The ultramicroscopy method allows for measuring the number concentration of nanoparticles in liquid media.
This experimental method was developed by Richard Zsigmondy, who received the Nobel Prize in Chemistry for this work in 1925.
This method is a type of optical microscopy with a special way of illuminating the sample using a laser.
The laser beam and the optical axis of the microscope’s observation are positioned perpendicular to each other. This allows for obtaining an image of the laser radiation scattered by the sample in the microscope’s field of view.
The image obtained in this way is recorded by a digital camera into a video file for further analysis. For liquids containing nanoparticles, such an image will appear as luminous points on a dark background, each representing light scattering from an individual particle. The nanoparticles themselves cannot be seen by this method; what is observed is the scattering from the particles. Thus, there are no limitations associated with the diffraction limit in this method.
Файл pdf – Метод ультрамикроскопии
Рисунок А (образец без наночастиц)
Рисунок Б (образец с наночастицами)
The laser beam is directed at a 90-degree angle to the optical axis of observation, i.e., it does not enter the lens and the digital camera’s sensor (Figure A). The field of view appears as a dark field. When nanoparticles are present in the sample, the laser radiation will scatter off them in all directions, including along the optical axis of the lens, reaching the digital camera’s sensor. In this case, the image will become a dark field with constantly moving (due to Brownian forces) luminous points (Figure B). The sizes of these luminous points are not directly the sizes of the nanoparticles: we see scattered light on the nanoparticles, not the particles themselves. Thus, there is no limitation associated with the diffraction limit. It is not possible to determine the particle size from a single such image, but it is possible to count the number of nanoparticles in the field of view. Knowing the volume in which the observation takes place, one can measure the concentration of nanoparticles. To increase the reliability of the measurement, concentrations are repeated on hundreds of frames obtained from the sample under study.
Special software (SW) NPVision allows for analyzing digital images obtained on the NPCounter device, identifying and counting objects of interest (OI) – luminous spherical objects (nanoparticles) – in each frame. From the image analysis, NPVision SW calculates the particle concentration in the sample under study.