Method of quantitative measurement of particle content using hydrated state imaging

Abstract

The method is for quantitative measurement of particle content using hydrated state imaging such as CryoTEM. A sample of virus-like particles (VLPs) or virus particles is provided. Preferably, the sample is rapidly frozen into a cryogenic liquid at a cryogenic temperature. While at the cryogenic temperature, the particle content of each VLP in the frozen sample is observed in the CryoTEM. An amount of the particle content of the VLPs is determined to assess whether the VLPs are empty or not.

We claim: 
     
         1 . A method for assessing a particle interior content using imaging, comprising:
 providing a sample of virus-like particles (VLPs) or virus particles;   maintaining the sample in a native unstained hydrated state;   imaging the sample in the native unstained hydrated state in an Electron Microscope;   observing the particle interior content of each virus-like particle (VLP) or virus AAV particle in the sample in acquired images in the Electron Microscope; and   based on the observation of the particle interior content, measuring an intensity of the particle interior content of each virus-like particle (VLP) or virus particle in the sample;   based on the measured intensity, determining an amount of the particle interior content of each virus-like particle (VLP) or virus particle in the sample and assessing whether the particles are empty, full or ambiguous.   
     
     
         2 . The method according to  claim 1  wherein the method further comprises the step of automatically or manually detecting particles in images and deleting particles that are smaller than a lower size limit and larger than an upper size limit. 
     
     
         3 . The method according to  claim 2  wherein the method further comprises displaying VLPs or virus particles and interactively deleting or adding VLPs or virus particles to the image. 
     
     
         4 . The method according to  claim 1  wherein the method further comprises the step of classifying a particle that has an inner density with no distinct boundary between a particle shell and a particle core as a filled particle. 
     
     
         5 . The method according to  claim 1  wherein the method further comprises the step of classifying a particle that has a distinct outer shell and a minute internal density as an empty particle. 
     
     
         6 . The method according to  claim 1  wherein the method further comprises the step of using Cryo Transmission Electron Microscopy to determine particle content of the VLPs or virus particles. 
     
     
         7 . The method according to  claim 1  wherein the method further comprises the step of determining the particle content of adeno associated virus (AAV) particles. 
     
     
         8 . The method according to  claim 7  wherein the method further comprises the step of classifying AAV particles that contain a gene as a filled particle. 
     
     
         9 . The method according to  claim 8  wherein the method further comprises the step of classifying AAV particles that contain no gene as an empty particle. 
     
     
         10 . The method according to  claim 1  wherein the method further comprises adding an ionic liquid to the sample to keep the VLPs) or virus particles in a hydrated state. 
     
     
         11 . The method according to  claim 1  wherein the method further comprises imaging the sample in a hydrated liquid state by using a liquid sample holder.