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Multi-photon microscopy to better treat diseases 

Chiara
Chiara Stringari
Researcher at the Laboratory of Optics and Biosciences (LOB*) at École Polytechnique (IP Paris)
Key takeaways
  • To understand fundamental physiological mechanisms and processes, as well as those of many pathologies, it is essential to observe cellular metabolic activity in real-time with subcellular resolution.
  • In biology, it is common to “tag” molecules with fluorescence markers, but this is an invasive technique, as one has to “enter” the cell.
  • However, there are fluorescent molecules already present in cells that can be stimulated without artificially integrating them and interfering with the living system.
  • Acquiring this type of knowledge opens the way to many applications in medicine, as it allows the metabolism of cells – an indicator of their activity – to be mapped. This, for example, allows the identification of the metabolism of cancer cells, which are likely to develop many forms of cancer.

Chiara Stringari, a researcher at the Lab­o­ra­to­ry of Optics and Bio­sciences (LOB) at École Poly­tech­nique, is skirt­ing at the fron­tier of sev­er­al dif­fer­ent sci­en­tif­ic dis­ci­plines. Her aim is to devel­op inno­v­a­tive micro­scop­ic obser­va­tion tech­niques to map the meta­bol­ic activ­i­ty of cells and tis­sues with greater pre­ci­sion. To do this, she uses a tech­nique based on two-pho­ton opti­cal microscopy; a method using two pho­tons pro­ject­ed onto a tar­get cell, through a laser, which make a mol­e­cule in that cell react so it becomes vis­i­ble by flu­o­res­cence. Cor­rect func­tion­ing of a healthy cell is deter­mined by activ­i­ty of the mol­e­cules with­in it. Observ­ing them and char­ac­ter­is­ing their flu­o­res­cence life­time with greater pre­ci­sion allows researchers to iden­ti­fy the mol­e­c­u­lar sig­na­ture, and thus nor­mal cel­lu­lar func­tion, so that they can estab­lish med­ical diagnoses. 

Observ­ing cel­lu­lar meta­bol­ic activ­i­ty in real-time with sub­cel­lu­lar res­o­lu­tion is cru­cial to under­stand­ing fun­da­men­tal phys­i­o­log­i­cal mech­a­nisms and process­es as well as those of sev­er­al patholo­gies. To do this, it is com­mon to “tag” mol­e­cules with flu­o­res­cence mark­ers, but this requires an inva­sive tech­nique, as one has to “enter” the cell. As such, an inva­sive tech­nique will always have an impact on the organ­ism being observed, how­ev­er small. Chiara Stringari, on the oth­er hand, relies on the flu­o­res­cent mol­e­cules already present in cells – in par­tic­u­lar NADH and FAD, which are present in all our liv­ing cells and pro­vide infor­ma­tion on metab­o­lism. They can be stim­u­lat­ed with­out being arti­fi­cial­ly inter­fer­ing with the liv­ing sys­tem. It is tech­nique that is still in the pre-clin­i­cal phase. So, tests on humans have there­fore not yet begun; exper­i­ments are still lim­it­ed to the in vit­ro (on arti­fi­cial organ­isms) and in vivo (in par­tic­u­lar on zebrafish and mice) stages.

Exam­ple of the use of two-pho­ton microscopy on a zebrafish. Source : Chiara Stringari1

Observing the molecule without tagging it

Bypass­ing the “tag­ging” stage there­fore makes this tech­nique, which is still under devel­op­ment, as inno­v­a­tive as it is use­ful. “It is the core of my research to devel­op new meth­ods using non-lin­ear optics,” states Chiara Stringari. It gives researchers access to new infor­ma­tion, while using con­trasts or endoge­nous bio­mark­ers – that remove the need for tag­ging – so that this tech­nique is as non-inva­sive as pos­si­ble.” The abil­i­ty to observe the activ­i­ty of mol­e­cules, like that of any oth­er cell in our body, as close­ly as pos­si­ble, thus allows us to under­stand their devel­op­ment, with­out dam­ag­ing the bio­log­i­cal sys­tems observed.

Two-pho­ton excit­ed Flu­o­res­cence Microscopy of NADH and FAD in the stem cell of the epi­der­mis of a recon­struct­ed skin. Source : Chiara Stringari2
Two-pho­ton excit­ed Flu­o­res­cence life­time Microscopy of NADH and FAD reveals meta­bol­ic gra­di­ents in recon­struct­ed skin. Source : Chiara Stringari3

Acquir­ing this type of knowl­edge opens the way to many appli­ca­tions in med­i­cine, as it allows researchers to map cel­lu­lar metab­o­lism – an indi­ca­tor of activ­i­ty in cells. “Espe­cial­ly since metab­o­lism, which is very impor­tant for devel­op­ment, affects epi­ge­net­ics,” she explains. “Map­ping allows us to cre­ate a mod­el and bet­ter under­stand the inter­est and role of the dif­fer­ent con­nec­tions between cells. And there­fore, to under­stand the inter­ac­tions they have with their envi­ron­ment.” This, in turn, allows the iden­ti­fi­ca­tion of the metab­o­lism of can­cer cells, which are like­ly to devel­op many forms of can­cer. “The bio­mark­ers used allowed us to dis­so­ci­ate healthy cells from can­cer cells. The bio­mark­ers used have allowed us to sep­a­rate healthy cells from can­cer cells, allow­ing us to estab­lish phe­no­typ­ic meta­bol­ic sub­types of cancer.”

Better understand how brain cells work

“The aim is not to diag­nose, but this tech­nique does pro­vide many tools to facil­i­tate this. In my opin­ion, the most impor­tant thing is to bet­ter under­stand how our brain cells work.”

These obser­va­tions give us infor­ma­tion about the envi­ron­ment of the mol­e­cules. It allows us to under­stand how they inter­act with their sur­round­ings, and what acti­vates them. The researchers make an exper­i­men­tal mea­sure­ment on a sub-cel­lu­lar scale (< 1 µm), in order to estab­lish a map of the metab­o­lism and iden­ti­fy the lack (or abun­dance) of cer­tain mol­e­cules in our cells. Espe­cial­ly since “each cell has a sort of fin­ger­print of its metab­o­lism”, she says. 

Using anoth­er non-labelling tech­nique – that of the third har­mon­ic gen­er­a­tion, which com­ple­ments two-pho­ton flu­o­res­cence – Chiara Stringari is work­ing in par­tic­u­lar on imag­ing myelin, a lipid sheath which is “very impor­tant in the con­nec­tion and meta­bol­ic sup­port of neu­rones,” she explains. Study­ing a 3D rep­re­sen­ta­tion of myelin allows her to under­stand the impact that its degra­da­tion can have on the metab­o­lism of neu­rones, in com­bi­na­tion with the data obtained using the two-pho­ton tech­nique. “This allows us to learn more about our brains, while also pro­vid­ing leads for research into diag­no­sis, both of which are very complementary.” 

Mul­ti­ple scle­ro­sis is a dis­ease that affects myelin, caus­ing its degra­da­tion (demyeli­na­tion) and neu­rode­gen­er­a­tion. Chiara Stringari and her team have there­fore under­tak­en research on this dis­ease, in ex vivo con­di­tions, with a view to estab­lish­ing “the bio­log­i­cal con­se­quences of myelin pathol­o­gy at the lev­el of cel­lu­lar metab­o­lism and neu­ronal net­works”. Com­par­ing the func­tion of dis­tinct phas­es known as myeli­na­tion and demyeli­na­tion makes it pos­si­ble to learn more about how it works, its cel­lu­lar activ­i­ty and, above all, its repair. 

Indeed, many dis­eases are caused by the degra­da­tion of brain cells; these are known as neu­rode­gen­er­a­tive dis­eases. The stud­ies under­tak­en by Chiara Stringari could help in the iden­ti­fi­ca­tion of effec­tive ther­a­peu­tic strate­gies. And it will allow us to go even fur­ther, by under­stand­ing how a cell ini­ti­ates its degra­da­tion, we could, per­haps one day, pre­vent this process. 

Interview by Pablo Andres
1Stringari C., Abde­ladim L., Malkin­son G., Mahou P., Soli­nas X., Lamarre I., Brizion S., Galey J.B, Supat­to W., Legouis R., Pena A.M., Beau­re­paire E. (2017)  Mul­ti­col­or two-pho­ton imag­ing of endoge­nous flu­o­rophores in liv­ing tis­sues by wave­length mix­ing Sci. Rep. 7(1):3792. https://www.nature.com/articles/s41598-017–03359‑8
2Ung T.P.L, Lim S., Soli­nas X., Mahou P., Ches­sel A., Mar­i­on­net C., Born­schlögl T., Beau­re­paire E., Bern­erd F., Pena A.M.*, Stringari C.* Simul­ta­ne­ous NAD(P)H and FAD Flu­o­res­cence Life­time Microscopy of long UVA–induced meta­bol­ic stress in recon­struct­ed human skin. Sci­en­tif­ic Report, Sci Rep. 2021 11(1):22171. https://www.nature.com/articles/s41598-021–00126‑8
3Stringari C., Abde­ladim L., Malkin­son G., Mahou P., Soli­nas X., Lamarre I., Brizion S., Galey J.B, Supat­to W., Legouis R., Pena A.M., Beau­re­paire E. (2017)  Mul­ti­col­or two-pho­ton imag­ing of endoge­nous flu­o­rophores in liv­ing tis­sues by wave­length mix­ing Sci. Rep. 7(1):3792. https://www.nature.com/articles/s41598-017–03359‑8