Enhancement of imaging depth of two-photon microscopy using pinholes: Analytical simulation and experiments

Abstract

Achieving a greater imaging depth with two-photon fluorescence microscopy (TPFM) is mainly limited by out-of-focus fluorescence generated from both ballistic and scattered light excitation. We report on an improved signal-to-noise ratio (SNR) in a highly scattering medium as demonstrated by analytical simulation and experiments for TPFM. Our technique is based on out-of-focus rejection using a confocal pinhole. We improved the SNR by introducing the pinhole in the collection beam path. Using the radiative transfer theory and the ray-optics approach, we analyzed the effects of different sizes of pinholes on the generation of the fluorescent signal in the TPFM system. The analytical simulation was evaluated by comparing its results with the experimental results in a scattering medium. In a combined confocal pinhole and two-photon microscopy system, the imaging depth limit of approximately 5 scattering mean free paths (MFP) was found to have improved to 6.2 MFP.

Oblique scanning 2‐photon light‐sheet fluorescence microscopy for rapid volumetric imaging

“…The overall FOV and the intensity of the OS‐2P‐LSFM have complex relationships defined by the axial extend and intensity of the Bessel beam, the scanning range of the GM, the spatial collection efficiency of the OBJ1—2 , and beam clipping associated with the tilted detection (PM) .…”

Zirconia dental implants degradation by confocal Raman microspectroscopy: analytical simulation and experiments

“…Pinhole function as rejecting out-of focus signals, enables thin optical sectionning within thick samples [21]. …”

Two-photon image-scanning microscopy with SPAD array and blind image reconstruction

“…This latter situation is where non-descanned microscopy architecture -with no pinhole and large single-element detector -provides the best results. On the other hand, non-descanned detection fails in the case of high near-surface (out-of-focus) fluorescence background signal, since there is no pinhole to reject it [35,36]. In any case, as long as there are some ballistic photons, 2PE-ISM can provide a significant resolution increase.…”

“…will move this limit deeper and deeper. Moreover, one should not underestimate the benefit of having an effective optical pinhole (that is not detrimental to the SNR) in a 2PE system, as the effective working distance of a typical 2PE system is ultimately limited by near-surface (out-of-focus) fluorescence signal that the wide area detectors cannot reject [35,36]. Our simple comparison in Fig.…”