Refractive‐index‐induced aberrations in two‐photon confocal fluorescence microscopy

Abstract

The effect of refractive index mismatch on the image quality in two-photon confocal fluorescence microscopy is investigated by experiment and numerical calculations. The results show a strong decrease in the image brightness using high-aperture objectives when the image plane is moved deeper into the sample. When exciting at 740 nm and recording the fluorescence around 460 nm in a glycerol-mounted sample using a lens of a numerical aperture of 1.4 (oil immersion), a 25% decrease in the intensity is observed at a depth of 9 pm. In an aqueous sample, the same decrease is observed at a depth of 3 pm. By reducing the numerical aperture to 1.0, the intensity decrease can be avoided at the expense of the overall resolution and signal intensity. The experiments are compared with the predictions of a theory that takes into account the vectorial character of light and the refraction of the wavefronts according to Fermat's principle. Advice is given concerning how the effects can be taken into account in practice.

Improving signal levels in intravital multiphoton microscopy using an objective correction collar

“…Second, MPM will be much more sensitive to any factor in tissue that broadens the focused spot. Consistent with this, studies in model systems have shown MPM is highly sensitive to spherical aberration [12,22,24,25].…”

“…This has been indirectly demonstrated by the fact that both signal level [12,24] and resolution [12] are significantly improved by more closely matching sample refractive index to that of the objective immersion medium.…”

“…In addition, signal levels can decline with depth when the refractive index of the tissue is not matched to that of the immersion fluid of the objective. [22][23][24][25] This "refractive index mismatch" results in spherical aberration, a condition in which paraxial light rays and peripheral light rays focus to different places, broadening the focal point of the objective lens. While both confocal and multiphoton microscopy are susceptible to each of these factors, the magnitude of their effects is much reduced by the unique design of the multiphoton microscope, as described below.…”

Strategies to overcome photobleaching in algorithm-based adaptive optics for nonlinearin-vivoimaging

“…Aberrations introduced either by the system's optical setup or the sample under investigation cause an elongation and broadening of the diffraction limited focal spot, shown to severely degrade nonlinear microscopy. [4][5][6] In nonlinear microscopy, an aberrated focal spot not only results in decreased resolution but also a considerable loss of signal intensity as signal scales with the square of laser intensity. 7 The amount of aberrations introduced by a sample can be manipulated in various ways.…”

Clearing of fixed tissue: a review from a microscopist’s perspective

“…This fact contributes to render confocal and two-photon microscopy sensible to optical aberrations. 43 Thus, in general, these techniques require objective lenses corrected for the refractive index of the clearing solution employed.…”