Effects of aberrations and specimen structure in conventional, confocal and two‐photon fluorescence microscopy

Abstract

SummarySpecimen-induced aberrations cause a reduction in signal levels and resolution in fluorescence microscopy. Aberrations also affect the image contrast achieved by these microscopes. We model the effects of aberrations on the fluorescence signals acquired from different specimen structures, such as pointlike, linear, planar and volume structures, when imaged by conventional, confocal and two-photon microscopes. From this we derive the image contrast obtained when observing combinations of such structures. We show that the effect of aberrations on the visibility of fine features depends upon the specimen morphology and that the contrast is less significantly affected in microscopes exhibiting optical sectioning. For example, we show that point objects become indistinguishable from background fluorescence in the presence of aberrations, particularly when imaged in a conventional fluorescence microscope. This demonstrates the significant advantage of using confocal or two-photon microscopes over conventional instruments when aberrations are present.

Focal-plane wavefront estimation and control using the extended Kalman filter

“…However, the later is more accurate due to the treatment of the nonlinearities present in the model. We compute in this appendix the Jacobian and the Hessian of the negative logarithm of the posterior distribution given by the index in (17). We want to find the zeros of …”

“…2b shows that the MAP estimator behaves relatively constant for increasing aberrations, although for aberration with rms higher than 0.68 outliers occur. These outliers are local minima of (17), which are obtained because of a badly chosen initial estimate. For high aberrations we can see an increase in the bias of the EKF due to the fact that the approximation of the PSF is not valid anymore.…”

“…Z,J t,l -Ai,j (Xk)) + �(Xk -fJx) T � ;;; l (Xk -fJx), (17) where c is the constant term. We want to minimize (17) using the Newton-Raphson method (see Appendix A) to obtain X, � a,g, :,, 'n ti n [ IT p (Xk IYk,;,j, I'" , E,) ]).…”

“…If only one image is used, there is an indetermination in the sign of the even part of the phase of the wavefront aberration. We use the images in an iterative algorithm in order to estimate and control the specimen induced aberrations [17] present in that sample point using a deformable mirror. We model our optical system with the indexes k, g, i, j as in Table I and Fig.…”

Adaptive optical microscopy: the ongoing quest for a perfect image

“…Aberrations affect not only resolution, but also image brightness and image contrast, and these effects vary according to microscope modality and the specimen structures under observation. 17,18 The effects of common aberrations, such as the spherical aberration introduced by a refractive index mismatch of the lens immersion medium and specimen, had been well studied long before the first adaptive microscope implementations. Specimeninduced aberrations, arising from the structure of tissues, are more complex and a statistical description of their nature is not yet available.…”