Abstract
Multiphoton laser scanning microscopy (MPM) has opened up an optical window into biological tissues; however, imaging is primarily qualitative. Cell morphology and tissue architectures can be clearly visualized but quantitative analysis of actual concentration and fluorophore distribution is indecisive. Fluorescence correlation spectroscopy (FCS) is a highly sensitive photophysical methodology employed to study molecular parameters such as diffusion characteristics on the single molecule level. In combination with laser scanning microscopy, and MPM in particular, FCS has been referred to as a standard and highly useful tool in biomedical research to study diffusion and molecular interaction with subcellular precision. Despite several proof-of-concept reports on the topic, the implementation of MPM-FCS is far from straightforward. This practical guideline aims to clarify the conceptual principles and define experimental operating conditions when implementing MPM-FCS. Validation experiments in Rhodamine solutions were performed on an experimental MPM-FCS platform investigating the effects of objective lens, fluorophore concentration and laser power. An approach based on analysis of time-correlated single photon counting data is presented. It is shown that the requirement of high numerical aperture (NA) objective lenses is a primary limitation that restricts field of view, working distance and concentration range. Within these restrictions the data follows the predicted theory of Poisson distribution. The observed dependence on laser power is understood in the context of perturbation on the effective focal volume. In addition, a novel interpretation of the effect on measured diffusion time is presented. Overall, the challenges and limitations observed in this study reduce the versatility of MPM-FCS targeting biomedical research in complex and deep tissue—being the general strength of MPM in general. However, based on the systematic investigations and fundamental insights this report can serve as a practical guide and inspire future research, potentially overcoming the technical limitations and ultimately allowing MPM-FCS to become a highly useful tool in biomedical research.
Highlights
In this paper we focus on time-correlated single photon counting (TCSPC) technology; but there are alternative solutions one being hard-ware correlation
It is evident from the figure that fluorescence correlation spectroscopy (FCS) curves can be acquired using both the 63× and 40× objective lenses; the amplitude of the correlation function is higher for the 63× objective, i.e., G(0) = 0.6; compared to G(0) = 0.035 for the 40× objective
This is expected as a higher numerical aperture (NA) will generate a smaller excitation volume, thereby comprising less molecules giving rise to a higher G(0) value according to Equation (3) in theory section
Summary
To the best of our knowledge there are no studies systematically comparing how FCS performance will be affected by the choice of objective lens under the same conditions using the same experimental system, which is a central part of the present investigation Another important factor is the concentration range in which the FCS method can operate. We in this paper take a “ground-zero” approach, developing a protocol starting from the raw data photon counts using TCSPC where the choice of data binning is of importance This practical guideline aims to clarify the conceptual principles and define experimental operating conditions in order to facilitate implementation of MPM-FCS using TSCPC. A protocol for converting raw data photon counts to autocorrelated signal is presented, the effect of binning explored and the effect of excitation power on autocorrelation data is investigated
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