Heejin Choi

Improvement of axial resolution and contrast in temporally focused widefield two-photon microscopy with structured light illumination

Although temporally focused wide-field two-photon microscopy (TFM) can perform depth resolved wide field imaging, it cannot avoid the image degradation due to scattering of excitation and emission photons when imaging in a turbid medium. Further, its axial resolution is inferior to standard point-scanning two-photon microscopy. We implemented a structured light illumination for TFM and have shown that it can effectively reject the out-of-focus scattered emission photons improving image contrast. Further, the depth resolution of the improved system is dictated by the spatial frequency of the structure light with the potential of attaining depth resolution better than point-scanning two-photon microscopy.

3D-resolved fluorescence and phosphorescence lifetime imaging using temporal focusing wide-field two-photon excitation

Fluorescence and phosphorescence lifetime imaging are powerful techniques for studying intracellular protein interactions and for diagnosing tissue pathophysiology. While lifetime-resolved microscopy has long been in the repertoire of the biophotonics community, current implementations fall short in terms of simultaneously providing 3D resolution, high throughput, and good tissue penetration. This report describes a new highly efficient lifetime-resolved imaging method that combines temporal focusing wide-field multiphoton excitation and simultaneous acquisition of lifetime information in frequency domain using a nanosecond gated imager from a 3D-resolved plane. This approach is scalable allowing fast volumetric imaging limited only by the available laser peak power. The accuracy and performance of the proposed method is demonstrated in several imaging studies important for understanding peripheral nerve regeneration processes. Most importantly, the parallelism of this approach may enhance the imaging speed of long lifetime processes such as phosphorescence by several orders of magnitude.

Depth-resolved cellular microrheology using HiLo microscopy

It is increasingly important to measure cell mechanical properties in three-dimensional environments. Particle tracking microrheology (PTM) can measure cellular viscoelastic properties; however, out-of-plane data can introduce artifacts into these measurements. We developed a technique that employs HiLo microscopy to reduce out-of-plane contributions. This method eliminated signals from 90% of probes 0.5 μm or further from the focal plane, while retaining all in-plane probes. We used this technique to characterize live-cell bilayers and found that there were significant, frequency-dependent changes to the extracted cell moduli when compared to conventional analysis. Our results indicate that removal of out-of-plane information is vital for accurate assessments of cell mechanical properties.

Wide-field two-photon microscopy with temporal focusing and HiLo background rejection

Scanningless depth-resolved microscopy is achieved through spatial-temporal focusing and has been demonstrated previously. The advantage of this method is that a large area may be imaged without scanning resulting in higher throughput of the imaging system. Because it is a widefield technique, the optical sectioning effect is considerably poorer than with conventional spatial focusing two-photon microscopy. Here we propose wide-field two-photon microscopy based on spatio-temporal focusing and employing background rejection based on the HiLo microscope principle. We demonstrate the effects of applying HiLo microscopy to widefield temporally focused two-photon microscopy.

Ultrafast optical pulse delivery with fibers for nonlinear microscopy

Nonlinear microscopies including multiphoton excitation fluorescence microscopy and multiple‐harmonic generation microscopy have recently gained popularity for cellular and tissue imaging. The optimization of these imaging methods for minimally invasive use requires optical fibers to conduct light into tight space, where free‐space delivery is difficult. The delivery of high‐peak power laser pulses with optical fibers is limited by dispersion resulting from nonlinear refractive index responses. In this article, we characterize a variety of commonly used optical fibers in terms of how they affect pulse profile and imaging performance of nonlinear microscopy; the following parameters are quantified: spectral bandwidth and temporal pulse width, two‐photon excitation efficiency, and optical resolution. A theoretical explanation for the measured performance of these fibers is also provided. Microsc. Res. Tech., 2008.

Three-dimensional image cytometer based on widefield structured light microscopy and high-speed remote depth scanning

A high throughput 3D image cytometer have been developed that improves imaging speed by an order of magnitude over current technologies. This imaging speed improvement was realized by combining several key components. First, a depth‐resolved image can be rapidly generated using a structured light reconstruction algorithm that requires only two wide field images, one with uniform illumination and the other with structured illumination. Second, depth scanning is implemented using the high speed remote depth scanning. Finally, the large field of view, high NA objective lens and the high pixelation, high frame rate sCMOS camera enable high resolution, high sensitivity imaging of a large cell population. This system can image at 800 cell/sec in 3D at submicron resolution corresponding to imaging 1 million cells in 20 min. The statistical accuracy of this instrument is verified by quantitatively measuring rare cell populations with ratio ranging from 1:1 to 1:105.

Improving femtosecond laser pulse delivery through a hollow core photonic crystal fiber for temporally focused two-photon endomicroscopy

In this paper, we present a strategy to improve delivery of femtosecond laser pulses from a regenerative amplifier through a hollow core photonic crystal fiber for temporally focused wide-field two-photon endomicroscopy. For endomicroscope application, wide-field two-photon excitation has the advantage of requiring no scanning in the distal end. However, wide-field two-photon excitation requires peak power that is 104–105 times higher than the point scanning approach corresponding to femtosecond pulses with energy on the order of 1–10 μJ at the specimen plane. The transmission of these high energy pulses through a single mode fiber into the microendoscope is a significant challenge. Two approaches were pursued to partially overcome this limitation. First, a single high energy pulse is split into a train of pulses with energy below the fiber damage threshold better utilizing the available laser energy. Second, stretching the pulse width in time by introducing negative dispersion was shown to have the dual benefit of reducing fiber damage probability and compensating for the positive group velocity dispersion induced by the fiber. With these strategy applied, 11 fold increase in the two photon excitation signal has been demonstrated.

Improving signal-to-noise ratio of structured light microscopy based on photon reassignment.

In this paper, we report a method for 3D visualization of a biological specimen utilizing a structured light wide-field microscopic imaging system. This method improves on existing structured light imaging modalities by reassigning fluorescence photons generated from off-focal plane excitation, improving in-focus signal strength. Utilizing a maximum likelihood approach, we identify the most likely fluorophore distribution in 3D that will produce the observed image stacks under structured and uniform illumination using an iterative maximization algorithm. Our results show the optical sectioning capability of tissue specimens while mostly preserving image stack photon count, which is usually not achievable with other existing structured light imaging methods.

Depth resolved hyperspectral imaging spectrometer based on structured light illumination and Fourier transform interferometry

A depth resolved hyperspectral imaging spectrometer can provide depth resolved imaging both in the spatial and the spectral domain. Images acquired through a standard imaging Fourier transform spectrometer do not have the depth-resolution. By post processing the spectral cubes (x, y, λ) obtained through a Sagnac interferometer under uniform illumination and structured illumination, spectrally resolved images with depth resolution can be recovered using structured light illumination algorithms such as the HiLo method. The proposed scheme is validated with in vitro specimens including fluorescent solution and fluorescent beads with known spectra. The system is further demonstrated in quantifying spectra from 3D resolved features in biological specimens. The system has demonstrated depth resolution of 1.8 μm and spectral resolution of 7 nm respectively.

6 Principles and applications of temporal-focusing wide-field two-photon microscopy

Temporal focusing allows for rapid optically sectioned two-photon widefieldmicroscopy. Depth sectioning is provided in a wide-fieldmanner, without spatial focusing, by controlling the temporal width of femtosecond laser pulses near the focal plane. This spatial control of the temporal pulse width is achieved by diffracting the light off a grating resulting in spectral component separation and temporal broadening; these spectral components are only recombined at the focal plane to reproduce short, femtosecond pulses. Applications include (i) high speed functional imaging in the brain, (ii) fast FLIM and PLIM, (iii) cell-selective optogenetic excitation, and (iv) temporal focusing photodynamic therapy that may allow selective killing of cancer cells.