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

Abstract

Multiphoton microscopy has enabled biologists to collect high-resolution images hundreds of microns into biological tissues, including tissues of living animals. While the depth of imaging exceeds that possible from any other form of light microscopy, multiphoton microscopy is nonetheless generally limited to depths of less than a millimeter. Many of the advantages of multiphoton microscopy for deep tissue imaging accrue from the unique nature of multiphoton fluorescence excitation. However, the quadratic relationship between illumination level and fluorescence excitation makes multiphoton microscopy especially susceptible to factors that degrade the illumination focus. Here we examine the effect of spherical aberration on multiphoton microscopy in fixed kidney tissues and in the kidneys of living animals. We find that spherical aberration, as evaluated from axial asymmetry in the point spread function, can be corrected by adjustment of the correction collar of a water immersion objective lens. Introducing a compensatory positive spherical aberration into the imaging system decreased the depth-dependence of signal levels in images collected from living animals, increasing signal by up to 50%.

Adaptive optical versus spherical aberration corrections for in vivo brain imaging

“…The approach that is most accessible to routine microscope users is adjusting the correction collar of some microscope objective lenses, which axially translates a movable lens group within the objective and was originally developed to correct SA introduced by coverglasses [16]. The functionality of correction collars was extended to the empirical removal of tissue-induced SA in multiphoton imaging [17]. This type of adjustment was sometimes automated by attaching to the correction collar a belt linked to a stepper motor [18].…”

Molecular mobility and activity in an intravital imaging setting – implications for cancer progression and targeting

“…In IVM, penetration depth is often limited by aberration caused by the sample itself, which may be compensated for by using adaptive optics, for instance using a correction collar (Muriello and Dunn, 2008), a deformable membrane mirror (Caroline Müllenbroich et al, 2014) or segmented pupil illumination (Ji et al, 2012;Wang et al, 2014), which can correct for sample aberrations by increasing signal at depth. Alternatively, image-based metrics can be used to estimate the sample aberrations (Burke et al, 2015;Débarre et al, 2009;Song et al, 2010).…”

Imaging the pharmacology of nanomaterials by intravital microscopy: Toward understanding their biological behavior

“…Heuristic computational approaches can be taken to optimize the time it takes to acquire images [257]. Although discussion of microscope optics extends beyond the scope of this review, several adjustments including objective collars [258], real-time wavefront detection and feedback [259,260], and increased inter-pinhole distance [261] all correct aberration in thick specimens and have been developed for deep-tissue IVM applications. IVM often involves imaging structures that are large and bright next to structures that are equally important yet small and dim, for example in the case of neuron nuclear bodies and their thin axon processes; high-dynamic-range setups have been described to simultaneously capture these disparate features [262].…”