Temporal Focusing Microscopy Research Articles

Multiline orthogonal scanning temporal focusing (mosTF) microscopy for scattering reduction in in vivo brain imaging

Temporal focusing two-photon microscopy has been utilized for high-resolution imaging of neuronal and synaptic structures across volumes spanning hundreds of microns in vivo. However, a limitation of temporal focusing is the rapid degradation of the signal-to-background ratio and resolution with increasing imaging depth. This degradation is due to scattered emission photons being widely distributed, resulting in a strong background. To overcome this challenge, we have developed multiline orthogonal scanning temporal focusing (mosTF) microscopy. mosTF captures a sequence of images at each scan location of the excitation line. A reconstruction algorithm then reassigns scattered photons back to their correct scan positions. We demonstrate the effectiveness of mosTF by acquiring neuronal images of mice in vivo. Our results show remarkable improvements in in vivo brain imaging with mosTF, while maintaining its speed advantage.

Adaptive optics with spatio-temporal lock-in detection for temporal focusing microscopy.

Wavefront distortion in temporal focusing microscopy (TFM) results in a distorted temporal profile of the excitation pulses owing to spatio-temporal coupling. Since the pulse duration is dramatically changed in the excitation volume, it is difficult to correct the temporal profile for a thick sample. Here, we demonstrate adaptive optics (AO) correction in a thick sample. We apply structured illumination microscopy (SIM) to an AO correction in wide-field TFM to decrease the change in the pulse duration in the signal detection volume. The AO correction with SIM was very successful in a thick sample for which AO correction with TFM failed.

HiLo Based Line Scanning Temporal Focusing Microscopy for High-Speed, Deep Tissue Imaging.

High-speed, optical-sectioning imaging is highly desired in biomedical studies, as most bio-structures and bio-dynamics are in three-dimensions. Compared to point-scanning techniques, line scanning temporal focusing microscopy (LSTFM) is a promising method that can achieve high temporal resolution while maintaining a deep penetration depth. However, the contrast and axial confinement would still be deteriorated in scattering tissue imaging. Here, we propose a HiLo-based LSTFM, utilizing structured illumination to inhibit the fluorescence background and, thus, enhance the image contrast and axial confinement in deep imaging. We demonstrate the superiority of our method by performing volumetric imaging of neurons and dynamical imaging of microglia in mouse brains in vivo.

Enhancement of optical sectioning capability of temporal focusing microscopy by using time-multiplexed multi-line focusing

Temporal focusing (TF) microscopy has a trade-off between temporal resolution, optical sectioning capability, and field of view. To improve this trade-off, we demonstrate time-multiplexed multi-line temporal focusing (TM-ML-TF) microscopy, which combines the time-multiplexed TF technique with multi-line focusing using a digital micromirror device. Compared with those for TF microscopy, the optical sectioning capability and the signal-to-background ratio were improved by factors of 2.5 and 4.17, respectively. In addition, by applying post-processing for structured illumination microscopy to TM-ML-TF microscopy, the optical sectioning capability and the signal-to-background ratio were further enhanced by factors of 1.2 and 4.63, respectively.

Holography-based structured light illumination for temporal focusing microscopy.

In this Letter, we present a holography-based structured light illumination (SLI) method to enhance the resolution of widefield temporal focusing microscopy (TFM). In the system, a digital micromirror device is employed to simultaneously disperse the incoming femtosecond laser to induce temporal focusing at the focal plane and generate designed structured patterns via a Lee hologram. As the generated structured patterns do not contain the zeroth order beam, it improves the contrast and modulation frequency. Mathematical models have been derived to calculate the electric fields at the focal plane and to explain the effects of improved optical cross-sectioning capability. Imaging experiments have been devised and performed on fluorescent beads and mouse kidney sections; the results demonstrate enhanced axial confinement and improved suppression of out-of-focus fluorescence. The new SLI method realizes high-resolution TFM and can be readily applied to other microscopy platforms for biophotonics applications.

Photobleaching Imprinting Enhanced Background Rejection in Line-Scanning Temporal Focusing Microscopy.

Compared with two-photon point-scanning microscopy, two-photon temporal focusing microscopy (2pTFM) provides a parallel high-speed imaging strategy with optical sectioning capability. Owing to out-of-focus fluorescence induced by scattering, 2pTFM suffers deteriorated signal-to-background ratio (SBR) for deep imaging in turbid tissue, Here, we utilized the photobleaching property of fluorophore to eliminate out-of-focus fluorescence. According to different decay rates in different focal depth, we extract the in-focus signals out of backgrounds through time-lapse images. We analyzed the theoretical foundations of photobleaching imprinting of the line-scanning temporal focusing microscopy, simulated implementation for background rejection, and demonstrated the contrast enhancement in MCF-10A human mammary epithelial cells and cleared Thy1-YFP mouse brains. More than 50% of total background light rejection was achieved, providing higher SBR images of the MCF-10A samples and mouse brains. The photobleaching imprinting method can be easily adapted to other fluorescence dyes or proteins, which may have application in studies involving relatively large and nontransparent organisms.

Overcoming tissue scattering in wide-field two-photon imaging by extended detection and computational reconstruction.

Compared to point-scanning multiphoton microscopy, line-scanning temporal focusing microscopy (LTFM) is competitive in high imaging speed while maintaining tight axial confinement. However, considering its wide-field detection mode, LTFM suffers from shallow penetration depth as a result of the crosstalk induced by tissue scattering. In contrast to the spatial filtering based on confocal slit detection, here we propose the extended detection LTFM (ED-LTFM), the first wide-field two-photon imaging technique to extract signals from scattered photons and thus effectively extend the imaging depth. By recording a succession of line-shape excited signals in 2D and reconstructing signals under Hessian regularization, we can push the depth limitation of wide-field imaging in scattering tissues. We validate the concept with numerical simulations, and demonstrate the performance of enhanced imaging depth in in vivo imaging of mouse brains.

Hybrid spatio-spectral coherent adaptive compensation for line-scanning temporal focusing microscopy

Line-scanning temporal focusing microscopy (LTFM) is promising for various biomedical studies, due to its higher imaging speed compared to the gold-standard point-scanning two-photon microscopy while maintaining the same axial confinement. However, LTFM is susceptible to deteriorated performances in excitation efficiency, penetration depth, and resolutions, as a result of wavefront distortions in deep-tissue imaging. Here, we report the hybrid spatio-spectral coherent adaptive compensation (HSSCAC), the first reported technique to fully correct the wavefront distortions in LTFM. Compared with the conventional hill-climbing method, HSSCAC can fully compensate the aberrations. We demonstrate the performance of HSSCAC in deep imaging of neurons in cleared mouse brains and in vivo dynamic imaging of microglias in living mouse brains.

Scattering reduction by structured light illumination in line-scanning temporal focusing microscopy.

Line-scanning temporal focusing microscopy (LineTFM) is capable of imaging biological samples more than 10 times faster than two-photon laser point-scanning microscopy (TPLSM), while achieving nearly the same lateral and axial spatial resolution. However, the image contrast taken by LineTFM is lower than that by TPLSM because LineTFM is severely influenced by biological tissue scattering. To reject the scattered photons, we implemented LineTFM using both structured illumination and uniform illumination combined with the HiLo post-processing algorithm, called HiLL microscopy (HiLo-Line-scanning temporal focusing microscopy). HiLL microscopy significantly reduces tissue scattering and improves image contrast. We demonstrate HiLL microscopy with in vivo brain imaging. This approach could potentially find applications in monitoring fast dynamic events and in mapping high resolution structures over a large volume.

Resistance to optical distortions in three-dimensional interferometric temporal focusing microscopy

Abstract We analyze how optical distortions in deep imaging or dense scattering media influence three-dimensional interferometric temporal focusing (3D-ITF) imaging, and derive the distorted point spread function (PSF) and optical transfer function (OTF). Upon this, we find that 3D-ITF microscopy is less affected by optical distortions in terms of spatial resolution, artifacts and sectioning ability. Temporal focusing (TF) illumination supports a robust spatial modulation in 3D-ITF microscopy with distorted optics. As the modulation spatial frequencies are unique, they offer a redundancy in 3D-ITF microscopy. The redundancy in the spatial frequency of 3D-ITF images compensates for the loss of spatial frequency caused by the optical distortion in the emission OTF. The redundancy recovers the high spatial frequencies to prevent the degradation of spatial resolution. Moreover, the redundancy recovers the distortion in the emission OTF spectrum, which causes distorted shapes in the PSF such as side lobes and double peaks. Thus, there is a relatively lossless OTF spectrum in 3D-ITF compared to TF, and this leads to better trade-off between remaining noise artifacts and resolution after the Wiener filter correction.

Enhancing axial resolution and background rejection in line-scanning temporal focusing microscopy by focal modulation.

Compared with two-photon point-scanning microscopy, line-scanning temporal focusing microscopy breaks the limitation on imaging rate and maintains the axial resolution, which makes it promising for various biomedical studies. However, for deep tissue imaging, it suffers from reduced axial resolution and increased background noise due to sample induced wavefront distortion. Here, we propose a spatio-spectral focal modulation technique to enhance axial resolution and background rejection by simply subtracting an aberrated image, which is induced by a spatial light modulator, from an unaberrated image. The proposed technique could improve the axial resolution by a factor of 1.3 in our implementation, verified by both simulations and experiments. Besides, we show that compared with spatial modulation alone, spatio-spectral modulation induces less peak intensity loss caused by image subtraction. We further demonstrate the performance of our technique on the enhanced axial resolution and background rejection by deep imaging of cleared mouse brains and in vivo imaging of living mouse brains.

Interferometric temporal focusing microscopy using three-photon excitation fluorescence.

Super-resolution microscopy has become a powerful tool for biological research. However, its spatial resolution and imaging depth are limited, largely due to background light. Interferometric temporal focusing (ITF) microscopy, which combines structured illumination microscopy and three-photon excitation fluorescence microscopy, can overcome these limitations. Here, we demonstrate ITF microscopy using three-photon excitation fluorescence, which has a spatial resolution of 106 nm at an imaging depth of 100 µm with an excitation wavelength of 1060 nm.

Fast two-snapshot structured illumination for temporal focusing microscopy with enhanced axial resolution

We present a new two-snapshot structured light illumination (SLI) reconstruction algorithm for fast image acquisition. The new algorithm, which only requires two mutually π phase-shifted raw structured images, is implemented on a custom-built temporal focusing fluorescence microscope (TFFM) to enhance its axial resolution via a digital micromirror device (DMD). First, the orientation of the modulated sinusoidal fringe patterns is automatically identified via spatial frequency vector detection. Subsequently, the modulated in-focal-plane images are obtained via rotation and subtraction. Lastly, a parallel amplitude demodulation method, derived based on Hilbert transform, is applied to complete the decoding processes. To demonstrate the new SLI algorithm, a TFFM is custom-constructed, where a DMD replaces the generic blazed grating in the system and simultaneously functions as a diffraction grating and a programmable binary mask, generating arbitrary fringe patterns. The experimental results show promising depth-discrimination capability with an axial resolution enhancement factor of 1.25, which matches well with the theoretical estimation, i.e, 1.27. Imaging experiments on pollen grain and mouse kidney samples have been performed. The results indicate that the two-snapshot algorithm presents comparable contrast reconstruction and optical cross-sectioning capability than those adopting the conventional root-mean-square (RMS) reconstruction method. The two-snapshot method can be readily applied to any sinusoidally modulated illumination systems to realize high-speed 3D imaging as less frames are required for each in-focal-plane image restoration, i.e., the image acquisition speed is improved by 2.5 times for any two-photon systems.

Temporal focusing microscopy using three-photon excitation fluorescence with a 92-fs Yb-fiber chirped pulse amplifier.

Temporal focusing (TF) microscopy is a wide-field two-photon excitation fluorescence (2PEF) microscopy technique, the optical sectioning capability of which is lower than that of point-scanning 2PEF microscopy. Here we demonstrate TF microscopy using three-photon excitation fluorescence (3PEF), which enhances the optical sectioning capability. As an excitation light source for the 3PEF, we developed an Yb-fiber chirped pulse amplifier, which produces 92-fs 9.0-μJ 1060-nm pulses at a repetition rate of 200 kHz. The optical sectioning capability was improved by a factor of 1.3 compared with that of 2PEF-TF microscopy. We also demonstrate dual-color imaging with both 2PEF and 3PEF.

Temporal focusing microscopy combined with three-dimensional structured illumination

Temporal focusing microscopy provides the optical sectioning capability in wide-field two-photon fluorescence imaging. Here, we demonstrate temporal focusing microscopy combined with three-dimensional structured illumination, which enables us to enhance the three-dimensional spatial resolution and reject the background fluorescence. Experimentally, the periodic pattern of the illumination was produced not only in the lateral direction but also in the axial direction by the interference between three temporal focusing pulses, which were easily generated using a digital micromirror device. The lateral resolution and optical sectioning capability were successfully enhanced by factors of 1.6 and 3.6, respectively, compared with those of temporal focusing microscopy. In the two-photon fluorescence imaging of a tissue-like phantom, the out-of-focus background fluorescence and the scattered background fluorescence could also be rejected.

Fast 3-D temporal focusing microscopy using an electrically tunable lens.

In this paper, we present a 3-D temporal focusing microscope based on an electrically tunable lens (ETL) and a femtosecond regenerative laser amplifier. The focus-tunable lens provides a fast and compact way to perform non-mechanical z-scanning and resolves the blurry image issue compared with GVD-based z-scanning methods. The optical performance of the temporal focusing system, including z-scanning characteristics, the associated the magnification variation, and the lateral and axial resolution, has been studied and characterized using calibrated Rhodamine-6G thin film sample, fluorescent beads, and pollen samples. Lastly, we demonstrate the optical cross-sectioning and z-scanning capability with an in vivo experiment, where Ca(2+) imaging of neurons in GaCamp6 labeled zebrafish was performed.

Enhancement of lateral resolution and optical sectioning capability of two-photon fluorescence microscopy by combining temporal-focusing with structured illumination

We demonstrate super-resolution imaging with background fluorescence rejection by interferometric temporal focusing microscopy, in which temporal focusing is combined with structured illumination. The lateral resolution and the optical sectioning capability are simultaneously improved by factors of 1.6 and 1.4, respectively, compared to conventional temporal focusing microscopy. Fluorescent beads (200 nm diameter) that are difficult to distinguish from the background fluorescence in conventional temporal focusing microscopy, are clearly visualized by interferometric temporal focusing microscopy.