Introduction

Observing viral morphology is essential in virology, for which transmission electron microscopy (TEM) is the most widely used technique because it allows direct visualization at the nanometer scale. Currently, advanced TEM techniques such as cryogenic TEM and electron tomography are being rapidly developed for constructing precise three-dimensional images of viruses and small proteins1,2,3,4,5, which require expensive TEM equipment and advanced expertise. Thus, methods for simple, rapid, and clear observations using traditional TEM are needed worldwide.

Generally, negative staining methods using heavy metals are required for traditional TEM observations6,7,8,9; the procedure is illustrated in Fig. 1. Initially, virions are adsorbed on a carbon support film (Fig. 1a). A small drop of staining reagent containing heavy metals is dropped onto the film (Fig. 1b). After removing excess solution, the sample is dried, leading to coating of the virions by heavy metals (Fig. 1c). Finally, reverse-contrast images are generated via enhanced electron scattering from the heavy elements coating the virions (Fig. 1d,e).

Figure 1
figure 1

Negative staining method. (a) Virions are attached to the carbon support film. (b) A solution containing heavy metals (negative-staining reagent) is dropped onto the film. (c) Excess solution is removed, and the sample is dried. (d) Transmission electron microscopy (TEM) of heavy-metal-coated virions produces (e) a reverse-contrast image of the virus.

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Without using staining reagents, it is difficult to obtain sufficient contrast between the virions and carbon support, resulting in unclear observations of virus morphology because the fragments of virions dispersed on the carbon support film are smaller than the thickness of the film. Therefore, viruses must be coated with heavy metals that have high electron-scattering constants.

Uranyl acetate ((CH3CO2)2UO2) is among the most commonly used negative staining reagents6,7,8,9. However, because uranyl compounds are internationally controlled nuclear materials, their purchase and storage entail complicated procedures10,11. Therefore, alternatives to uranyl acetate must be developed. Phosphotungstic acid (PTA) is a substitute, and the commercially used one is Keggin-type PTA (H3PW12O40) (Supplementary Figs. S1 and S2). It is a protonated Keggin-type phosphotungstate ([PW12O40]3): a ball-shaped molecule having one central tetragonal PO4 unit surrounded by 12 octahedral WO6 units with Td symmetry (Fig. 2a)12. Keggin-type PTA is a highly acidic compound and is mainly used after neutralization with NaOH or KOH6,7,8,9. TEM images obtained using this PTA are less clear than those obtained using uranyl acetate. Therefore, other tungsten reagents such as sodium silicotungstate and methylamine tungstate13 have been considered as alternative negative-staining reagents7.

Figure 2
figure 2

(a) Polyhedral representation of Keggin-type phosphotungstate ([PW12O40]3−). (b) Polyhedral and (c) ball-and-stick representation of Preyssler-type phosphotungstate, ([P5W30O110Mn+](15−n)−). Blue and green polyhedra represent tetragonal PO4 and octahedral WO6, respectively. Blue, green, grey, red, and black balls represent P, W, encapsulated cation M (Na+, Ca2+, Bi3+, Y3+, or Eu3+), O, and O (H2O), respectively.

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These tungsten reagents, along with Keggin-type phosphotungstate, belong to the class of polyoxotungstates, which are anionic tungsten oxide clusters12. Polyoxotungstates have diverse molecular structures and physico-chemical properties such as stability, solubility, acidity, and crystallinity. Based on this information, we examined a new high-performance negative staining reagent using members of this family of compounds.

Preyssler-type phosphotungstate is a doughnut-shaped molecule with one encapsulated cation (M = Na+, Ca2+, Eu3+, Bi3+, or Y3+) and 5 tetragonal PO4 surrounded by 30 octahedral WO6 with C5v symmetry (Fig. 2b,c). Preyssler-type phosphotungstate is stable over a wide pH range (pH 1–12) and is produced as a potassium salt; thus, it can be used without neutralization14. We previously reported that Preyssler-type phosphotungstate (K12[P5W30O110Eu]) can be used as a negative staining reagent to observe the approximately 9-nm-thick fimbriae of a bacterium (Edwardsiella tarda)15. In the current study, we demonstrate that Preyssler-type polyoxotungstates (K(15-n)[P5W30O110Mn+], M = Na+, Ca2+, Eu3+, Bi3+, or Y3+) are high performance negative-staining reagents for visualizing viruses, which are much smaller than bacteria. We observed the stained virion samples using TEM, scanning electron microscopy (SEM), and atomic force microscopy (AFM) to determine the staining ability of each negative staining reagent.

Results and discussion

Comparison of negative-staining reagents

Figure 3 shows the TEM images of T4 phages obtained using two common negative staining reagents, uranyl acetate and neutralized Keggin-type PTA, and the potassium salt of Na-encapsulated Preyssler-type phosphotungstate ((K14[P5W30O110Na]) as negative staining reagents. Enterobacteria phage T4 (family Myoviridae) was selected as a model virus because its detailed morphology has been established16. The T4 phage is constructed from a head with an elongated icosahedron shape, tail part, and base plate. In addition, it has six whisker-like short fibers and long tail fibers (Fig. 3a)17. Although the head and tail part were clearly visible using uranyl acetate, the short and long fibers were not observed (Fig. 3b,c), which are similar to reported TEM images18,19,20,21. The head, tail part, and long tail fibers were observable using the neutralized Keggin-type PTA; however, the background was not homogeneous (Fig. 3d). In contrast, the background, head, tail part, and long tail fibers were all clearly observed using the potassium salt of Na-encapsulated Preyssler-type phosphotungstate (Fig. 3e,f). It has been reported that the long tail fibers were also observed by using uranyl acetate17,22. However, it is worth to note that we could observe clear images without radioactive uranyl acetate.

Figure 3
figure 3

(a) Morphology of T4 phage. (b–e) Transmission electron microscopy (TEM) images of T4 phages using negative staining reagents (b, c) uranyl acetate, (d) neutralized Keggin-type PTA (H3[PW12O40]–KOH), and (e, f) potassium salt Na-encapsulated Preyssler-type phosphotungstate (K14[P5W30O110Na(H2O)]). (g) Atomic force microscopy (AFM) image of the TEM grid.

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Concentration effect of staining reagents

The concentration of K14[P5W30O110Na(H2O)] in the negative staining solution is an important factor affecting image clarity. We examined the concentration effect of K14[P5W30O110Na(H2O)] (Supplementary Fig. S3) and the neutralized Keggin-type PTA (Supplementary Fig. S4) in the staining solution. When the concentration of K14[P5W30O110Na(H2O)] was high (for example, 2.0 wt%), crystal-like plates of staining reagent were observed, and the phage shape was unclear. Moreover, crystal formation was observed in the SEM image. These images were categorized as image A. Only the heads were observable in image A, which was obtained from approximately 57% of the TEM grid area (Supplementary Fig. S3). From the other 40% of the grid, we obtained images categorized as image B, in which staining reagents coated the T4 phages and parts of the carbon film. In image B, no long tail fibers were observed. However, instances of staining reagents coating T4 phages and clear phage images from only a few percent of the grid were obtained (categorized as image C). In image C, the long tail fibers were clearly observable. The concentration of Preyssler compound in the staining solution was directly proportional to the area where image A was observed and inversely proportional to the areas of images B and C. Moreover, the area ratio of image C reached a maximum when the concentration was 0.3 wt%. However, an area with low contrast was also observed (categorized as image D) that was inversely proportional to the concentration.

Furthermore, an AFM image obtained from image area C on the TEM grid (Fig. 3g and Supplementary Fig. S5) after TEM observation revealed head and tails. The observed length (230 and 225 nm) of the T4 body (head and tail together) was close to that expected for a T4 phage (Fig. 3a). However, the observed height of the head (47 nm) (Supplementary Fig. S5) was less than its thickness (78 nm in Fig. 3a), indicating that the head shrank under TEM vacuum conditions.

In the case of the neutralized Keggin-type PTA (Supplementary Fig. S4), no crystal formation was observed in the high-concentration samples. Although decreasing the concentration improved the images, better images were obtained using Preyssler-type phosphotungstate.

Difference between Preyssler-type and Keggin-type phosphotungstates

Keggin-type and Preyssler-type phosphotungstates exhibit greatly different stabilities in neutral solution. Supplementary Fig. S6 shows the pH titration curves of Keggin-type PTA and Na-encapsulated Preyssler-type PTA in aqueous solution. For Preyssler-type PTA, the pH rapidly increased when equal moles of NaOH were added to 14 protons, indicating that the Preyssler-type phosphotungstate was stable in aqueous solution with a pH range of 1–12. The reaction in question is as follows:

$${\text{H}}_{{{14}}} \left[ {{\text{P}}_{{5}} {\text{W}}_{{{3}0}} {\text{O}}_{{{11}0}} {\text{Na}}} \right] + {\text{14NaOH}} = {\text{Na}}_{{{14}}} \left[ {{\text{P}}_{{5}} {\text{W}}_{{{3}0}} {\text{O}}_{{{11}0}} } \right] + {\text{14H}}_{{2}} {\text{O}}$$
(1)

In contrast, the Keggin-type PTA solution remained acidic (pH almost unchanged) after adding equal moles of NaOH to 3 acidic protons. This result indicates that the Keggin-type phosphotungstate molecule ([PW12O40]3−) was decomposed. Moreover, it has been reported that [PW12O40]3− is stable only under very acidic conditions (pH < 2), and its neutralization produces a complex mixture of phosphotungstate and tungstate species depending on the solution pH23,24. In an aqueous solution of pH 7, the main phosphotungstate species detected by phosphorus-31 nuclear magnetic resonance (31P NMR) was mono-defective (lacunary) phosphotungstate ([PW11O39]7−) (Supplementary Fig. S2), in which the one moiety [W = O]4+ removed from [PW12O40]3− forms tungsten oxide clusters:

$${\text{H}}_{{3}} \left[ {{\text{PW}}_{{{12}}} {\text{O}}_{{{4}0}} } \right] + {\text{7NaOH}} = {\text{Na}}_{{7}} \left[ {{\text{PW}}_{{{11}}} {\text{O}}_{{{39}}} } \right] + {\text{H}}_{{2}} \left[ {{\text{WO}}_{{4}} } \right]\left( {\text{tungsten oxide cluster}} \right) + {\text{4H}}_{{2}} {\text{O}},$$
(2)

These species may produce an inhomogeneous background. In contrast, the Preyssler-type phosphotungstate molecule is stable over a wide range (pH 1–12), which might be attributable to the homogeneous background.

Further advantages of Preyssler-type compounds

Preyssler-type phosphotungstates have several advantages. They are prepared as a potassium salt (K14[P5W30O110Na]), which is a neutral compound, and therefore do not require prior neutralization. Moreover, the potassium counter-cation can be protonated by a reaction with H+-resin,

$${\text{K}}_{{{14}}} \left[ {{\text{P}}_{{5}} {\text{W}}_{{{3}0}} {\text{O}}_{{{11}0}} {\text{Na}}} \right] + {\text{14 H}}^{ + }-{\text{resin}} = {\text{H}}_{{{14}}} \left[ {{\text{P}}_{{5}} {\text{W}}_{{{3}0}} {\text{O}}_{{{11}0}} {\text{Na}}} \right] + {\text{14 K}}^{ + }-{\text{resin}},$$
(3)

allowing for easy exchange of protons with other cations by the acid–base reaction

$${\text{H}}_{{{14}}} \left[ {{\text{P}}_{{5}} {\text{W}}_{{{3}0}} {\text{O}}_{{{11}0}} {\text{Na}}} \right] + {\text{14AOH}} = {\text{A}}_{{{14}}} \left[ {{\text{P}}_{{5}} {\text{W}}_{{{3}0}} {\text{O}}_{{{11}0}} {\text{Na}}} \right] + {\text{14H}}_{{2}} {\text{O}},$$
(4)

where (A = Li+, Na+, NH4+, Bu4N+, and Bu4P+). Thus, it is possible to change the counter-cations. Clear images with a homogeneous background were obtained using the salts of lithium (Li14[P5W30O110Na]), sodium (Na14[P5W30O110Na]), and ammonium ((NH4)14[P5W30O110Na]) (Supplementary Fig. S7). However, long tail fibers were not obtained using tetrabutylammonium ((Bu4N)14[P5W30O110Na]) and tetrabutylphosphonium ((Bu4P)14[P5W30O110Na]) salts (Supplementary Fig. S7).

Furthermore, the encapsulated Na+ is exchangeable with other cations that have different charges, such as Ca2+, Bi3+, Y3+, and lanthanoid cations. Such exchange alters the negative charge of the Preyssler molecule without affecting its shape. The change in the negative charge affects the crystallinity of Preyssler molecules and their interaction with the virus surface and carbon film support, changing the performance of the negative staining reagent. Clear TEM images were obtained using these Preyssler-type compounds with different encapsulated cations, such as Ca2+25, Y3+25, Bi3+26, Ce3+27, and Eu3+25 (Supplementary Fig. S8). The Eu3+-encapsulated compound (K12[P5W30O110Eu(H2O)]) was the best negative staining reagent among these compounds (Fig. 4) and clear images were obtained from more than 75 area% of grid (Supplementary Fig. S8g).

Figure 4
figure 4

Transmission electron microscopy (TEM) images of T4 phages using Preyssler-type phosphotungstate with encapsulated cation Eu3+, K12[P5W30O110Eu(H2O)], as a negative staining reagent. Concentration of the staining reagent was 0.3 wt% in water.

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Staining performance with other viruses

The Preyssler-type phosphotungstate was a good negative staining reagent for T4 and other phages examined in the present study. The lambda phage (family Siphoviridae) has an icosahedral head with a diameter of ca. 60 nm, a long flexible tail with a length of ~ 150 nm, a short terminal fiber, and four tail fibers17,28, which were all observed (Fig. 5). The T7 phage (family Podoviridae) has an icosahedral head with a diameter of ~ 60 nm, a short tail, and six short fibers17,29, which were clearly visible (Fig. 6).

Figure 5
figure 5

(a) Morphology of the lambda phage. (b) Transmission electron microscopy (TEM) image of lambda phage using Preyssler-type phosphotungstate with encapsulated cation Eu3+, K12[P5W30O110Eu(H2O)], as a negative staining reagent. Concentration of the staining reagent was 0.3 wt% in water.

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Figure 6
figure 6

(a) Morphology of the T7 phage. (b) Transmission electron microscopy (TEM) images of T7 phage using Preyssler-type phosphotungstate with encapsulated cation Eu3+, K12[P5W30O110Eu(H2O)], as a negative staining reagent. Concentration of staining reagent was 0.3 wt% in water.

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Conclusions

Negative staining has been widely used to observe the morphologies of viruses6, other biological particles, lipid vesicles, micelles, liposomes, and polymer particles30. Our results indicate that Preyssler-type phosphotungstates are good negative staining reagents for virus observations. Furthermore, tungsten forms a variety of metal oxide clusters known as polyoxotungstate in an aqueous solution, depending on the other elements present and pH12. Polyoxotungstates are promising tools for developing negative staining reagents for TEM observations.

Methods

Materials

The potassium salt of Preyssler-type phosphotungstate with different encapsulated cations (K(15-n)[P5W30O110Mn+], M = Na+, Ca2+, Bi3+, Y3+, Eu3+), and Preyssler-type phosphotungstic acid with encapsulated sodium, (H14[P5W30O110Na]), were prepared and purified according to a previously reported method25. The obtained potassium salts were dissolved in water and used as the staining solution. An aqueous solution of uranyl acetate (3 wt%) and commercial phosphotungstic acid (TAAB Laboratories Equipment Ltd., Berks, England) were used for comparison. The phosphotungstic acid solution was neutralized using 2 M KOH solution.

Preparation of phages

Enterobacteria phages T4 (NBRC20004), T7 (NBRC20007), and lambda (NBRC20016) and their host bacteria Escherichia coli (NBRC13168 and NBRC12713) were obtained from the NITE Biological Resource Center (NBRC, Chiba, Japan). The phages were propagated by the agar overlay method31 using peptone yeast medium (1% polypeptone, 0.2% yeast extract, and 0.1% MgSO4·7H2O) and purified as previously reported32. Additionally, the phage titers (PFU mL−1) of the purified phage were determined by the agar overlay method.

Virus observation

For TEM analysis, the phage solution (1011 PFU mL−1, 5 μL) was placed in contact with a glow-discharged (JEOL HDT-400, Tokyo, Japan) carbon-coated collodion film on a Cu grid (Nisshin EM, Tokyo, Japan) for 3 min. Excess solution was removed using a filter paper. Subsequently, a drop (5 μL) of staining solution was placed on the grid for 3 min. The staining solution was removed using a filter paper, and the grid was air-dried. TEM (JEOL, JEM-1200EX) with a tungsten filament was employed at 80 kV. The sample grid prepared for the TEM observation was fixed using a carbon tape on a sample holder and observed using SEM (S-4800, Hitachi, Tokyo, Japan) and AFM (SPM-9600, Shimadzu, Kyoto, Japan).