Measurement of protein concentration in bacteria and small organelles under a light transmission microscope

Abstract

Protein concentration (PC) is an essential characteristic of cells and organelles; it determines the extent of macromolecular crowding effects and serves as a sensitive indicator of cellular health. A simple and direct way to quantify PC is provided by brightfield-based transport-of-intensity equation (TIE) imaging combined with volume measurements. However, since TIE is based on geometric optics, its applicability to micrometer-sized particles is not clear. Here, we show that TIE can be used on particles with sizes comparable to the wavelength. At the same time, we introduce a new ImageJ plugin that allows TIE image processing without resorting to advanced mathematical programs. To convert TIE data to PC, knowledge of particle volumes is essential. The volumes of bacteria or other isolated particles can be measured by displacement of an external absorbing dye ("transmission-through-dye" or TTD microscopy), and for spherical intracellular particles, volumes can be estimated from their diameters. We illustrate the use of TIE on Escherichia coli, mammalian nucleoli, and nucleolar fibrillar centers. The method is easy to use and achieves high spatial resolution.

Keywords: bacteria; cell volume; fibrillar centers; macromolecular crowding; nucleoli; refractive index; transport‐of‐intensity equation.

Figure 1.

The effect of the choice of BF1 and BF2 (top rows) on the TIE results (bottom rows). Each TIE image is based on two BF1 and BF2 that are centered on the brightfield image across a given TIE and are separated by Δz = 1 μm. For example, the TIE image indicated by an upward arrow was computed from the two brightfield images indicated by the downward arrows. The numbers over the brightfield images indicate the vertical position z_m of the objective (oil-immersion 60x/1.42) relative to the best focus position; positive numbers indicate a shift of the objective toward the sample. The numbers under TIE images show the cumulative intensity of the bead or of the cell. (A) The darker images of beads and negative numbers result from a higher refractive index of the oil (1.4745 in this case) compared to silica (1.454, according to our measurements; see Fig. 2). Scale bar, 3 μm. (B) TIE images of E. coli. (C) The intensity profile through a bead in an oil with n = 1.4054 compared to the theoretical curve based on a 1.56 μm diameter). The TIE curve was scaled to match the bead diameter.

Figure 2.

An example of TIE calibration with silica beads. TIE plotted on the vertical scale is the cumulative value normalized to the average volume of the beads. Between 9 and 19 beads or groups of beads clustered together were analyzed per experimental point. Cluster of beads gave the same results per bead as individual ones.

Figure 3.

Distribution of the intensity relative to the midline of E. coli (the top cell). The units on the horizontal scale are micrometers; the units on the vertical scale are micrometers for the TTD line and for the curve best-fit to a circle with the same diameter (1.27 μm); the TIE curve was scaled to the same height for profile comparison. The upper 2/3 of the TTD profile almost perfectly matches the theoretical curve (the bottom is distorted by the Becke lines); the TIE curve is noticeably wider, which may reflect a higher E. coli density away from the center. Scale bar, 2.5 μm.

Figure 4.

Nucleoli in HeLa and their internal structure. (A) One of the bright-field images showing nucleoli and the darker spots corresponding to FCs. (B) Pseudocolored TIE image.