Confocal supercritical angle microscopy for cell membrane imaging

Sivankutty, Siddharth; Barroca, Thomas; Mayet, Céline; Dupuis, Guillaume; Fort, Emmanuel; Lévêque‐Fort, Sandrine

doi:10.1364/ol.39.000555

Cited by 8 publications

(6 citation statements)

References 15 publications

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“…The difference image, vSAF = (UAF + SAF) − UAF, maintains lateral spatial resolution, but it shows only those fluorophores close to the interface, producing an optical sectioning that is similar to, if not better than, 15 an image of the same sample acquired with azimuthal beam-spinning TIRF (Figure 1b). 21 Due to its axial sectioning and background-suppression properties, SAF has been used for ultrasensitive detection 16 and fluorescence correlation spectroscopy (FCS) in tiny volumes 22,23 as well as for improving the axial optical sectioning of confocal, 24,25 stimulated emission depletion (STED), 26,27 and even TIRF microscopy. 15…”

Section: Supercritical-angle Fluorescencementioning

confidence: 99%

Decoding the Information Contained in Fluorophore Radiation Patterns

2018

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Dipole radiation patterns change when a fluorescent molecule comes close to the boundary between media of different refractive indices. Near-interface molecules emit mostly into the higher-index medium, predominantly around the critical angle. The radiation pattern encodes information about the emitter distance, orientation, and the refractive index of the embedding medium. Analyses of the supercritical angle fluorescence on pupil plane images can retrieve this information and have been applied both for refractometry with subcellular resolution and for the detection of metabolically active cancerous cells. In this issue of ACS Nano, Ferdman et al. employ this strategy in a label-free assay for detecting single bacteria, based on measuring the refractive-index change produced by bacterial growth in a fluorophorecoated microfluidic channel.

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Section: Supercritical-angle Fluorescencementioning

confidence: 99%

Decoding the Information Contained in Fluorophore Radiation Patterns

2018

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“…The SAF effect has been exploited to obtain TIRF-like optical sectioning in confocal scanning [18,19], fluorescence widefield [20][21][22] and lately also STED imaging [23]. The first use of SAF in the context of SMLM was reported about five years ago [24,25].…”

Section: Introductionmentioning

confidence: 99%

Defocused imaging exploits supercritical-angle fluorescence emission for precise axial single molecule localization microscopy

Zelger¹,

Bodner²,

Veľas³

et al. 2020

Biomed. Opt. Express

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Single molecule localization microscopy (SMLM) is one of the key techniques that break the classical resolution limit in optical imaging. It is based on taking multiple recordings of a sample, each showing only a sparse arrangement of spatially well separated fluorescent molecules which can be localized at nanometer precision. While localizing along the lateral directions is usually straightforward, estimating axial positions at a comparable precision is known to be much harder, which is due to the relatively large depth of focus provided by the microscope optics. Whenever a molecule is sufficiently close to the coverslip, it becomes feasible to draw additional information from near field coupling effects: super-critical angle fluorescence (SAF) appears and can be exploited to boost the axial localization precision. Here we propose defocused imaging as a SMLM strategy that is capable of leveraging the information contained in SAF. We show that, regarding axial localization precision, our approach is superior to established SAF-based approaches. At the same time it is simple and can be conducted on any research-grade microscope where controlled defocusing on the order of a few hundred nanometers is possible.

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“…Of course, SAF does not require TIRF excitation, and SAF detection has been combined with many other excitation geometries, including EPI, confocal-spot excitation without (1,18,19) and with stimulated emission depletion (20). A growing body of spot excitation and confocal SAF detection is devoted to supercritical fluorescence correlation spectroscopy (FCS) (21)(22)(23)(24).…”

Section: Selective Saf Detection Permits the Acquisition Of Tirf-like Imagesmentioning

confidence: 99%

“…In objective-type TIRF (15), several studies have documented an unwanted 5-10% of non-evanescent (long-range) excitation components that adds to the localized EW excitation (4,12,16,17). Advantageously, TIRF excitation and SAF Of course, SAF does not require TIRF excitation and SAF detection has been combined with many other excitation geometries, including epi-fluorescence, confocal-spot excitation without (1,18,19) and with stimulated-emission depletion (STED) (20). A growing body of spot-excitation and confocal SAF detection is devoted to supercritical fluorescence correlation spectroscopy (FCS) (21)(22)(23)(24).…”

Section: Selective Saf Detection Permits the Acquisition Of Tirf-like...mentioning

confidence: 99%

Supercritical Angle Fluorescence Microscopy and Spectroscopy

Oheim

Salomon

Brunstein

2020

Biophysical Journal

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Fluorescence detection, either involving propagating or near-field emission, is widely being used in spectroscopy, sensing, and microscopy. Total internal reflection fluorescence (TIRF) confines fluorescence excitation by an evanescent (near) field, and it is a popular contrast generator for surface-selective fluorescence assays. Its emission equivalent, supercritical angle fluorescence (SAF), is comparably less established, although it achieves a similar optical sectioning as TIRF does. SAF emerges when a fluorescing molecule is located very close to an interface and its near-field emission couples to the higher refractive index medium (n 2 > n 1 ) and becomes propagative. Then, most fluorescence is detectable on the side of the higher-index substrate, and a large fraction of this fluorescence is emitted into angles forbidden by Snell's law. SAF, as well as the undercritical angle fluorescence (UAF; far-field emission) components, can be collected with microscope objectives having a high-enough detection aperture (numerical aperture > n 2 ) and be separated in the back focal plane by Fourier filtering. The back focal plane image encodes information about the fluorophore radiation pattern, and it can be analyzed to yield precise information about the refractive index in which the emitters are embedded, their nanometric distance from the interface, and their orientation. A SAF microscope can retrieve this near-field information through wide-field optics in a spatially resolved manner, and this functionality can be added to an existing inverted microscope. Here, we describe the potential underpinning of SAF microscopy and spectroscopy, particularly in comparison with TIRF. We review the challenges and opportunities that SAF presents from a biophysical perspective, and we discuss areas in which we see potential.

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Confocal supercritical angle microscopy for cell membrane imaging

Cited by 8 publications

References 15 publications

Decoding the Information Contained in Fluorophore Radiation Patterns

Decoding the Information Contained in Fluorophore Radiation Patterns

Defocused imaging exploits supercritical-angle fluorescence emission for precise axial single molecule localization microscopy

Supercritical Angle Fluorescence Microscopy and Spectroscopy

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