Urany-Less Low Voltage Transmission Electron Microscopy: A Powerful Tool for Ultrastructural Studying of Cyanobacterial Cells

Ci a ion: M azo a, K.; Baco sky, J.; Sed lo a, Z.; Slanino a, E.; Ob uca, S.; F i z, I.; K zyzanek, V. U any-Less Low Vol age T ansmission Elec on Mic oscopy: A Powe ul Tool o Ul as uc u al S udying o Cyanobac e ial Cells. Mic oo ganisms 2023,11, 888. h ps://doi.o g/ 10.3390/mic oo ganisms11040888 Academic Edi o : Juan M. Gonzalez Recei ed: 7 Ma ch 2023 Re ised: 27 Ma ch 2023 Accep ed: 28 Ma ch 2023 Published: 29 Ma ch 2023 Copy igh : © 2023 by he au ho s. Licensee MDPI, Basel, Swi ze land. This a icle is an open access a icle dis ibu ed unde he e ms and condi ions o he C ea i e Commons A ibu ion (CC BY) license (h ps:// c ea i ecommons.o g/licenses/by/ 4.0/). mic oo ganisms A icle U any-Less Low Vol age T ansmission Elec on Mic oscopy: A Powe ul Tool o Ul as uc u al S udying o Cyanobac e ial Cells Ka e ina M azo a 1, Ja omi Baco sky 2, Zuzana Sed lo a 3, E a Slanino a 3, S anisla Ob uca 3, Ines F i z 4 and Vladisla K zyzanek 1,* 1Ins i u e o Scien i ic Ins umen s o he Czech Academy o Sciences, . .i., K alo opolska 147, 612 64 B no, Czech Republic; [email p o ec ed] 2Delong Ins umen s a.s., Palackeho T ida 3019/153 b, 612 00 B no, Czech Republic; ja omi [email p o ec ed] 3Depa men o Food Chemis y and Bio echnology, Facul y o Chemis y, B no Uni e si y o Technology, Pu kyno a 118, 612 00 B no, Czech Republic; [email p o ec ed] (Z.S.); [email p o ec ed] (E.S.); [email p o ec ed] (S.O.) 4 Ins i u e o En i onmen al Bio echnology, Depa men o Ag obio echnology, IFA-Tulln, Uni e si y o Na u al Resou ces and Li e Sciences, Kon ad-Lo enz-S ase 20, 3430 Tulln an de Donau, Aus ia; [email p o ec ed] *Co espondence: [email p o ec ed] Abs ac : Sample p epa a ion p o ocols o con en ional high ol age ansmission elec on mi- c oscopy (TEM) hea ily ely on he usage o s aining agen s con aining a ious hea y me als, mos commonly u anyl ace a e and lead ci a e. Howe e high oxici y, ising legal egula ions, and p oblema ic was e disposal o u anyl ace a e ha e inc eased calls o he educ ion o e en comple e eplacemen o his s aining agen . One o he s a egies o u anyless imaging is he employmen o low- ol age ansmission elec on mic oscopy. To in es iga e he in luence o di e en imaging and s aining s a egies on he inal image o cyanobac e ial cells, samples s ained by u anyl ace a e wi h lead ci a e, as well as uns ained samples, we e obse ed using TEM and accele a ing ol ages o 200 kV o 25 kV. Mo eo e , o examine he possibili ies o educing ch oma ic abe a ion, which o en causes issues when imaging using elec ons o lowe ene gies, samples we e also imaged using a scanning ansmission elec on mic oscopy a 15 kV accele a ing ol ages. The esul s o his s udy demons a e ha low- ol age elec on mic oscopy o e s g ea po en ial o u anyless elec on mic oscopy. Keywo ds: low ol age elec on mic oscopy; u anyl ace a e; con as ing agen s; ansmission elec on mic oscopy; Synechocys is; polyhyd oxyalkanoa es 1. In oduc ion To da e, ansmission elec on mic oscopy (TEM) is commonly he numbe one me hod used o obse ing bo h he shape and in acellula space o cells [ 1 , 2 ]. The cu en mi- c oscopes ha e e ealed specimen de ails igh down o he a omic s uc u e [ 3 , 4 ], ye he esolu ion is no he only pa ame e used o e alua e image quali y. The second pa ame e , which is o equal impo ance as he spa ial esolu ion, is image con as . This pa ame e s a s o be highly c ucial, especially when imaging biological sec ions o o he specimens composed o ligh elemen s. B ie ly, he inciden elec ons in he ansmission elec on mic oscope ha in e ac wi h he sample a e dependen on he hickness and chemical composi ion o he ma e [ 5 , 6 ]. Since biological samples a e composed o ligh elemen s and a e also embedded in ca bon- based esins, he in e ac ions be ween he p ima y elec on beam and he sample a e no su icien o p o ide sa is ac o y con as s [6]. Mic oo ganisms 2023,11, 888. h ps://doi.o g/10.3390/mic oo ganisms11040888 h ps://www.mdpi.com/jou nal/mic oo ganisms Mic oo ganisms 2023,11, 888 2 o 13 The e a e se e al ways o deal wi h low image con as . Con en ional imaging o biological samples equi es specimen s aining. [ 7 , 8 ]. Howe e , such sample p epa a ion o TEM includes he use o a ious oxic subs ances, namely osmium e oxide, o o ganic sal s o hea y me als, such as u anium and lead, which a e used as pos ixa ion o con- as ing agen s [ 7 , 9 ]. Speci ically high oxici y, oge he wi h ising legal egula ions and p oblema ic was e disposal o u anyl ace a e, is egula ly used as a con as ing agen o ul a hin sec ions alongside a nega i e s aining agen , ha e aised he need o changes in con en ional biological sample p epa a ion p ocedu es o TEM [10,11]. Se e al s a egies on how o eplace u anyl ace a e a e being es ed. Sal s o he lan hanoid se ies o elemen s pose as he mos equen ly sui able subs i u es [ 11 – 13 ]. In e es ingly, some publica ions also sugges oolong ea ex ac as a s aining agen o ul a hin sec ions [14–16]. A di e en app oach, how o inc ease he con as o he image wi hou hea y me al s aining, is o al e he imaging echnique i sel . I can be ealized by cons uc ion imp o emen s o elec on op ics, such as a mic oscope ha can be equipped wi h a phase pla e [ 17 , 18 ], o ano he s a egy is o lowe he accele a ing ol age o he p ima y elec on beam [19,20]. To explain he o igin o he image con as , he p e iously men ioned elec on in e ac- ions wi h he sample ha e o be conside ed. The e a e se e al mechanisms in ol ed in he con as o ma ion o TEM images, connec ed o he a ious scena ios o he inciden elec- on beam in e ac ion wi h he sample. Elec ons can be elas ically o inelas ically sca e ed o hey can go h ough he sample wi hou any in e ac ion, while all o hese phenomena mani es di e en ly in he inal image. Elec ons ansmi ed h ough he sample wi hou any in e ac ion con ibu e o he b igh backg ound o he image. Elas ically sca e ed elec ons a e il e ed by he ape u es and, hus, inc ease he image con as . The mos p oblema ic a e inelas ically sca e ed elec ons, which lose some ene gy in e ac ing wi h he sample, because hey b oaden he ene gy spec um and, hus, b ing an addi ional con i- bu ion o he ch oma ic abe a ion. The p obabili y o each kind o in e ac ion is dependen on he alue o he c oss-sec ion, which is speci ic o a pa icula ype o in e ac ion, sample composi ion, and elec on ene gy [5,6]. Fo con en ional ansmission elec on mic oscopes ha use accele a ing ol ages be ween 60 and 300 kV [ 6 , 19 ], phase con as is essen ial [ 6 ]. Howe e , going o he lowe beam ene gy, phase con as is no longe as necessa y and he sca e ing con as becomes he mos impo an componen o he con as . Reducing he elec on beam ene gy leads o highe sca e ing and, hus, o highe image con as s. In o he wo ds, he in ensi y o he elec on in e ac ion wi h he sample is dependen on he hickness and chemical composi ion o he ma e [6,20,21]. The dependence o he con as o he esul ing image on he accele a ing ol age o he p ima y elec on beam in Figu e 1shows ha he con as o he image dec eases wi h highe elec on ene gies, while he esolu ion inc eases [ 20 ]. As was desc ibed o a ious laye s o polyme s and polyme blends, imaging using lowe ene gies p o ides he possibili y o e en dis inguish a eas o di e en composi ions in ca bon-based samples [20,22,23]. S udies employing low- ol age ansmission elec on mic oscopy o he examina ion o biological samples ha e al eady p o en he bene i s o imaging a an accele a ing ol age o 5 kV [ 19 , 24 ]. Howe e , using such low ene gies comes oge he wi h he need o e en hinne samples han he con en ional ~70 nm. Speci ically, o obse e he panc ea ic issue o a a , Bendayan e al. chemically ixed he samples using glu a aldehyde wi hou any pos ixa ion p ocedu e, while o compa ison osmi ica ed samples we e also p epa ed. Samples embedded in Epon esin we e cu o ul a hin sec ions o 30–40 nm hickness. Obse a ion using a low ol age elec on mic oscope (LVEM) e ealed ha imaging using a 5 kV elec on beam p o ided su icien con as o he esul ing image and was able o expose s uc u es, which we e o he wise co e ed by osmium o o he hea y me als. [ 24 ] Ne e heless, he p epa a ion o sec ions 30–40 nm hin is qui e challenging o common use s o ul amic o ome, e en when using a diamond kni e [ 25 ]. Howe e , using mode - Mic oo ganisms 2023,11, 888 3 o 13 a ely highe accele a ing ol ages o 25 kV, he elec ons o he p ima y elec on beam had su icien ene gy o pene a e he hicke common sec ions o 70 nm [26]. Mic oo ganisms 2023, 11, x FOR PEER REVIEW 3 o 14 me als. [24] Ne e heless, he p epa a ion o sec ions 30–40 nm hin is qui e challenging o common use s o ul amic o ome, e en when using a diamond kni e [25]. Howe e , using mode a ely highe accele a ing ol ages o 25 kV, he elec ons o he p ima y elec- on beam had sufficien ene gy o pene a e he hicke common sec ions o 70 nm [26]. Figu e 1. The dependence o con as and esolu ion on he accele a ing ol age o he p ima y elec on beam o TEM imaging o 20 nm ca bon laye s [20,27]. The model o ganism o choice, wi h well-desc ibed in acellula s uc u es o ou s udy, was he unicellula cyanobac e ium o PHA-p oducing s ain Synechocys is sp., PCC 6803. Cyanobac e ia belong o o ganisms signi ican in bo h he ields o science and indus y. They a e capable o g owing in di e se en i onmen s and can adap o a ious ad e se g ow h condi ions (e.g., hype saline en i onmen [28], high o low empe a u es [29,30], UV i adia ion [31], e c.). Mo eo e , many cyanobac e ial s ains pose as p oduc- e s o bio echnologically aluable subs ances [32–34]. Speci ically, membe s o he genus Synechocys is a e, along wi h some he e o ophic mic oo ganisms, capable o p oducing polyhyd oxyalkanoa es (PHAs), which a e polyes e s o hyd oxy acids ha o m g anules inside a ious mic obial cells [35–37]. In addi ion o se ing as s o age o ca bon and ene gy, PHAs ha e a signi ican ole in he enhanced obus ness o cells and, he e o e, in he capabili y o he cells o su i e ha sh en i onmen al condi ions [38,39]. The abili y o cope wi h un a o able en i onmen al condi ions o en comes oge he wi h he possibili y o mo phological changes o he cells, as was p e iously p o en o bo h he e o ophic bac e ia and cyanobac e ia. Fo a ious mic oo ganisms, he obse ed changes in he shape and size o cells, as well as he o ma ion o p ecipi a es and o he changes in he cy oplasm we e ela ed o exposu e o a ious s ess condi ions [40–42]. In ou s udy, we aimed o p o e ha an elec on beam o 15–25 kV was s ill capable o p o iding high con as o ligh e elemen s and, he e o e, i was possible o obse e biological samples wi hou he necessi y o hea y me als s aining. Samples we e ixed us- ing he high-p essu e eezing me hod, ollowed by eeze subs i u ion. Then, samples we e obse ed using TEMs ope a ing a 200 kV and 25 kV accele a ing ol ages. Bo h mi- c oscopes we e used o he analysis o he samples s ained using sal s o u anyl ace a e and lead ci a e alongside samples wi hou s aining wi h hea y me als. To demons a e he possibili y o how o minimize ch oma ic abe a ion a lowe accele a ing ol ages, imaging wi h a scanning ansmission elec on mic oscope (STEM) mode was also pe - o med. Figu e 1. The dependence o con as and esolu ion on he accele a ing ol age o he p ima y elec on beam o TEM imaging o 20 nm ca bon laye s [20,27]. The model o ganism o choice, wi h well-desc ibed in acellula s uc u es o ou s udy, was he unicellula cyanobac e ium o PHA-p oducing s ain Synechocys is sp., PCC 6803. Cyanobac e ia belong o o ganisms signi ican in bo h he ields o science and indus y. They a e capable o g owing in di e se en i onmen s and can adap o a ious ad e se g ow h condi ions (e.g., hype saline en i onmen [ 28 ], high o low em- pe a u es [ 29 , 30 ], UV i adia ion [ 31 ], e c.). Mo eo e , many cyanobac e ial s ains pose as p oduce s o bio echnologically aluable subs ances [ 32 – 34 ]. Speci ically, membe s o he genus Synechocys is a e, along wi h some he e o ophic mic oo ganisms, capable o p oducing polyhyd oxyalkanoa es (PHAs), which a e polyes e s o hyd oxy acids ha o m g anules inside a ious mic obial cells [ 35 – 37 ]. In addi ion o se ing as s o age o ca bon and ene gy, PHAs ha e a signi ican ole in he enhanced obus ness o cells and, he e o e, in he capabili y o he cells o su i e ha sh en i onmen al condi ions [38,39]. The abili y o cope wi h un a o able en i onmen al condi ions o en comes oge he wi h he possibili y o mo phological changes o he cells, as was p e iously p o en o bo h he e o ophic bac e ia and cyanobac e ia. Fo a ious mic oo ganisms, he obse ed changes in he shape and size o cells, as well as he o ma ion o p ecipi a es and o he changes in he cy oplasm we e ela ed o exposu e o a ious s ess condi ions [40–42]. In ou s udy, we aimed o p o e ha an elec on beam o 15–25 kV was s ill capable o p o iding high con as o ligh e elemen s and, he e o e, i was possible o obse e biological samples wi hou he necessi y o hea y me als s aining. Samples we e ixed using he high-p essu e eezing me hod, ollowed by eeze subs i u ion. Then, samples we e obse ed using TEMs ope a ing a 200 kV and 25 kV accele a ing ol ages. Bo h mic oscopes we e used o he analysis o he samples s ained using sal s o u anyl ace a e and lead ci a e alongside samples wi hou s aining wi h hea y me als. To demons a e he possibili y o how o minimize ch oma ic abe a ion a lowe accele a ing ol ages, imaging wi h a scanning ansmission elec on mic oscope (STEM) mode was also pe o med. 2. Ma e ials and Me hods 2.1. Mic oo ganisms and Thei Cul i a ion In his s udy cul u es o he cyanobac e ium Synechocys is sp., PCC 6803, we e ob ained om he Pas eu cul u e collec ion (Pa is, F ance) and used. Cul u es we e cul i a ed in a mine al medium based on BG-11 [ 43 , 44 ]. The con en o ni ogen and phospho us Mic oo ganisms 2023,11, 888 4 o 13 was adjus ed o enable ea ly g ow h o biomass ollowed by s a a ion o ni ogen and phospho us o induce PHA p oduc ion. Cul u es in E lenmeye lasks we e cul i a ed in a anspa en box wi h con olled ai low, empe a u e, and dayligh illumina ion simula ion using a 16/8 h day/nigh cycle o illumina ion. 2.2. Sample P epa a ion o Elec on Mic oscopy Cyanobac e ial cul u es we e cen i uged o 4 min a 4000 pm and p ocessed using c yogenic me hods o sample p epa a ion. The cell pelle was pipe ed on 3 mm Au/Cu ca ie s ype A wi h 1% solu ion o soy leci hin in chlo o o m and co e ed wi h he la side o 3 mm Au/Cu ca ie ype B. Samples we e ixed using high-p essu e eezing (EM ICE, Leica Mic osys ems, Vienna, Aus ia), ollowed by eeze subs i u ion (EM AFS2, Leica Mic osys ems, Vienna, Aus ia). The subs i u ion solu ion con ained 1.5% OsO 4 in ace one and he p ocedu e was se o − 90 ◦ C o 72 h, hen, he samples we e wa med up o − 20 ◦ C o 24 h and he p ocedu e inished, wi h he inal phase a 4 ◦ C o 18 h, as p e iously desc ibed [ 45 ]. Fixed samples we e in il a ed wi h epoxy esin (Epoxy Embedding Medium ki , Sigma Ald ich, Da ms ad , Ge many ) and cu ed o 48 h a 62 ◦ C. Embedded samples we e cu in o ul a hin sec ions (~75 nm) using a diamond kni e (Ul a 45 ◦ , DiATOME, Nidau, Swi ze land) and ul amic o ome (EM UC7, Leica Mic osys ems, Vienna, Aus ia). Hal o he sec ions we e obse ed wi hou any pos s aining p ocedu es, while he o he hal we e s ained using con en ional s aining agen s: u anyl ace a e and lead ci a e. 2.3. Elec on Mic oscopy Cyanobac e ial cells we e obse ed using a ious ypes o elec on mic oscopes. As p e iously men ioned, he con en ional ansmission elec on mic oscope ope a es wi hin he ange o 60–300 kV accele a ing ol ages o he elec on beam [ 6 , 19 ]. In ou s udy, we compa ed wo di e en ansmission elec on mic oscopes using di e en accele a ing ol ages o he elec on beam: Talos F200C ope a ing a 200 kV and LVEM 25 ope a ing a 25 kV. Mo eo e , con en ional TEM imaging equi es s aining o ul a hin sec ions commonly using u anyl ace a e and lead ci a e [ 7 , 8 ], which should no be necessa y o low- ol age imaging [ 24 ]. Thus, o p o ide a ho ough s udy o he in luence o a ious imaging condi ions we compa ed no only imaging using di e en elec on beam ene gies bu also imaging o samples pos s ained using only osmium e oxide, p esen in he eeze subs i u ion solu ion, and also con en ionally s ained samples, using u anyl ace a e and lead ci a e. Con en ional high- ol age imaging was ca ied ou on a ansmission elec on mic o- scope Talos F200C (The mo Fishe Scien i ic, Wal ham, MA, USA), equipped wi h a Ce a-D Came a, using an elec on beam ol age o 200 kV. Low- ol age images we e ob ained using a ansmission elec on mic oscope LVEM 25 (Delong Ins umen s, B no, Czech Republic) equipped wi h an sCMOS came a BSI Teledyne o TEM mode, using a ol age o 25 kV. Mo eo e , o explo e he possibili ies o educing he ch oma ic abe a ion when imaging using low ene gies o he elec on beam, he STEM mode o LVEM 25, using a YAG sc een, ollowed by a pho omul iplie o egis e STEM signal a 15 kV elec on beam ol age, was employed as well as imaging a he same ol age using a scanning elec on mic oscope Helios G4 HP (The moFishe Scien i ic, Wal ham, MA, USA) equipped wi h a STEM3+ de ec o , while he b igh ield segmen o he de ec o was selec ed o bo h mic oscopes. 3. Resul s 3.1. High Vol age T ansmission Elec on Mic oscopy The in luence o he s aining p ocedu e on con en ional TEM imaging was subs an ial. As seen in Figu e 2A, e en hough he OsO 4 p o ided sub le con as in he in acellula Mic oo ganisms 2023,11, 888 5 o 13 s uc u es o he cyanobac e ial cells, compa ed o he s ained sample in Figu e 2B, he ul as uc u e is almos indis inguishable. Mic oo ganisms 2023, 11, x FOR PEER REVIEW 5 o 14 3. Resul s 3.1. High Vol age T ansmission Elec on Mic oscopy The in luence o he s aining p ocedu e on con en ional TEM imaging was subs an- ial. As seen in Figu e 2A, e en hough he OsO4 p o ided sub le con as in he in acel- lula s uc u es o he cyanobac e ial cells, compa ed o he s ained sample in Figu e 2B, he ul as uc u e is almos indis inguishable. Figu e 2. Compa ison o he in luence o diffe en s aining p ocedu es and ene gies o he elec on beam in he inal TEM image on cyanobac e ial cells. (A) A 200 kV accele a ing ol age o elec on beam wi hou s aining, (B) 200 kV accele a ing ol age o elec on beam and s aining wi h u anyl ace a e and lead ci a e, (C) 25 kV accele a ing ol age o elec on beam wi hou s aining, (D) 25 kV accele a ing ol age o elec on beam and s aining wi h u anyl ace a e and lead ci a e. Scale ba : 1 µm. Figu e 2. Compa ison o he in luence o di e en s aining p ocedu es and ene gies o he elec on beam in he inal TEM image on cyanobac e ial cells. ( A ) A 200 kV accele a ing ol age o elec on beam wi hou s aining, ( B ) 200 kV accele a ing ol age o elec on beam and s aining wi h u anyl ace a e and lead ci a e, ( C ) 25 kV accele a ing ol age o elec on beam wi hou s aining, ( D ) 25 kV accele a ing ol age o elec on beam and s aining wi h u anyl ace a e and lead ci a e. Scale ba : 1µm. E en he mo e de ailed images o he uns ained sample obse ed using a 200 kV elec on beam p o ided minimal con as wi h which o ecognize he in acellula s uc- u es. As shown in Figu e 3, he hylakoid memb anes o he cyanobac e ial cell a e ha dly dis inguishable wi hin he in acellula space and could e en be conside ed ‘noise’ in he image, while o he s uc u es, possibly occu ing in he cyanobac e ial cells, a e mos ly un ecognizable. Mic oo ganisms 2023,11, 888 6 o 13 Mic oo ganisms 2023, 11, x FOR PEER REVIEW 6 o 14 E en he mo e de ailed images o he uns ained sample obse ed using a 200 kV elec- on beam p o ided minimal con as wi h which o ecognize he in acellula s uc u es. As shown in Figu e 3, he hylakoid memb anes o he cyanobac e ial cell a e ha dly dis- inguishable wi hin he in acellula space and could e en be conside ed ‘noise’ in he image, while o he s uc u es, possibly occu ing in he cyanobac e ial cells, a e mos ly un ecognizable. Figu e 3. TEM image o uns ained cyanobac e ial cells ob ained using a 200 kV elec on beam. Thylakoid memb anes ma ked by a ow. Scale ba : 500 nm. On he o he hand, when ollowing he con en ional p ocedu e, he de ailed images o he s ained sample, obse ed using a 200 kV elec on beam (Figu e 4A), p o ide us wi h balanced con as and, he e o e, he in acellula s uc u es such as hylakoid memb anes (ma ked by a ow), ca boxysome (ma ked by iangle), glycogen g anules (ma ked by “gg”) a e easily dis inguishable, while i is e en possible o ecognize he diffe ence be- ween he elec on-lucen PHA g anules (ma ked by a owhead) and he holes in he spec- imen owing o he emains o he washed-away g anules (ma ked by c oss) [46]. Figu e 3. TEM image o uns ained cyanobac e ial cells ob ained using a 200 kV elec on beam. Thylakoid memb anes ma ked by a ow. Scale ba : 500 nm. On he o he hand, when ollowing he con en ional p ocedu e, he de ailed images o he s ained sample, obse ed using a 200 kV elec on beam (Figu e 4A), p o ide us wi h balanced con as and, he e o e, he in acellula s uc u es such as hylakoid memb anes (ma ked by a ow), ca boxysome (ma ked by iangle), glycogen g anules (ma ked by “gg”) a e easily dis inguishable, while i is e en possible o ecognize he di e ence be ween he elec on-lucen PHA g anules (ma ked by a owhead) and he holes in he specimen owing o he emains o he washed-away g anules (ma ked by c oss) [46]. Mic oo ganisms 2023, 11, x FOR PEER REVIEW 7 o 14 Figu e 4. In acellula s uc u es o cyanobac e ial cells we e acqui ed using diffe en imaging condi ions. (A) Sample s ained using u anyl ace a e and lead ci a e imaged using a 200 kV elec- on beam, (B) sample wi hou s aining imaged using a 25 kV elec on beam. In acellula s uc- u es ma ked by an a ow: hylakoid memb anes; a owhead: PHA g anules; iangle: ca boxy- some; c oss: washed-ou g anule; gg: glycogen g anule. Scale ba : 500 nm. 3.2. Low Vol age T ansmission Elec on Mic oscopy As shown in he p e ious sec ion, o ob ain sa is ac o y con as s in he images ac- qui ed by a high ol age ansmission elec on mic oscope, i is necessa y o use con- as ing agen s, mos commonly using u anyl ace a e and lead ci a e o s ain ul a hin sec ions on he g id. Howe e , i he accele a ing ol age o he elec on beam is lowe ed, he uns ained sample (Figu e 2C) p o ides an easily ecognizable ul as uc u e o he s udied cells, which is, on he o he hand, in e es ingly a he co e ed by he s aining agen s (Figu e 2D), esul ing in e y da k images o he cells wi h ha dly any dis inguish- able in acellula s uc u es. Fu he mo e, as seen in Figu e 4B, he de ailed image o an uns ained sample ob- se ed using a 25 kV elec on beam p o ides clea ly ecognizable in acellula s uc u es, and in addi ion, when ocusing on he esin su ounding he cells, he low ol age image p o ides minimum noise, whe eby only he lines le by he kni e du ing sec ioning a e sligh ly isible, while he glycogen g anules a e easily dis inguishable om he ‘noise’. 3.3. Low Vol age Scanning T ansmission Elec on Mic oscopy In scanning ansmission elec on mic oscopes (STEMs), he elec on beam is ocused on a e y small p obe and is scanned o e he sample; o each posi ion (pixel) ansmi ed elec ons a e eco ded by a sui able STEM de ec o . Mos TEM and scanning elec on mi- c oscopes (SEMs), i hey a e equipped wi h he necessa y ha dwa e, can also enable STEM image eco ding. This echnique is also e y use ul o imaging ul a hin sec ions o biological samples [47,48]. As shown in Figu e 5C, he low- ol age STEM p o ides a sha p image o he ul as uc u e o he cyanobac e ial cells wi h a sa is ac o y con as . Simila ly o he low- ol age TEM, he s ained sec ions o he samples p o ided a highe con as han he uns ained samples (Figu e 5D). Howe e , in he STEM images, i is s ill possible o ecognize he elemen al s uc u es o he cells. Figu e 4. In acellula s uc u es o cyanobac e ial cells we e acqui ed using di e en imaging condi ions. ( A ) Sample s ained using u anyl ace a e and lead ci a e imaged using a 200 kV elec on beam, ( B ) sample wi hou s aining imaged using a 25 kV elec on beam. In acellula s uc u es ma ked by an a ow: hylakoid memb anes; a owhead: PHA g anules; iangle: ca boxysome; c oss: washed-ou g anule; gg: glycogen g anule. Scale ba : 500 nm. Mic oo ganisms 2023,11, 888 7 o 13 3.2. Low Vol age T ansmission Elec on Mic oscopy As shown in he p e ious sec ion, o ob ain sa is ac o y con as s in he images ac- qui ed by a high ol age ansmission elec on mic oscope, i is necessa y o use con as ing agen s, mos commonly using u anyl ace a e and lead ci a e o s ain ul a hin sec ions on he g id. Howe e , i he accele a ing ol age o he elec on beam is lowe ed, he uns ained sample (Figu e 2C) p o ides an easily ecognizable ul as uc u e o he s udied cells, which is, on he o he hand, in e es ingly a he co e ed by he s aining agen s (Figu e 2D), esul ing in e y da k images o he cells wi h ha dly any dis inguishable in acellula s uc u es. Fu he mo e, as seen in Figu e 4B, he de ailed image o an uns ained sample obse ed using a 25 kV elec on beam p o ides clea ly ecognizable in acellula s uc u es, and in addi ion, when ocusing on he esin su ounding he cells, he low ol age image p o ides minimum noise, whe eby only he lines le by he kni e du ing sec ioning a e sligh ly isible, while he glycogen g anules a e easily dis inguishable om he ‘noise’. 3.3. Low Vol age Scanning T ansmission Elec on Mic oscopy In scanning ansmission elec on mic oscopes (STEMs), he elec on beam is ocused on a e y small p obe and is scanned o e he sample; o each posi ion (pixel) ansmi ed elec ons a e eco ded by a sui able STEM de ec o . Mos TEM and scanning elec on mic oscopes (SEMs), i hey a e equipped wi h he necessa y ha dwa e, can also enable STEM image eco ding. This echnique is also e y use ul o imaging ul a hin sec ions o biological samples [ 47 , 48 ]. As shown in Figu e 5C, he low- ol age STEM p o ides a sha p image o he ul as uc u e o he cyanobac e ial cells wi h a sa is ac o y con as . Simila ly o he low- ol age TEM, he s ained sec ions o he samples p o ided a highe con as han he uns ained samples (Figu e 5D). Howe e , in he STEM images, i is s ill possible o ecognize he elemen al s uc u es o he cells. To p o e ha he use ulness o he low- ol age imaging is no bound solely h ough he specialized low- ol age TEM, he same samples we e also imaged using a SEM equipped wi h a STEM de ec o also ope a ing a a 15 kV accele a ing ol age. As seen in Figu e 6A, he o e all con as o he uns ained sample is compa able o bo h mic oscopes, while he specialized low- ol age TEM p o ided sha pe images. Howe e , he image o he s ained sample (Figu e 6B) shows mo e a balanced con as han in he specialized low- ol age TEM, while he image is also sha pe han in he uns ained sample imaged by a SEM equipped wi h a STEM de ec o , which is demons a ed by he s uc u e o glycogen g anules. Ne e heless, he p oblem o possible con amina ion o he sample by p ecipi a ed con as ing agen s also emains o STEM imaging, simila o he isk o con en ional TEM imaging. In Figu e 7B, he impu i ies caused by he p ecipi a ed con as ing agen s a e clea ly isible, as well as he impu i ies in Figu e 7A, whe e he esidual osmium e oxide om he eeze subs i u ion solu ion could ha e c ys alized and le he elec on-dense impu i ies in he sample. Howe e , he ex en o he con amina ion by he impu i ies is sub- s an ially highe o he con en ional s aining p ocedu e han o he osmium-only s aining. Mic oo ganisms 2023,11, 888 8 o 13 Mic oo ganisms 2023, 11, x FOR PEER REVIEW 8 o 14 Figu e 5. Compa ison o he images o cyanobac e ial cells ob ained using a low ol age ansmis- sion elec on mic oscope in diffe en imaging modes. (A) Uns ained sample imaged in TEM mode using a 25 kV accele a ing ol age elec on beam, (B) sample s ained using u anyl ace a e and lead ci a e imaged in TEM mode using a 25 kV accele a ing ol age elec on beam, (C) uns ained sam- ple imaged in STEM mode using a 15 kV accele a ing ol age elec on beam, (D) sample s ained using u anyl ace a e and lead ci a e imaged in TEM mode using a 15 kV accele a ing ol age elec- on beam. Scale ba : 1 µm. To p o e ha he use ulness o he low- ol age imaging is no bound solely h ough he specialized low- ol age TEM, he same samples we e also imaged using a SEM equipped wi h a STEM de ec o also ope a ing a a 15 kV accele a ing ol age. As seen in Figu e 6A, he o e all con as o he uns ained sample is compa able o bo h mic o- scopes, while he specialized low- ol age TEM p o ided sha pe images. Howe e , he Figu e 5. Compa ison o he images o cyanobac e ial cells ob ained using a low ol age ansmission elec on mic oscope in di e en imaging modes. ( A ) Uns ained sample imaged in TEM mode using a 25 kV accele a ing ol age elec on beam, ( B ) sample s ained using u anyl ace a e and lead ci a e imaged in TEM mode using a 25 kV accele a ing ol age elec on beam, ( C ) uns ained sample imaged in STEM mode using a 15 kV accele a ing ol age elec on beam, ( D ) sample s ained using u anyl ace a e and lead ci a e imaged in TEM mode using a 15 kV accele a ing ol age elec on beam. Scale ba : 1 µm. Mic oo ganisms 2023,11, 888 9 o 13 Mic oo ganisms 2023, 11, x FOR PEER REVIEW 9 o 14 image o he s ained sample (Figu e 6B) shows mo e a balanced con as han in he spe- cialized low- ol age TEM, while he image is also sha pe han in he uns ained sample imaged by a SEM equipped wi h a STEM de ec o , which is demons a ed by he s uc u e o glycogen g anules. Figu e 6. Cyanobac e ial cells we e imaged using SEM equipped wi h a STEM de ec o wi h a 15 kV accele a ing ol age elec on beam. (A) Uns ained sample, (B) sample s ained using u anyl ace a e and lead ci a e. Glycogen g anules ma ked by “gg”. Scale ba : 1 µm. Ne e heless, he p oblem o possible con amina ion o he sample by p ecipi a ed con as ing agen s also emains o STEM imaging, simila o he isk o con en ional TEM imaging. In Figu e 7B, he impu i ies caused by he p ecipi a ed con as ing agen s a e clea ly isible, as well as he impu i ies in Figu e 7A, whe e he esidual osmium e- oxide om he eeze subs i u ion solu ion could ha e c ys alized and le he elec on- dense impu i ies in he sample. Howe e , he ex en o he con amina ion by he impu i- ies is subs an ially highe o he con en ional s aining p ocedu e han o he osmium- only s aining. Figu e 6. Cyanobac e ial cells we e imaged using SEM equipped wi h a STEM de ec o wi h a 15 kV accele a ing ol age elec on beam. ( A ) Uns ained sample, ( B ) sample s ained using u anyl ace a e and lead ci a e. Glycogen g anules ma ked by “gg”. Scale ba : 1 µm. Mic oo ganisms 2023, 11, x FOR PEER REVIEW 9 o 14 image o he s ained sample (Figu e 6B) shows mo e a balanced con as han in he spe- cialized low- ol age TEM, while he image is also sha pe han in he uns ained sample imaged by a SEM equipped wi h a STEM de ec o , which is demons a ed by he s uc u e o glycogen g anules. Figu e 6. Cyanobac e ial cells we e imaged using SEM equipped wi h a STEM de ec o wi h a 15 kV accele a ing ol age elec on beam. (A) Uns ained sample, (B) sample s ained using u anyl ace a e and lead ci a e. Glycogen g anules ma ked by “gg”. Scale ba : 1 µm. Ne e heless, he p oblem o possible con amina ion o he sample by p ecipi a ed con as ing agen s also emains o STEM imaging, simila o he isk o con en ional TEM imaging. In Figu e 7B, he impu i ies caused by he p ecipi a ed con as ing agen s a e clea ly isible, as well as he impu i ies in Figu e 7A, whe e he esidual osmium e- oxide om he eeze subs i u ion solu ion could ha e c ys alized and le he elec on- dense impu i ies in he sample. Howe e , he ex en o he con amina ion by he impu i- ies is subs an ially highe o he con en ional s aining p ocedu e han o he osmium- only s aining. Figu e 7. Cyanobac e ial cells we e imaged using SEM equipped wi h a STEM de ec o wi h a 15 kV accele a ing ol age elec on beam. ( A ) Uns ained sample, ( B ) sample s ained using u anyl ace a e and lead ci a e. Impu i ies ma ked by a ow. Scale ba : 1 µm. 4. Discussion The compa ison be ween he impac o he s aining p ocedu e and he accele a ing ol age o he elec on beam used o imaging he cyanobac e ial cells shows subs an ial di e ences o each echnique. The clea di e ence is al eady obse ed in he lowe magni ica ion images. As p e iously men ioned, he uns ained samples obse ed using he 200 kV elec on beam show almos no con as ; he e o e, he ul as uc u e o he cells is ha dly isible. On he o he hand, imaging he same uns ained sample using low accele a ing ol ages o he elec on beam p o ided a balanced con as and, he e o e, easily ecognizable in acellula s uc u es.