Incoherent superposition of polychromatic light enables single-shot nondiffracting light-sheet microscopy.

We demonstrate single-shot nondiffracting light-sheet microscopy by the incoherent superposition of dispersed polychromatic light sources. We characterized our technique by generating a Bessel light-sheet with a supercontinuum light-source and a C-light-sheet using a diode laser, and demonstrated its applicability to fluorescence microscopy. We emphasize that our method is easily implementable and compatible with the requirements of high-resolution microscopy.

Single-shot, shadowless total internal reflection fluorescence microscopy via annular fiber bundle.

We demonstrate a method of generating instantaneous and uniform total internal reflection fluorescence (TIRF) excitation by using an annular fiber bundle and spatially incoherent light sources. We show the flexibility of our method in that it can generate TIRF excitation with either a laser light source or an LED of different wavelengths, and facilitate switching between TIRF and epi illumination. In this report we detail the design of the fiber bundle, then demonstrate the performance via single-molecule imaging in the presence of high background and high throughput, and uniform TIRF imaging of cells over a large field of view. Our versatile method will enable quantitative shadowless TIRF imaging.

Optimizing the performance of multiline-scanning confocal microscopy.

Line-scanning confocal microscopy provides high imaging speed and moderate optical sectioning strength, which makes it a useful tool for imaging various biospecimens ranging from living cells to fixed tissues. Conventional line-scanning systems have only used a single excitation line and slit, and thus have not fully exploited benefits of parallelization. Here we investigate the optical performance of multi-line scanning confocal microscopy (mLS) by employing a digital micro-mirror that provides programmable patterns of the illumination beam and the detection slit. Through experimental results and optical simulations, we assess the depth discrimination of mLS under different optical parameters and compare it with multi-point systems such as scanning disk confocal microscopy (SDCM). Under the same illumination duty cycle, we find that mLS has better optical sectioning than SDCM at a high degree of parallelization. The optimized mLS provides a low photobleaching rate and video-rate imaging while its optical sectioning is similar to single line-scanning confocal microscopy.

Instantaneous non-diffracting light-sheet generation by controlling spatial coherence.

We demonstrate single-shot non-diffracting light-sheet generation by controlling the spatial coherence of light. A one-dimensional coherent beam, created by either increasing the spatial coherence of an LED or decreasing the spatial coherence of a laser, makes it unnecessary to scan non-diffracting beams to generate light-sheets. We theoretically and experimentally demonstrate the equivalence between our method and a scanned light-sheet, and investigate the characteristics of the light-sheet in detail. Our method is easily implementable and universally applicable for high-resolution multicolor light-sheet fluorescence imaging.

Flat-field illumination for quantitative fluorescence imaging.

The uneven illumination of a Gaussian profile makes quantitative analysis highly challenging in laser-based wide-field fluorescence microscopy. Here we present flat-field illumination (FFI) where the Gaussian beam is reshaped into a uniform flat-top profile using a high-precision refractive optical component. The long working distance and high spatial coherence of FFI allows us to accomplish uniform epi and TIRF illumination for multi-color single-molecule imaging. In addition, high-throughput borderless imaging is demonstrated with minimal image overlap.

Endogenous Alpha-Synuclein Protein Analysis from Human Brain Tissues Using Single-Molecule Pull-Down Assay.

Alpha-synuclein (α-SYN) is a central molecule in Parkinson's disease pathogenesis. Despite several studies, the molecular nature of endogenous α-SYN especially in human brain samples is still not well understood due to the lack of reliable methods and the limited amount of biospecimens. Here, we introduce α-SYN single-molecule pull-down (α-SYN SiMPull) assay combined with in vivo protein crosslinking to count individual α-SYN protein and assess its native oligomerization states from biological samples including human postmortem brains. This powerful single-molecule assay can be highly useful in diagnostic applications using various specimens for neurodegenerative diseases including Alzheimer's disease and Parkinson's disease.

Fabrication of an integrated high-quality-factor (high-Q) optofluidic sensor by femtosecond laser micromachining.

We report on fabrication of a microtoroid resonator of a high-quality factor (i.e., Q-factor of ~3.24 × 10(6) measured under the critical coupling condition) integrated in a microfluidic channel using femtosecond laser three-dimensional (3D) micromachining. Coupling of light into and out of the microresonator has been realized with a fiber taper that is reliably assembled with the microtoroid. The assembly of the fiber to the microtoroid is achieved by welding the fiber taper onto the sidewall of the microtoroid using CO2 laser irradiation. The integrated microresonator maintains a high Q-factor of 3.21 × 10(5) as measured in air, which should still be sufficient for many sensing applications. We test the functionality of the integrated optofluidic sensor by performing bulk refractive index sensing of purified water doped with tiny amount of salt. It is shown that a detection limit of ~1.2 × 10(-4) refractive index unit can be achieved. Our result showcases the capability of integration of high-Q microresonators with complex microfluidic systems using femtosecond laser 3D micromachining.

Low-photobleaching line-scanning confocal microscopy using dual inclined beams.

Confocal microscopy is an indispensable tool for biological imaging due to its high resolution and optical sectioning capability. However, its slow imaging speed and severe photobleaching have largely prevented further applications. Here, we present dual inclined beam line-scanning (LS) confocal microscopy. The reduced excitation intensity of our imaging method enabled a 2-fold longer observation time of fluorescence compared to traditional LS microscopy while maintaining a good sectioning capability and single-molecule sensitivity. We characterized the performance of our method and applied it to subcellular imaging and three-dimensional single-molecule RNA imaging in mammalian cells.

Fluorescence imaging with tailored light.

Fluorescence microscopy has long been a valuable tool for biological and medical imaging. Control of optical parameters such as the amplitude, phase, polarization and propagation angle of light gives fluorescence imaging great capabilities ranging from super-resolution imaging to long-term real-time observation of living organisms. In this review, we discuss current fluorescence imaging techniques in terms of the use of tailored or structured light for the sample illumination and fluorescence detection, providing a clear overview of their working principles and capabilities.

A Guide to Build a Highly Inclined Swept Tile Microscope for Extended Field-of-view Single-molecule Imaging.

Single-molecule imaging has greatly advanced our understanding of molecular mechanisms in biological studies. However, it has been challenging to obtain large field-of-view, high-contrast images in thick cells and tissues. Here, we introduce highly inclined swept tile (HIST) microscopy that overcomes this problem. A pair of cylindrical lenses was implemented to generate an elongated excitation beam that was scanned over a large imaging area via a fast galvo mirror. A 4f configuration was used to position optical components. A scientific complementary metal-oxide semiconductor camera detected the fluorescence signal and blocked the out-of-focus background with a dynamic confocal slit synchronized with the beam sweeping. We present a step-by-step instruction on building the HIST microscope with all basic components.

Spatially encoded fast single-molecule fluorescence spectroscopy with full field-of-view.

We report a simple single-molecule fluorescence imaging method that increases the temporal resolution of any type of array detector by >5-fold with full field-of-view. We spread single-molecule spots to adjacent pixels by rotating a mirror in the detection path during the exposure time of a single frame, which encodes temporal information into the spatial domain. Our approach allowed us to monitor fast blinking of an organic dye, the dissociation kinetics of very short DNA and conformational changes of biomolecules with much improved temporal resolution than the conventional method. Our technique is useful when a large field-of-view is required, for example, in the case of weakly interacting biomolecules or cellular imaging.