I'm looking to compare a few fluorescent dyes for cell permeability. I'm interested to see how much of the dye actually enters the cell, but would specifically like to quantify this data. Any thoughts on how this could be achieved, preferably in bacterial cells?
It is possible to do flow cytometry with bacteria to measure fluorescent dye uptake. Fluorescence microscopy can also be used. The problem is to relate the amount of fluorescence to the amount of dye.
In principle, you could soak the bacteria in the dye, separate the bacteria from the external dye by centrifugation or filtration, then extract the bacteria to get the dye out and measure how much you recovered by fluorescence or by mass spectrometry. I say "in principle" because the internal volume of bacteria is so minuscule that it is difficult to get enough dye in them and to assure yourself that you have not included dye stuck to the outside of the bacteria. It has been done, but it's challenging.
Check these methods and with a few tweaks would work for your settings:
- Analysis of Antimicrobial-Triggered Membrane Depolarization Using Voltage Sensitive Dyes. J. Derk te Winkel, Declan A. Gray, Kenneth H. Seistrup, Leendert W. Hamoen, and Henrik Strahl. Front Cell Dev Biol. 2016; 4: 29.
- Viability states of bacteria—Specific mechanisms of selected probes. Heike Sträuber Susann Müller. Article Viability States of Bacteria-Specific Mechanisms of Selected Probes
Make sure you have all the +ve and -ve controls. Good luck.
@Sathya Srinivasan
Mrs/Miss Campbell,
Please, consider the content of the following discussion [A] together with the reference section shown, therein, devoted to uncertainty of measurements of optical spectrometric methods. The error contributions due to chemometric treatment of UV-VIS and Fs to the mean values of the spectroscopic bands are comparable.
Precise, accurate, selective, sensitive and even exact quantification of the analytes in living cells, currently, can be achieved only by mass spectrometry. However, within the framework of our own-authored stochastic dynamic theory and model formulas (mine and my co-author's ones, according to the authorship shown in the references) applicable to account accurately for the fluctuations of the experimental mass spectrometric measurable variable 'intensity.' Please, consider references [1-3].
The method has been tested so far at analyte mixtures at pg.(mL)-1 and ng.(L)-1 concentration levels, yielding to coefficients of linear correlations between theory and the experiment /r/ = 0.99997 - 1.
[A] [https://www.researchgate.net/post/Sensitivity_Coefficient_in_Uncertainty_Calculations_for_HPLC_assay_method_UV_assay_methods_and_pH_measurement]
[1] Steroids, 164 (2020) 108750
Stochastic dynamic mass spectrometric quantification of steroids in mixture — Part II; Bojidarka Ivanova, Michael Spiteller
[2] B.Ivanova, M. Spiteller, Stochastic dynamic mass spectrometric approach to quantify reserpine in solution, (2020) in press.
[3] Bioorganic Chemistry, 93 (2019) 103308
A mass spectrometric stochastic dynamic diffusion approach to selective quantitative and 3D structural analyses of native cyclodextrins by electrospray ionization and atmospheric pressure chemical ionization methods
Bojidarka Ivanova, Michael Spiteller
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