned point by point by this elec­tron beam in a rect­angle. Vari­ous ac­cel­er­a­tion voltages (500 V to 30 kV) can be used for the elec­trons. When the elec­tron beam (primary elec­trons, PE) strikes the sur­face of the sample, vari­ous in­ter­ac­tions oc­cur with the atoms of the sample, and vari­ous sig­nals are cre­ated – in­clud­ing sec­ond­ary elec­trons (SE), which can be used for ima­ging.

Be­cause the SE are low-en­ergy elec­trons, they ori­gin­ate only from a thin layer from the sur­face of the sample. SE im­ages thus con­tain in­form­a­tion about the to­po­graphy con­trast. In or­der to be able to see fine struc­tures in minute mi­cro-or­gan­isms, a gentle and three-di­men­sional, struc­ture-pre­serving sample pre­par­a­tion with crit­ical point dry­ing (CPD) is re­quired. A CPD device is avail­able for this pur­pose.

An­other sig­nal, the back scattered elec­trons (BSE), are higher in en­ergy than the SE. BSE im­ages con­tain in­form­a­tion about the com­pos­i­tion of the sample through the ma­ter­ial con­trast. Ele­ments with higher nuc­lear charge num­bers ap­pear brighter on the im­age than ele­ments with lower nuc­lear charge num­bers.

Two SE images of polished grains from marine sediments from the island of Elba. The image on the right is a BSE image of the same grains as in the image on the left. Because of the higher nuclear charge number of iron, the iron oxide-rich areas in the grains appear brighter in contrast to the silicate matrix. (© Max Planck Institute for Marine Microbiology, S. Littmann)
Two SE images of polished grains from marine sediments from the island of Elba. The image on the right is a BSE image of the same grains as in the image on the left. Because of the higher nuclear charge number of iron, the iron oxide-rich areas in the grains appear brighter in contrast to the silicate matrix. (© Max Planck Institute for Marine Microbiology, S. Littmann)
Element mapping of marine sediment grains from the island of Elba using EDS. The differences between the silicate matrix and Fe–Cr-rich phases are clearly visible. (© Max Planck Institute for Marine Microbiology, S. Littmann)
Element mapping of marine sediment grains from the island of Elba using EDS. The differences between the silicate matrix and Fe–Cr-rich phases are clearly visible. (© Max Planck Institute for Marine Microbiology, S. Littmann)

The SEM is equipped with dif­fer­ent de­tect­ors so that SE and BSE can be de­tec­ted in all va­cuum work­ing ranges. De­pend­ing on the nature of the sample, in­vest­ig­a­tions can be car­ried out in a high va­cuum (6 × 10−4 Pa), low va­cuum (10–130 Pa), and ESEM mode (for li­quids, 10–4000 Pa).

X-ray ra­di­ation also arises from the in­ter­ac­tion of the PE with the atoms of the sample. This can be used for the chem­ical ana­lysis of a sample be­cause each chem­ical ele­ment has a char­ac­ter­istic X-ray ra­di­ation. For the de­tec­tion of the char­ac­ter­istic X-rays, our SEM is equipped with a double de­tector sys­tem from Bruker Nano Ana­lyt­ics for en­ergy dis­pers­ive X-ray spec­tro­scopy (EDS or EDX). The EDS sys­tem has two Xflash 6/​30 de­tect­ors (30 mm2 de­tector area) with an en­ergy res­ol­u­tion < 123 eV for the man­ganese Kα-spec­tral line. The data is eval­u­ated us­ing the Bruker Es­prit 2.1 soft­ware. Both point ana­lyses and ele­ment map­ping can be car­ried out with this sys­tem.

The SEM can also be used for scan­ning trans­mis­sion elec­tron mi­cro­scopy (STEM). The sample is im­aged and ana­lysed by means of trans­mit­ted elec­trons. Thin sec­tions as low as 100 nm thick can be ex­amined with this method. For this pur­pose, a spe­cial de­tector (STEM de­tector) is placed un­der­neath the sample. Ac­cel­er­at­ing voltages between 20 and 30 kV are gen­er­ally used.

STEM image of a thin section of a piece of seagrass root. (© Max Planck Institute for Marine Microbiology, S. Littmann)
STEM image of a thin section of a piece of seagrass root. (© Max Planck Institute for Marine Microbiology, S. Littmann)

The SEM in the De­part­ment of Biogeo­chem­istry at the MPI Bre­men can also be equipped with a SECOM plat­form. SECOM en­ables cor­rel­at­ive ima­ging of a sample us­ing scan­ning elec­tron and fluor­es­cence mi­cro­scopy. For this, the sample must be pre­pared on a glass slide coated with in­dium tin ox­ide (ITO). Scan­ning elec­tron mi­cro­scopy is per­formed on the con­duct­ive top side (ITO) of the glass slide and fluor­es­cence mi­cro­scopy from the bot­tom side through the glass slide. This makes it pos­sible to identify cer­tain mi­cro-or­gan­isms by fluor­es­cence and to de­scribe sur­face struc­tures us­ing SEM.

SEM equipped with the SECOM platform (blue). (© Max Planck Institute for Marine Microbiology, S. Littmann)
SEM equipped with the SECOM platform (blue). (© Max Planck Institute for Marine Microbiology, S. Littmann)

The scan­ning elec­tron mi­cro­scope in ac­tion

Illustration of the endosymbiont ‘Candidatus Azoamicus ciliaticola’ and its ciliate host from Lake Zug. The figure is a composite of a scanning electron microscope image (SEM, grey) and fluorescence images.. Visible is the ‘Candidatus Azoamicus ciliaticola’ endosymbiont (visualized by FISH, yellow) and bacterial prey in food vacuoles as well as the large cell nucleus (stained by DAPI, blue). The outer structure of the weakly fluorescent ciliate as well as the cilia are also visible. (© Max Planck Institute for Marine Microbiology, S. Ahmerkamp)
Illustration of the endosymbiont ‘Candidatus Azoamicus ciliaticola’ and its ciliate host from Lake Zug. The figure is a composite of a scanning electron microscope image (SEM, grey) and fluorescence images.. Visible is the ‘Candidatus Azoamicus ciliaticola’ endosymbiont (visualized by FISH, yellow) and bacterial prey in food vacuoles as well as the large cell nucleus (stained by DAPI, blue). The outer structure of the weakly fluorescent ciliate as well as the cilia are also visible. (© Max Planck Institute for Marine Microbiology, S. Ahmerkamp)

New form of symbiosis discovered

They are also called power plants of the cells: the mi­to­chon­dria. They are present in al­most all eu­k­a­ryotic cells and they sup­ply the cells with en­ergy. Un­til now, it was as­sumed that only mi­to­chon­dria can act as the cells’ en­ergy pro­viders. Sci­ent­ists at the Max Planck In­sti­tute for Mar­ine Mi­cro­bi­o­logy have now dis­covered that sym­bi­otic bac­teria can ful­fil this func­tion too. Their find­ings shed a com­pletely new light on the sur­vival of simple eu­k­a­ryotes in oxy­gen-free en­vir­on­ments.

The sci­ent­ists have found a unique bac­terium that lives in­side a uni­cel­lu­lar eu­k­a­ryote and provides it with en­ergy. Un­like mi­to­chon­dria, this so-called en­dosym­biont de­rives en­ergy from the res­pir­a­tion of ni­trate, not oxy­gen. Such part­ner­ship is com­pletely new.

For this study, also the scan­ning elec­tron mi­cro­scope was used.

 

You can find more in­form­atin about the study in the press re­lease "New form of symbiosis discovered"

 

Here you find the ori­ginal pub­lic­a­tion:

Jon S. Graf, Sina Schorn, Kath­ar­ina Kitzinger, So­eren Ah­merkamp, Chris­tian Woehle, Bruno Huettel, Carsten J. Schubert, Mar­cel M. M. Kuypers, Jana Milucka: An­aer­obic en­dosym­biont gen­er­ates en­ergy for cili­ate host by de­ni­tri­fic­a­tion. Nature, 2021

https://dx.doi.org/10.1038/s41586-021-03297-6

 

 

Who uses the scan­ning elec­tron mi­cro­scope?

It is mainly used by sci­ent­ists of the Department of Biogeochemistry. However, it is also open to all other sci­ent­ists of the in­sti­tute as well as to ex­ternal re­search­ers in the con­text of col­lab­or­at­ive pro­jects.

Con­tact

Scientist

Biogeochemistry Group

Sten Littmann

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

Room: 

3136

Phone: 

+49 421 2028-6720

Sten Littmann
 
 
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