Three dimensional live-cell STED microscopy at increased depth using a water immersion objective

Abstract

Modern fluorescence superresolution microscopes are capable of imaging living cells on the nanometer scale. One of those techniques is stimulated emission depletion (STED) which increases the microscope's resolution many times in the lateral and the axial directions. To achieve these high resolutions not only close to the coverslip but also at greater depths, the choice of objective becomes crucial. Oil immersion objectives have frequently been used for STED imaging since their high numerical aperture (NA) leads to high spatial resolutions. But during live-cell imaging, especially at great penetration depths, these objectives have a distinct disadvantage. The refractive index mismatch between the immersion oil and the usually aqueous embedding media of living specimens results in unwanted spherical aberrations. These aberrations distort the point spread functions (PSFs). Notably, during z- and 3D-STED imaging, the resolution increase along the optical axis is majorly hampered if at all possible. To overcome this limitation, we here use a water immersion objective in combination with a spatial light modulator for z-STED measurements of living samples at great depths. This compact design allows for switching between objectives without having to adapt the STED beam path and enables on the fly alterations of the STED PSF to correct for aberrations. Furthermore, we derive the influence of the NA on the axial STED resolution theoretically and experimentally. We show under live-cell imaging conditions that a water immersion objective leads to far superior results than an oil immersion objective at penetration depths of 5-180 μm.

Coherent-hybrid STED: a tunable photo-physical pinhole for super-resolution imaging at high contrast

“…Although very efficient in improving axial resolution, z-STED provides a lower lateral resolution than 2D-STED, while being relatively noisy due to the spurious signal emitted by secondary excitation lobes ('ghosts') that are not well covered by the z-STED beam geometry. In addition, an intensity-zero will only form if the central disc of the top-hat has the correct dimension relative to the microscope objective's back-aperture (Keller et al, 2007, Heine et al, 2018 or, more precisely, relative to the effective aperture covered by the laser beam. A scale mismatch will unbalance the contribution of the -shifted electric fields, leading to a filling of the central zero that depletes the signal of interest, compromising resolution and in particular the signal-to-noise ratio (SNR).…”

High photon count rates improve the quality of super-resolution fluorescence fluctuation spectroscopy

“…Yet, penetration through the aqueous cellular environment over such a distance causes spherical aberrations when employing a traditional oilimmersion STED microscope objective (due to the refractive index mismatch between water and oil) 45 , having detrimental effects on STED-FCS experiments ( Figure SI 5). Such aberrations can either be corrected for using adaptive optics 46,47 or employing a water immersion objective 48 , which shows constant signal levels and performance of for example STED and STED-FCS experiments in a wide range of focal depths (5-100 µm above the coverslip) without the need for depth-dependent readjustments of the correction collar ( Figure SI 5). Taking the observation spot size as determined from calibration data ( Figure SI 6, diameter of 100 nm), we could calculate apparent values of the diffusion coefficient D for both the confocal and STED mode (spot diameters of 280 and 100 nm, respectively; see Materials and Methods), which were both in the same range (D ≈ 0.45 µm²/s, Figure 5D) as observed before for the basal membrane 44 , highlighting free diffusion and indicating similar diffusion characteristics of the probe in the apical and basal plasma membrane 44 , i.e.…”

3D super-resolution deep-tissue imaging in living mice

“…If left uncorrected, aberrations can raise the intensity minimum in the center of the depletion focus, causing fluorescence emission to be depleted entirely, rather than being confined to the center of the depletion ring. Recently, Heine et al replaced the oil or glycerol immersion objective lens of a typical 3D-STED microscope with a water immersion objective to minimize the spherical aberration induced when imaging aqueous living specimens [17]. Despite the lower NA of water immersion objectives, the authors resolved 153 nm structures axially.…”