Measuring vesicle loading with holographic microscopy and bulk light scattering

TL;DR

This work introduces a label-free, holography-based method to quantify solute loading inside vesicles by fitting in-line holograms to a Lorenz-Mie scattering model, enabling extraction of the interior refractive index $n$ and radius $r$ without tracers. It demonstrates that a homogeneous-sphere approximation is robust for giant vesicles (recovering $n$ to about $1e-4$ RIU when $r>1 μm$ and $6 μm<z<15 μm$) and can track loading and leakage, with nanoscale vesicles better served by bulk turbidimetry under certain conditions. The approach provides a non-invasive, scalable means to monitor vesicle content and membrane transport, with practical implications for drug delivery and membrane biophysics; future work extends to other solutes and membrane perturbations. Key findings include successful measurement of sucrose loading and leakage, and a quantified permeability on the order of $2×10^{-11}$ cm/s, validated against known glucose permeability, underscoring the method’s relevance for membrane transport studies.

Abstract

We report efforts to quantify the loading of cell-sized lipid vesicles using in-line digital holographic microscopy. This method does not require fluorescent reporters, fluorescent tracers, or radioactive tracers. A single-color LED light source takes the place of conventional illumination to generate holograms rather than bright field images. By modelling the vesicle's scattering in a microscope with a Lorenz-Mie light scattering model, and comparing the results to data holograms, we are able to measure the vesicle's refractive index and thus loading. Performing the same comparison for bulk light scattering measurements enables retrieval of vesicle loading for nanoscale vesicles.

Figures (7)

• Figure 1: A. Phase contrast and B. holographic images of vesicles encapsulating sucrose (nominal concentration 0.5 M) diluted into an isotonic glucose solution. Under coherent illumination, changing the focal plane of the microscope results in changes in the diffraction pattern of the vesicle. When in focus, the vesicle (inside the white dotted circle) is barely visible in holographic mode.
• Figure 2: The light scattering model for a homogeneous sphere can be used to extract the refractive index of the contents of a vesicle (a core-shell scatterer with a very thin shell). A. The fitted error in the refractive index $n$ becomes negligible for vesicles larger than 1 $\mu$m in radius. B. The fitted error in $z$ remains below 5 nm and fluctuates with vesicle size. C. The fitted error in $r$ remains approximately 40 nm for all vesicle sizes. D. A schematic showing a core-shell scatterer with inner radius $r$ and shell thickness $t$ (left) and a homogeneous sphere with radius $r'$ = $r$+$t'$ (right).
• Figure 3: Holograms and the intensity values across the centre of the hologram were calculated for vesicles with varying A.$r$, B.$z$, and C. refractive index $n$. Varying $r$ and $z$ change the hologram fringe pattern and contrast, whereas varying $n$ only changes the fringe contrast. See also Videos S1--S3. Parameters used in A:$z$ = 10 $\mu$m, the internal refractive index $n$ = 1.35, the lipid refractive index $n_{lipid}$ = 1.47, and the lipid shell thickness $t$ = 3 nm; B:$r$ = 1 $\mu$m, $n$ = 1.35, $n_{lipid}$ = 1.47, and $t$ = 3 nm; C.$r$ = 1 $\mu$m, $z$ = 10 $\mu$m, $n_{lipid}$ = 1.47, and $t$ = 3 nm.
• Figure 4: A--B. Holograms of vesicles are captured at several different focal planes. C. The vesicles labelled in A--B were analysed at different $z$ distances to retrieve their refractive index $n$ as a function of $z$. See also Video S4.
• Figure 5: Vesicles encapsulating 500 mM sucrose were diluted into buffers containing either 250 mM glucose (resulting in an osmotic imbalance; number of vesicles $N$ = 23) or 500 mM glucose ($N$ = 14). The refractive indices of individual vesicles were measured with holography. For the vesicles exposed to a hypotonic solution, there was content loss of approximately 200 mM sucrose. Refractive indices of 250 mM and 500 mM encapsulated sucrose are shown as red squares. The vesicles analyzed were between 1--2 $\mu$m in radius.