Application notes

For any application questions feel free to contact us (contact), or maybe you can find an answer in one of these papers:

  • Protein aggregation counting (apn16-BT001)
  • Detection of silicone oil in protein suspension (apn16-BT002)
  • Analysis of small sample quantity for suspension application (apn16-BT003)
  • Particle tracking (apn16-BT004)
  • Routine analysis with pipetting robot and Occhio IPAC (apn16-BT005)
  • Active pharmaceutical ingredients dry dispersion and analysis (apn16-BT006)
  • Analysis of small sample quantity for dry powder application (apn16-BT007)

Our knowledge goes beyond this list so do not hesitate to ask more!

(Apn16-BT001) Characterization of Proteins Aggregation in Vaccine Suspension, Detection and Counting

One of the most challenging aspects in the production of vaccines is logistics, the cold chain transportation from manufacturer to recipient. Great care must be taken to ensure that the vaccines do not aggregate. A vaccine is a suspension which contains three types of particles, all smaller than 200nm: aggregated particles, folded proteins, and foreign particles known as pollutants. Aggregation of the folded proteins occurs during the lifetime of the suspension and is directly correlated to sample stress. To maintain the immunization ability of the vaccine, it is important to minimize aggregation; therefore, to guarantee the results and safety of the vaccine, it is important to verify the condition and quantify any aggregation that has occurred as well as pollutants that may be present during production and before the vaccine is administered.


For this sample, the Occhio Ipac2, flowing microscope was used. The measurement was taken from a 200µl sample of a vaccine. The sample was then passed through a calibrated flow cell where the particles are detected by the high-resolution camera and accompanying zoom lens with monochromatic backlighting. The Callisto software then sorts and uses this optical data to identify and quantify protein aggregation, pollutants, and folded proteins.

Sample preparation

A sterile pipette was used to gather and then inject the vaccine sample into the Occhio Ipac 2 via the specially designed pipette captor. The captor is connected to the viewing cell by a short PTFE micro-tubing. A precision syringe controls the flow of the sample through the cell and the cell thickness is adjusted to allow for minimal dilution of the sample. The integrated three-way valve ensures that there is no sample contamination due to reverse flow.

Sample measurement

During the measurement the instrument allows:

Flowing of the suspension
Acquiring images
Identifying all the particles in the images
Computing the size and density distribution (particles counting distribution)
Storing of the analyzed particles in a dedicated database
Finishing the analysis and rinsing the cell
Printing the report

All the images are processes in real time as well the displaying of the distribution data and graphs.

Sample VA AGG B01: Identification and counting of proteins aggregate

Instrument resolution 0.34 µm/pixel

Cell thickness 100µm

Sample quantity 200µl



We found that the quantity of the aggregates contained in the suspension was directly related to the lifetime and storage conditions of each sample. Our goal for this instrument was to provide an effective and precise instrument for the quality control of vaccines and general protein aggregation quantification; our solution was the Occhio Ipac 2. This instrument has been specifically designed for transparent, subvisible particles in a liquid suspension. It utilizes a high-resolution camera with monochrome backlighting to detect the particles and our patented Callisto software then deciphers the data and automatically files the images while creating graphs and analysis reports in real time.

(Apn16-BT006) Active pharmaceutical ingredients dry dispersion and analysis 


For the majority of applications, the most important product in a pharmaceutical preparation is the A.P.I. (active pharmaceutical ingredient). This powder is characterized by its crystal morphology, with size ranging from just a few microns up to hundreds of microns.

Occhio has developed a fully automated method for analyzing the shape and the size of A.P.I. powders.


The main Objectives for this instrument are:

  • Provide the user with a standard procedure to disperse and analyze the sample
  • Guarantee the integrity of the samples during the dispersion
  • Allow repeated analyses with a small quantity of powder
  • Reduce the contamination associated with the ambient atmosphere
  • Supply a statistical software to elucidate the structure and provide comparative size morphology studies.

Importance of dispersion:

The sample dispersion is the main part of every particle sizing instrument.  Requirements for a good dispersion are:

  • The particles must be separated homogenously
  • The size and the shape of the sample must not be modified during the dispersion process.
  • The smaller particles must be separated in the same way that the larger particles are separated.
  • The dispersion process can be repeated several times with identical results

If you look to the adjacent image in the left graph, you will see there is a difference between the blue line, analysis of air dispersion method, and the red line, analysis of Occhio vacuum dispersion method. Most notably, we see that the blue line, near the number 2, indicates far more particles being observed under 10µm. This is due to the micro crystalline structures being fractured upon introduction into the observation area. The Occhio vacuum dispersion system eliminates these fractures, as is indicated by the smooth, fast climbing curve of the red line. 

If you look to the right graph, you will see that due to agglomeration and fracturing of the A.P.I., the elongation cumulative distribution shows a significant difference between the two dispersions. With the air dispersion method, the crystals are much more elongated.

The images underneath of the two graphs are real images taken from three different, standard methods for introducing a sample into an observation area. Starting with Image A, Manuel dispersion, we see the presence of agglomerates and non-homogeneous distribution. Moving to image B, Pulse Air Dispersion, we can observe how the fragile crystals are broken due to the air pressure. Finally, we move to image 3, Occhio controlled vacuum dispersion, we see that the dispersion is homogeneous, and the fragile crystals remain undamaged.