As part of the development of nano-particles targeted to specific cell types, we have been studying the uptake of non-targeted fluorescent nano-particles by live PC3 cells. In order to compare the relative cell uptake rates of nano-particles, live cell confocal fluorescence microscopy is used to acquire images of the cells following incubation with nano-particles of various sizes. Quantitative measurement of nanoparticle uptake presents many challenges, some of which must be addressed during post-acquisition image processing.
Our experiments were carried out in 8-well multi-chambered slides. The medium autofluores- cence in the wavelength range 525±20nm was used, in order to establish a baseline against which fluorescence could be compared between wells. Although the measurement parameters were kept constant between wells, it was necessary in some cases to change the percent power of the laser excitation to avoid saturation when imaging the larger nano-particles. In addition, even small variations in height above the glass had a large effect on fluorescence intensity. We needed to determine whether the washing step, which was used in order to remove excess nano-particles following the incubation period, was effective. The medium autofluorescence in control wells was measured at various excitation intensities, and allowed us to create a calibration curve of average medium autofluorescence versus percent laser power (as set by the acousto-optic tunable filter). We were then able to show that the data from other wells were consistent with our calibration curve, thus demonstrating that the washing was effective, and that the autofluorescence behaved consistently across wells.
In order to clearly define the inside of the cells, an anionic dye (sulforhodamine B) was added to the medium. Over the time scale of our experiments, the inside of viable cells remained dark, while the medium was highly fluorescent in the spectral region above 600nm. This allowed us to use a simple intensity based segmentation method to mark the cell volume.
Finally, having clearly established the volume of the cell, we were able to measure total inte- grated fluorescence inside the cell, corrected for autofluorescence. Since the relative fluorescence of different sizes of nano-particles is known, we are able to compare uptake of different nano- particle sizes. We still cannot determine the absolute number of nano-particles, but even so, we have a useful method which will allow us to compare targeted to non-targeted nano-particles. Here, we present these methods, which were developed using the FIJI distribution of ImageJ in order to analyze and quantify these data.
fluorescence, confocal microscopy, nanoparticles
Presenting author: Victoria Machtey
Organisation: Bar Ilan University
co-authors: Raz Khan-Dadash, Gerardo Byk, and Aryeh M. Weiss