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. 2016 May 4:6:25275.
doi: 10.1038/srep25275.

Nanoparticle suspensions enclosed in methylcellulose: a new approach for quantifying nanoparticles in transmission electron microscopy

Christian Hacker  1 , Jalal Asadi  1 , Christos Pliotas  2 , Sophie Ferguson  1 , Lee Sherry  3 , Phedra Marius  2 , Javier Tello  1 , David Jackson  3 , James Naismith  2 , John Milton Lucocq  1
Affiliations

Nanoparticle suspensions enclosed in methylcellulose: a new approach for quantifying nanoparticles in transmission electron microscopy

Christian Hacker et al. Sci Rep. .

Abstract

Nanoparticles are of increasing importance in biomedicine but quantification is problematic because current methods depend on indirect measurements at low resolution. Here we describe a new high-resolution method for measuring and quantifying nanoparticles in suspension. It involves premixing nanoparticles in a hydrophilic support medium (methylcellulose) before introducing heavy metal stains for visualization in small air-dried droplets by transmission electron microscopy (TEM). The use of methylcellulose avoids artifacts of conventional negative stain-TEM by (1) restricting interactions between the nanoparticles, (2) inhibiting binding to the specimen support films and (3) reducing compression after drying. Methylcellulose embedment provides effective electron imaging of liposomes, nanodiscs and viruses as well as comprehensive visualization of nanoparticle populations in droplets of known size. These qualities facilitate unbiased sampling, rapid size measurement and estimation of nanoparticle numbers by means of ratio counting using a colloidal gold calibrant. Specimen preparation and quantification take minutes and require a few microliters of sample using only basic laboratory equipment and a standard TEM.

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Figures

Figure 1
Figure 1. Nanoparticles in methylcellulose films imaged in TEM.
(a) The stain mix contained 1.5 μl of 1% methylcellulose, 7.5 μl of nanoparticle suspension and 1 μl of 0.3% uranyl acetate (uranyl acetate was added after dialysis if required; see text and Fig. 3). 0.5 μl was loaded onto a standard pioloform coated EM grid support and allowed to dry before examination in the TEM. (b–i) A range of nanoparticles imaged at low magnification (b–e) and high magnification (f–i). Scale bars (b–e) 100 nm and (f–i) 50 nm.
Figure 2
Figure 2. Methylcellulose restricts binding to the substratum, allows freedom of orientation and prevents substantial collapse of nanoparticles.
(a) Binding to substratum. Liposomes were incubated in 1% methylcellulose or water before adsorbing the mix to an EM grid, contrasting, and density of liposomes determined. (b) Orientation. 12 nm diameter nanodiscs were mixed with methylcellulose solution varying between 0.1% or 0.5% before adding uranyl acetate. 0.5 μl droplets were loaded onto EM grids and dried before classifying orientation by TEM (flat, tilted and upright). Nanodiscs become progressively tilted with increasing concentration of methyl cellulose. Bars, 7nm. (c) Gold nanorods were mixed with 0.1% or 0.5% MC. 0.5 μl drops were loaded onto EM grids and nanorods situated in either the rim or center were measured. Data are presented as cumulative fractions. Nanorods in the thinnest films (0.1% MC center) were longer compared to those in thicker films at the 0.1% MC rim (Kolmogorov-Smirnov test, P 0.001; n 45 and 52 data points respectively) or the 0.5% MC center (Kolmogorov-Smirnov test, P 0.01; n 45 and 38 data points respectively). The data are consistent with increased freedom to rotate in thicker MC films. (d,e) Tilt analysis of vesicle compression in methylcellulose films. Vesicles containing (MscL) pore protein were embedded in methyl cellulose as described in the text and the degree of flattening (oblate spheroid; model) estimated from minor and major axes measured after 50° specimen tilt. Data are from the whole population (All), n = 38, 20–30 nm vesicles, n = 16 and 50–70 nm vesicles, n = 10. Bar = 50 nm.
Figure 3
Figure 3. Microdialysis procedure using semipermeable cellulose kidney dialysis capillaries.
(a) Three to five μl of sample was drawn into the kidney dialysis straw by capillary action. (b) Ends of the straw were sealed using an assembly made from interlocking truncated micropipette tips. (c) The assembly was transferred into the dialysate. (d) The dialyzed sample was extruded onto parafilm and added to methylcellulose/uranyl acetate mix before loading and drying on an EM support grid.
Figure 4
Figure 4. Liposome sampling and size estimation.
Liposomes were sampled using a scanning band to which an unbiased counting rule was applied (a). All particles passing completely between the lines or intersecting the acceptance line (green) were selected for analysis. Particles intersecting the forbidden line (red) were excluded. Selected particles were translocated through size a systematic series of dots the upper edge of each tracing a scanning line. The particle caliper diameter was calculated from the number of intersection of dot edges with the nanoparticle and the real spacing of the dots as described in the text. Data are from three experimental runs from the same liposome preparation (b). Approximately 100 vesicles were counted per experiment; error bars are standard error of the mean (n = 3; each estimation took approximately 10 minutes).
Figure 5
Figure 5. Quantification of nanoparticles using gold particle calibration.
(a) During scanning gold particles and test nanoparticles were imaged “live” using a digital camera and counted using a scanning band as described in the text and in Fig. 4. The tip of the green arrow traces the acceptance line (dashed green) and red arrow the forbidden line (dashed red) during translocation of the specimen. The liposome (nanoparticle) concentration was estimated from the ratio of golds to liposome (nanoparticle) multiplied by the gold calibrant concentration. (b) Estimates of gold/nanoparticle ratio from single equatorial scans closely match those obtained from multiple SUR scans. Number estimates were obtained from the same grid but in different locations for each type of sample (n = 3, mean value and bars standard error of mean). Scale bar, 500 nm.
Figure 6
Figure 6. Quantification of liposomes.
Scaling between liposome dilution and estimates of liposome particle number. In each case, approximately 100 each of gold calibration and liposomes were counted (time for each scan 10–15 minutes). Values are means of 4 separate dilution series derived from the same liposome preparation and error bars are coefficients of variance.
Figure 7
Figure 7. Influenza A virus quantification.
(a) The virus preparation was mixed with gold/methylcellulose calibration solution, dialyzed by the microdialysis method (see Fig. 3) and embedded in methylcellulose/uranyl acetate. Estimates of virus particle number using the suspensions in methylcellulose method were compared with plaque forming units (PFU assay), determined on the same samples (n = 3; error bars, standard error of the mean). (b) TEM image showing lack of gold (arrows) or virus particle interaction/aggregation. Scale bar = 100 nm.
Figure 8
Figure 8. Overview of suspension in methylcellulose method.
Nanoparticle (NP) suspension is added to gold calibrant (Au)/methylcellulose (MC) to make a calibration mix (NP/Cal mix). If necessary, NP/Cal mix is dialyzed in microdialysis straws and extruded before adding heavy metal stain (uranyl acetate). 0.5 μl is loaded onto the plastic coated TEM support grid for counting, sizing and further characterization.
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