6 research outputs found
Surface-enhanced coherent anti-Stokes Raman imaging of lipids
This work describes in detail a wide-field surface-enhanced coherent anti-Stokes Raman scattering (CARS) microscope, which enables enhanced detection of sample structures in close proximity (∼100  nm) of the substrate interface. Unlike conventional CARS microscopy, where the sample is illuminated with freely propagating light, the current implementation uses evanescent fields to drive Raman coherences across the entire object plane. By coupling the pump and Stokes excitation beams to the surface plasmon-polariton mode at the interface of a 30 nm thick gold film, we obtained strong CARS signals from cholesteryl oleate droplets adhered to the surface. The surface-enhanced CARS imaging system visualizes lipid structures with vibrational selectivity using illumination doses per unit area that are more than four orders of magnitude lower than in point-scanning CARS microscopy
Particle sensing with confined optical field enhanced fluorescence emission (Cofefe).
We describe the development and performance of a new type of optical sensor suitable for registering the binding/dissociation of nanoscopic particles near a gold sensing surface. The method shares similarities with surface plasmon resonance microscopy but uses a completely different optical signature for reading out binding events. This new optical read-out mechanism, which we call confined optical field enhanced fluorescence emission (Cofefe), uses pulsed surface plasmon polariton fields at the gold/liquid interface that give rise to confined optical fields upon binding of the target particle to the gold surface. The confined near-fields are sufficient to induce two-photon absorption in the gold sensor surface near the binding site. Subsequent radiative recombination of the electron-hole pairs in the gold produces fluorescence emission, which can be captured by a camera in the far-field. Bound nanoparticles show up as bright confined spots against a dark background on the camera. We show that the Cofefe sensor is capable of detecting gold and silicon nanoparticles, as well as polymer nanospheres and sub-μm lipid droplets in a label-free manner with average illumination powers of less than 10 μW/μm2
Multiplexed and Millimeter-Scale Fluorescence Nanoscopy of Cells and Tissue Sections via Prism-Illumination and Microfluidics-Enhanced DNA-PAINT
Fluorescence nanoscopy has become increasingly powerful for biomedical research, but it has
historically afforded a small field-of-view (FOV) of around 50 μm
× 50 μm at once and more recently up to ∼200 μm
× 200 μm. Efforts to further increase the FOV in fluorescence
nanoscopy have thus far relied on the use of fabricated waveguide
substrates, adding cost and sample constraints to the applications.
Here we report PRism-Illumination and Microfluidics-Enhanced DNA-PAINT
(PRIME-PAINT) for multiplexed fluorescence nanoscopy across millimeter-scale
FOVs. Built upon the well-established prism-type total internal reflection
microscopy, PRIME-PAINT achieves robust single-molecule localization
with up to ∼520 μm × 520 μm single FOVs and
25–40 nm lateral resolutions. Through stitching, nanoscopic
imaging over mm2 sample areas can be completed in as little
as 40 min per target. An on-stage microfluidics chamber facilitates
probe exchange for multiplexing and enhances image quality, particularly
for formalin-fixed paraffin-embedded (FFPE) tissue sections. We demonstrate
the utility of PRIME-PAINT by analyzing ∼106 caveolae
structures in ∼1,000 cells and imaging entire pancreatic cancer
lesions from patient tissue biopsies. By imaging from nanometers to
millimeters with multiplexity and broad sample compatibility, PRIME-PAINT
will be useful for building multiscale, Google-Earth-like views of
biological systems
Multiplexed and Millimeter-Scale Fluorescence Nanoscopy of Cells and Tissue Sections via Prism-Illumination and Microfluidics-Enhanced DNA-PAINT
Fluorescence nanoscopy has become increasingly powerful for biomedical research, but it has
historically afforded a small field-of-view (FOV) of around 50 μm
× 50 μm at once and more recently up to ∼200 μm
× 200 μm. Efforts to further increase the FOV in fluorescence
nanoscopy have thus far relied on the use of fabricated waveguide
substrates, adding cost and sample constraints to the applications.
Here we report PRism-Illumination and Microfluidics-Enhanced DNA-PAINT
(PRIME-PAINT) for multiplexed fluorescence nanoscopy across millimeter-scale
FOVs. Built upon the well-established prism-type total internal reflection
microscopy, PRIME-PAINT achieves robust single-molecule localization
with up to ∼520 μm × 520 μm single FOVs and
25–40 nm lateral resolutions. Through stitching, nanoscopic
imaging over mm2 sample areas can be completed in as little
as 40 min per target. An on-stage microfluidics chamber facilitates
probe exchange for multiplexing and enhances image quality, particularly
for formalin-fixed paraffin-embedded (FFPE) tissue sections. We demonstrate
the utility of PRIME-PAINT by analyzing ∼106 caveolae
structures in ∼1,000 cells and imaging entire pancreatic cancer
lesions from patient tissue biopsies. By imaging from nanometers to
millimeters with multiplexity and broad sample compatibility, PRIME-PAINT
will be useful for building multiscale, Google-Earth-like views of
biological systems