Comparison of single and two-photon fluorescence imaging using Monte Carlo techniques

Abstract

In the light of applications in medical diagnosis of skin dermal layers where incisions are difficult or impractical (e.g., in infant hemoglobin analysis), the development of a non-invasive technique is important. Two-photon fluorescence microscopy (2PFM) has proven to be a versatile approach in imaging through a multiple-scattering medium like the skin. In tandem, with the development of target-specific fluorescent dyes, it is capable of selectively imaging microscopic organic structures hidden underneath skin tissues by exciting particular fluorophores attached to the sample.
As of late, there has been no quantitative comparison between two-photon and single photon confocal fluorescence microscopy (1PFM). Confocal 1PFM utilizes a detector pinhole to reject the scattered photons that corrupt the detected fluorescent image. 2PFM on the other hand utilizes a unique intensity requirement that endows it with an intrinsic ability to discriminate against scattered light. We investigate these effects by utilizing the Monte Carlo method to simulate and compare the photon distributions in the 1PFM and 2PFM excitation, absorption and fluorescence processes.