Quantitative particle content assessment using hydrated state imaging and image analysis

A method for establishing a correlation between measured interior intensities of the particles using electron microscopy and the size of the genetic materials contained in virus or virus-like particles (VLPs), comprising:

• a) providing a first reference sample (200) of virus or virus-like particles (VLPs) (210, 212, 214) containing a first genetic material (302) having a known first size, a second reference sample (202) of virus or virus-like particles (VLPs) (216, 218, 220) containing a second genetic material (306) having a known second size, a third reference sample (204) of virus or virus-like particles (VLP) (222, 224, 226) containing a third genetic material (308) having a known third size, the first size being different from the second size and the second size being different from the third size;
• b) imaging the first, second and third samples (200, 202, 204) using a hydrated state imaging technique;
• c) measuring a particle interior intensity of each virus particle or VLP in images of each reference sample (200, 202, 204);
• d) determining a distribution of particle interior intensities of the virus particles or VLPs in each reference sample (200, 202, 204);
• e) fitting a curve to the distribution of the first, second and third reference samples (200, 202, 204), correlating the measured interior intensities to each respective known first, second and third size;
• f) the fitted curve describing a relation between the interior particle intensities and the first, second and third sizes of the first, second and third genetic materials, respectively;
• and determining the size of an unknown genetic material of a sample of an unknown virus or VLP by measuring a particle interior intensity of the genetic material of the unknown sample by applying the measured particle interior intensity on the curve to find the size of the genetic material that corresponds to the measured particle interior intensity of the unknown sample;
• g) wherein the measured particle interior intensities are normalized locally based on each particle's shell intensity and an intensity of the local background surrounding the particle excluding a closest background region corresponding to a bright defocus ring.