Video-rate remote refocusing through continuous oscillation of a membrane deformable mirror

This paper presents the use of a deformable mirror (DM) configured to rapidly refocus a microscope employing a high numerical aperture objective lens. An Alpao DM97-15 membrane DM was used to refocus a 40×/0.80 NA water-immersion objective through a defocus range of −50 to 50 μm at 26.3 sweeps per second. We achieved imaging with a mean Strehl metric of > 0.6 over a field of view in the sample of 200×200 μm2 over a defocus range of 77 μm. We describe an optimisation procedure where the mirror is swept continuously in order to avoid known problems of hysteresis associated with the membrane DM employed. This work demonstrates that a DM-based refocusing system could in the future be used in light-sheet fluorescence microscopes to achieve video-rate volumetric imaging.

In vivo imaging with a water immersion objective affects brain temperature, blood flow and oxygenation

Previously, we reported the first oxygen partial pressure (Po2) measurements in the brain of awake mice, by performing two-photon phosphorescence lifetime microscopy at micrometer resolution (Lyons et al., 2016). However, this study disregarded that imaging through a cranial window lowers brain temperature, an effect capable of affecting cerebral blood flow, the properties of the oxygen sensors and thus Po2 measurements. Here, we show that in awake mice chronically implanted with a glass window over a craniotomy or a thinned-skull surface, the postsurgical decrease of brain temperature recovers within a few days. However, upon imaging with a water immersion objective at room temperature, brain temperature decreases by ~2–3°C, causing drops in resting capillary blood flow, capillary Po2, hemoglobin saturation, and tissue Po2. These adverse effects are corrected by heating the immersion objective or avoided by imaging through a dry air objective, thereby revealing the physiological values of brain oxygenation.

On-Chip Real-Time Chemical Sensors Based on Water-Immersion-Objective Pumped Whispering-Gallery-Mode Microdisk Laser

Optical whispering-gallery-mode (WGM) microresonator-based sensors with high sensitivity and low detection limit down to single unlabeled biomolecules show high potential for disease diagnosis and clinical application. However, most WGM microresonator-based sensors, which are packed in a microfluidic cell, are a “closed” sensing configuration that prevents changing and sensing the surrounding liquid refractive index (RI) of the microresonator immediately. Here, we present an “open” sensing configuration in which the WGM microdisk laser is directly covered by a water droplet and pumped by a water-immersion-objective (WIO). This allows monitoring the chemical reaction progress in the water droplet by tracking the laser wavelength. A proof-of-concept demonstration of chemical sensor is performed by observing the process of salt dissolution in water and diffusion of two droplets with different RI. This WIO pumped sensing configuration provides a path towards an on-chip chemical sensor for studying chemical reaction kinetics in real time.

Aberration-corrected cryoimmersion light microscopy

Cryogenic fluorescent light microscopy of flash-frozen cells stands out by artifact-free fixation and very little photobleaching of the fluorophores used. To attain the highest level of resolution, aberration-free immersion objectives with accurately matched immersion media are required, but both do not exist for imaging below the glass-transition temperature of water. Here, we resolve this challenge by combining a cryoimmersion medium, HFE-7200, which matches the refractive index of room-temperature water, with a technological concept in which the body of the objective and the front lens are not in thermal equilibrium. We implemented this concept by replacing the metallic front-lens mount of a standard bioimaging water immersion objective with an insulating ceramic mount heated around its perimeter. In this way, the objective metal housing can be maintained at room temperature, while creating a thermally shielded cold microenvironment around the sample and front lens. To demonstrate the range of potential applications, we show that our method can provide superior contrast in Escherichia coli and yeast cells expressing fluorescent proteins and resolve submicrometer structures in multicolor immunolabeled human bone osteosarcoma epithelial (U2OS) cells at −140°C.