Rapid phenotypic analysis of uncoated Drosophila samples with low-vacuum scanning electron microscopy

Figures & data

Table 1. Optimized conditions for Drosophila LV-SEM using the FEI Quanta 450

Variable	Optimum	Notes
Sample fixation and dehydration	none	Adults are frozen live, then mounted (or can be mounted live)
Mounting medium	CCC (Leit-C)	Allows sample to be oriented on the stub
Chamber pressure	0.53 Torr (71 Pa)	Higher pressures may favor sample preservation, but increase noise
Working distance (sample to aperture)	10–12 mm	Closer distances result in shallower depth of field, although slightly better resolution
Accelerating voltage	7.5–15 kV	7.5 kV for wings; 10–12.5 kV for bristles; 15 kV if charging is not an issue
Spot size	3.5	Lower values may improve resolution at high mag, but increase noise. Higher values impart rapid sample damage.
Scan speed	30 µsec/pixel	Faster scans are noisy; 40 µsec is low-noise but may trigger charging or damage
Integration	None (setting = 1)	Small sample movements preclude averaging or integration
Sample lifetime	Up to 1 h	Longer times under vacuum may lead to dehydration-related artifacts. Avoid using a zoomed focusing window, due to beam damage.

Figure 1. Effects of spot size, working distance, and pressure on LV-SEM images. Bar = 10 µm for each set of images. The contrast was adjusted post-collection in this and the remaining figures, except where noted (see Materials and Methods). (A–E) Effect of spot size on the w¹¹¹⁸ eye; the same region is shown in each panel; shot at 1000× magnification. Spot sizes were 2.0 (A); 2.5 (B); 3.0 (C); 3.5 (D); and 4.5 (E) as indicated. Resolution is slightly better at lower spot sizes, but the signal is lower and noise more apparent. Spot = 3.5 balances good resolution with low noise and sample damage. (F-H) Thoracic bristles of w¹¹¹⁸ were scanned at 2000× and 15 kV, with spot sizes 2.5 (F), 3.5 (G), and 5.0 (H) as indicated. The same region is shown in each panel. Using spot = 5.0 leads to singed hairs (arrowhead) and charging on the bristle sockets, problems not observed with spot = 3.5. (I–J) Increased working distance (WD) provides increased depth of field. Focus is set on the upper surface of a w¹¹¹⁸ haltere in both panels, taken at 500×. Note the underlying thorax has better focus in (J) (WD = 14.8 mm) than in (I) (WD = 5.0 mm). (K–M) Variation in chamber pressure, using orbital bristles taken at 2500× and shown with original contrast. The same bristle socket is shown at 0.30 (K), 0.53 (L), and 0.68 Torr (M).

Figure 2. Effect of accelerating voltage on the w¹¹¹⁸ eye and the sex comb of the male foreleg. All panels were shot at 1000×; bar = 10 µm for all. A-D, w¹¹¹⁸ eye; the same region is shown in each panel. Voltages were 10 (A); 15 (B); 20 (C); and 25 kV (D). Appearance of ommatidia changes substantially as voltage varies; the 15 kV image most resembles a conventional high-vacuum SEM image. (E–F) the striations of the sex comb bristles are clearly seen at 7.5 kV (E), but not at 15 kV (F).

Figure 3. Examples of wild type structures at varying magnifications in w¹¹¹⁸ flies. (B, D and E) were scanned in large format (2048 pixel width). Beam = 15 kV for (A, B, E and F). (A) Whole female mounted in CCC; bar = 1 mm; shot at 20×. (B) Male genitalia (ventral); bar = 50 µm; shot at 250×. (C) Male wing margin (distal is up); bar = 10 µm; shot at 500× and 7.5 kV. (D) Male tarsal claw and pulvillus; bar = 10 µm; shot at 2000× and 10 kV. (E) Sensory hair bed on the ventral anterior side of the thorax near the head (male; anterior is up). Bar = 10 µm; shot at 1500×. (F) Proboscis; bar = 50 µm; shot at 340× and spot = 3.0.

Figure 4. Analysis of mutant phenotypes using LV-SEM. Bars = 10 µm for each pair of images. (A–B) orbital bristles from dorsal head of w¹¹¹⁸ (A) and sn (B) males shot at 750×, 10 kV. (C–D) retina of w¹¹¹⁸ (C) and GMR-GAL4 > Mi[Hto-WP]QYE (D), shot at 1000× and 2048 pixel width. (E–F) Equivalent regions of w¹¹¹⁸ (E) and ms1096w-GAL4 > Mi[Hto-WP]VRC male wings (F), shot at 1500×, 7.5 kV. Note multipronged wing hairs in (F). (G–H) Wing surface with L3 vein and campaniform sense organ (center), from w¹¹¹⁸ (G) and ms1096w-GAL4 > Mi[Hto-WP]NAW (H), shot at 1000×, 7.5 kV. In (H), note the hairs are eliminated from this region of the wing, and the cuticle is abnormal.