Another key to accurate measurements is the ability to control and measure the amount of fluid displaced by small changes in the syringe plunger's position. For sample volumes <1 mL, a micrometer was used to control plunger position. Volumes from ~0.1–1.0 mL were measured with a 2.0-mL Gilmont syringe with built-in micrometer (Figure 1B, nominal resolution: 2.0 µL/division; Cole Parmer). For volumes <0.1 mL, one of a series of smaller syringes (250, 50, 25, or 10 µL, 1700LT series; Hamilton, Reno NV, USA) was selected. The Hamilton syringes were interchangeably mounted on a plexiglass platform, with the plunger tightly coupled to a manually driven micrometer (Daedal, now Parker Hannifin, Irwin, PA, USA) (Figure 1C, 10 µm/division). The micrometer resolutions were improved at least 3-fold by visually interpolating the micrometer scale position to one additional digit.
As repeated handling of fragile samples should be minimized, it is important to choose syringe-micrometer combinations in advance that will be suitable for each approximate volume to be measured. The ability to discriminate small volume changes is largely determined by the syringe volume, the syringe length, and the micrometer resolution. These three parameters were combined as a single index that can be used to help select appropriate MVM configurations. Thus, for a known sample volume, Vs (in µL), an index of the MVM's overall resolution is Vs × (A ÷ B), where A is the syringe length per volume (µm/µL), and B (µm/division) is the distance the plunger moves per micrometer division. To illustrate its utility for correctly configuring the MVM, the accuracy of volume estimates for selected small reference standards was compared while using different syringes to change the value of this index.Comparing MVM and confocal volumes
To help demonstrate the effectiveness of the MVM for measuring biological specimens, volumes of freshly dissected ant brains (Atta colombica [Formicidae]) were measured both by using the MVM and by confocal microscopy. Small worker ants were selected to provide brain volumes close to the smallest volume for which highly accurate measurements have been established (0.066 µL; see “Results and discussion” section). Brains were dissected in physiological saline and treated for 10–15 min with 2.5×10-5 M propidium iodide (Invitrogen, Carlsbad, CA, USA) to highlight cell nuclei. Osmotic stress can alter tissue volumes, so the saline should emulate the osmotic properties of fluids that surround the specimen in vivo. Since the ionic environment of ant brains is poorly known, we used a saline (150 mM NaCl, 24 mM KCl, 7.0 mM CaCl2, 4.0 mM MgCl2, 5.0 mM HEPES buffer, and 131 mM sucrose, pH 6.9–7.0) that emulates the ionic composition and osmolarity of culture media (13,14) known to keep dissociated insect neurons alive (including those of hymenopterans).
Using the 559-nm laser of a confocal microscope (FV1000; Olympus, Miami, FL, USA), serial optical sections of a freshly dissected brain were obtained every 10 µm at 10× magnification. 3-D reconstruction software (15) was used subsequently to trace brain profiles in each optical section and compute the brain volume. Next, the brain was transferred with a small amount of saline to a small platform positioned ~1 mm from the fluid-filled MVM receptacle and visible under the dissecting microscope. The platform was covered with Parafilm (Pechiney Plastic Packaging Company, Chicago, IL, USA) to provide a hydrophobic surface, facilitating the removal of excess fluid and minimizing tissue adhesion. As an alternative to Parafilm, synthetic hydrophobic coatings (e.g., Gelest, Inc., Morrisville, PA, USA) may be useful. To avoid tissue shrinkage due to evaporation, a humid microchamber was created with a small piece of saline-soaked sponge, with a notch cut into one side and covered with a microscope coverslip. After first obtaining the pre-addition data from the MVM (see Figure 2B), the wet brain was transferred from the saline solution to the dry floor of the humidified microchamber. Excess saline was carefully removed under magnification by using a fine-tipped suction pipet pulled from a 1-mm capillary and connected to a Gilmont syringe. The isolated tissue was then quickly transferred with sharp forceps (Dumont no. 55; Fine Science Tools, Foster City, CA, USA) into the MVM receptacle.