skip to main content


Title: Scattered‐light‐sheet microscopy with sub‐cellular resolving power
Abstract

Since its first demonstration over 100 years ago, scattering‐based light‐sheet microscopy has recently re‐emerged as a key modality in label‐free tissue imaging and cellular morphometry; however, scattering‐based light‐sheet imaging with subcellular resolution remains an unmet target. This is because related approaches inevitably superimpose speckle or granular intensity modulation on to the native subcellular features. Here, we addressed this challenge by deploying a time‐averaged pseudo‐thermalized light‐sheet illumination. While this approach increased the lateral dimensions of the illumination sheet, we achieved subcellular resolving power after image deconvolution. We validated this approach by imaging cytosolic carbon depots in yeast and bacteria with increased specificity, no staining, and ultralow irradiance levels. Overall, we expect this scattering‐based light‐sheet microscopy approach will advance single, live cell imaging by conferring low‐irradiance and label‐free operation towards eradicating phototoxicity.

 
more » « less
Award ID(s):
2041523
PAR ID:
10423811
Author(s) / Creator(s):
 ;  ;  ;  ;  
Publisher / Repository:
Wiley Blackwell (John Wiley & Sons)
Date Published:
Journal Name:
Journal of Biophotonics
Volume:
16
Issue:
9
ISSN:
1864-063X
Format(s):
Medium: X
Sponsoring Org:
National Science Foundation
More Like this
  1. Reflection phase imaging provides label-free, high-resolution characterization of biological samples, typically using interferometric-based techniques. Here, we investigate reflection phase microscopy fromintensity-only measurements under diverse illumination. We evaluate the forward and inverse scattering model based on the first Born approximation for imaging scattering objects above a glass slide. Under this design, the measured field combineslinearforward-scattering and height-dependentnonlinearback-scattering from the object that complicates object phase recovery. Using only the forward-scattering, we derive a linear inverse scattering model and evaluate this model’s validity range in simulation and experiment using a standard reflection microscope modified with a programmable light source. Our method provides enhanced contrast of thin, weakly scattering samples that complement transmission techniques. This model provides a promising development for creating simplified intensity-based reflection quantitative phase imaging systems easily adoptable for biological research.

     
    more » « less
  2. Abstract

    Fluorescence super-resolution microscopy has, over the last two decades, been extensively developed to access deep-subwavelength nanoscales optically. Label-free super-resolution technologies however have only achieved a slight improvement compared to the diffraction limit. In this context, we demonstrate a label-free imaging method, i.e., hyperbolic material enhanced scattering (HMES) nanoscopy, which breaks the diffraction limit by tailoring the light-matter interaction between the specimens and a hyperbolic material substrate. By exciting the highly confined evanescent hyperbolic polariton modes with dark-field detection, HMES nanoscopy successfully shows a high-contrast scattering image with a spatial resolution around 80 nm. Considering the wavelength at 532 nm and detection optics with a 0.6 numerical aperture (NA) objective lens, this value represents a 5.5-fold resolution improvement beyond the diffraction limit. HMES provides capabilities for super-resolution imaging where fluorescence is not available or challenging to apply.

     
    more » « less
  3. Quantitative oblique back-illumination microscopy (qOBM) is an emerging label-free optical imaging technology that enables 3D, tomographic quantitative phase imaging (QPI) with epi-illumination in thick scattering samples. In this work, we present a robust optimization of a flexible, fiber-optic-based qOBM system. Our approach enables in silico optimization of the phase signal-to-noise-ratio over a wide parameter space and obviates the need for tedious experimental optimization which could easily miss optimal conditions. Experimental validations of the simulations are also presented and sensitivity limits for the probe are assessed. The optimized probe is light-weight (∼40g) and compact (8mm in diameter) and achieves a 2µm lateral resolution, 6µm axial resolution, and a 300µm field of view, with near video-rate operation (10Hz, limited by the camera). The phase sensitivity is <20nm for a single qOBM acquisition (at 10Hz) and a lower limit of ∼3 nm via multi-frame averaging. Finally, to demonstrate the utility of the optimized probe, we image (1) thick, fixed rat brain samples from a 9L gliosarcoma tumor model and (2) freshly excised human brain tissues from neurosurgery. Acquired qOBM images using the flexible fiber-optic probe are in excellent agreement with those from a free-space qOBM system (both in-situ), as well as with gold-standard histopathology slices (after tissue processing).

     
    more » « less
  4. Abstract

    Multiple scattering and absorption limit the depth at which biological tissues can be imaged with light. In thick unlabeled specimens, multiple scattering randomizes the phase of the field and absorption attenuates light that travels long optical paths. These obstacles limit the performance of transmission imaging. To mitigate these challenges, we developed an epi-illumination gradient light interference microscope (epi-GLIM) as a label-free phase imaging modality applicable to bulk or opaque samples. Epi-GLIM enables studying turbid structures that are hundreds of microns thick and otherwise opaque to transmitted light. We demonstrate this approach with a variety of man-made and biological samples that are incompatible with imaging in a transmission geometry: semiconductors wafers, specimens on opaque and birefringent substrates, cells in microplates, and bulk tissues. We demonstrate that the epi-GLIM data can be used to solve the inverse scattering problem and reconstruct the tomography of single cells and model organisms.

     
    more » « less
  5. Optogenetics has emerged as an exciting tool for manipulating neural activity, which in turn, can modulate behavior in live organisms. However, detecting the response to the optical stimulation requires electrophysiology with physical contact or fluorescent imaging at target locations, which is often limited by photobleaching and phototoxicity. In this paper, we show that phase imaging can report the intracellular transport induced by optogenetic stimulation. We developed a multimodal instrument that can both stimulate cells with subcellular spatial resolution and detect optical pathlength (OPL) changes with nanometer scale sensitivity. We found that OPL fluctuations following stimulation are consistent with active organelle transport. Furthermore, the results indicate a broadening in the transport velocity distribution, which is significantly higher in stimulated cells compared to optogenetically inactive cells. It is likely that this label‐free, contactless measurement of optogenetic response will provide an enabling approach to neuroscience.

     
    more » « less