Simultaneous multiplane confocal microscopy using acoustic tunable lenses (original) (raw)
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Enhanced volumetric imaging in 2-photon microscopy via acoustic lens beam shaping
Journal of Biophotonics, 2018
Three-dimensional imaging at high-spatiotemporal resolutions and over large penetration depths is key for unmasking the dynamics and structural organization of complex biological systems. However, the need to axially shift the focus, with consequent limitations in imaging speed, and signal degradation at large depths due to scattering effects, makes this task challenging. Here, we present a novel approach in 2- photon excitation microscopy that allows fast volumetric imaging and enhanced signal-to-background (S/B) in thick tissue. Our technique is based on ultrafast beam shaping at each pixel by means of an acoustic optofluidic lens. Shaping the excitation beam with different phase profiles enables both high-speed axial focus shifting, for continuous volumetric imaging, and controlled aberrated imaging, advantageous for out-of-focus background removal. We provide a theoretical description of our approach, and demonstrate volumetric imaging of neuronal cells from a mouse brain slice with enhancements in S/B up to a factor of 10 over a depth of 600 μm.
Integrated multimodal photoacoustic microscopy with OCT-guided dynamic focusing
Optical Society of America, 2019
Combining different contrast mechanisms to achieve simultaneous multimodal imaging is always desirable but is challenging due to the various optical and hardware requirements for different imaging systems. We developed a multimodal microscopic optical imaging system with the capability of providing comprehensive structural, functional and molecular information of living tissues. This imaging system integrated photoacoustic microscopy (PAM), optical coherence tomography (OCT), optical Doppler tomography (ODT) and confocal fluorescence microscopy in one platform. By taking advantage of the depth resolving capability of OCT, we developed a novel OCT-guided surface contour scanning methodology for dynamic focusing adjustment. We have conducted phantom, in vivo, and ex vivo tests to demonstrate the capability of the multimodal imaging system for providing comprehensive microscopic information of biological tissues. Integrating all the aforementioned imaging modalities with OCT-guided dynamic focusing for simultaneous multimodal imaging has promising potential for preclinical research and clinical practice in the future.
We present a combined multiphoton-acoustic microscope giving collocated access to the local morphological as well as mechanical properties of living cells. Both methods relay on intrinsic contrast mechanisms and dispense with the need of staining. In the acoustic part of the microscope, a gigahertz ultrasound wave is generated by an acoustic lens and the reflected sound energy is detected by the identical lens in a confocal setup. The achieved lateral resolution is in the range of 1 lm. Contrast in the images arises mainly from the local absorption of sound in the cells related to viscose damping. Additionally, acoustic microscopy can access the sound speed as well as the acoustic impedance of the cell membrane and the cell shape, as it is an intrinsic volume scanning technique. The multiphoton image formation bases on the detection of autofluorescence due to endogenous fluorophores. The nonlinearity of two-photon absorption provides submicron lateral and axial resolution without the need of confocal optical detection. In addition, in the near-IR cell damages are drastically reduced in comparison with direct excitation in the visible or UV. The presented setup was aligned with a dedicated procedure to ensure identical image areas. Combined multiphoton/acoustic images of living myoblast cells are discussed focusing on the reliability of the method.
Converting lateral scanning into axial focusing to speed up three-dimensional microscopy
Light: Science & Applications
In optical microscopy, the slow axial scanning rate of the objective or the sample has traditionally limited the speed of volumetric imaging. Recently, by conjugating either a movable mirror to the image plane in a remote-focusing geometry or an electrically tuneable lens (ETL) to the back focal plane, rapid axial scanning has been achieved. However, mechanical actuation of a mirror limits the axial scanning rate (usually only 10–100 Hz for piezoelectric or voice coil-based actuators), while ETLs introduce spherical and higher-order aberrations that prevent high-resolution imaging. In an effort to overcome these limitations, we introduce a novel optical design that transforms a lateral-scan motion into a spherical aberration-free axial scan that can be used for high-resolution imaging. Using a galvanometric mirror, we scan a laser beam laterally in a remote-focusing arm, which is then back-reflected from different heights of a mirror in the image space. We characterize the optical p...