Near-Membrane Refractometry Using Supercritical Angle Fluorescence

Abstract

Total internal reflection fluorescence (TIRF) microscopy and its variants are key technologies for visualizing the dynamics of single molecules or organelles in live cells. Yet, truely quantitative TIRF remains problematic. One unknown hampering the interpretation of evanescent-wave excited fluorescence intensities is the undetermined cell refractive index (RI). Here, we use a combination of TIRF excitation and supercritical angle fluorescence emission detection to directly measure the average RI in the 'footprint' region of the cell, during imaging. Our RI measurement is based on the determination on a back-focal plane image of the critical angle separating supercritical and undercritial fluorescence emission components. We validate our method by imaging mouse embryonic fibroblasts. By targeting various dyes and fluorescent-protein chimerae to vesicles, the plasma membrane as well as mitochondria and the ER, we demonstrate local RI measurements with subcellular resolution on a standard TIRF microscope with a removable Bertrand lens as the only modification. Our technique has important applications for imaging axial vesicle dynamics, mitochondrial energy state or detecting cancer cells.Total internal fluorescence (TIRF) has evolved from an ex-pert technique to a routine contrast mode used for singlemolecule and single-organelle tracking at or near the basal plasma membrane of cells adherent to a glass substrate [1]. PALM/STORM localization-based super-resolution micro-scopies have further broadened the range of TIRF applica-tions. The emission counterpart of TIRF, supercritical angle fluorescence (SAF) [2] is increasingly being used for sur-face microscopies [3, 4 , 5]. For TIRF, the presence of a re-fractive-index (RI) boundary between the glass substrate (of index n 2 ) and the aqueous sample (n 1 ) results in the emergence of an evanescent wave that provides excitation-light confinement. In SAF microscopies, the otherwise un-detected evanescent emission component of surface-proxi-mal fluorophores couples to the interface where it becomes propagative and detectable in the far field, provided the ob-jective has a sufficiently large numerical aperture (NA= n 2 sin NA , with  NA > c ). The critical angles at the excitation or emission wavelength ,  c =asin[n 2 ()/n 1 ()], the axial decay length =/[4(n 2 2 sin-n 1 2 ) 1/2 ] of the evanescent-wave intensity and the dipole radiation pattern are all modi-fied by the local sample RI n 1 (x,y), which is generally un-determined. Knowledge, even of the average near-mem-brane RI, n 1 , for the very cell under study, would greatly enhance our capacity to chose appropriate incidence and detection angles, to better understand and eliminate image imperfections and to interpret TIRF and SAF in quantita-tive terms, e.g., for axial profilometry, size-or concentra-tion measurements, or for axial single-vesicle tracking. Hilbert-phase microscopy [6], digital confocal microscopy (DCM) [7 , 8] and full-field optical coherence tomography (OCT) [9] all allow RI m...

Label-free classification of neurons and glia in neural stem cell cultures using a hyperspectral imaging microscopy combined with machine learning

“…Recent studies have reported differences in the refractive index (RI) among subcellular components, i.e., nuclei have a lower refractive index than cytoplasm 29 , and the mitochondria, lysosomes and endoplasmic reticulum have their own unique RIs 30–32 , respectively. In addition to intracellular RI differences, cell type dependent differences 33,34 and cell state dependent differences 34,35 have also been demonstrated.…”

Breaking the Axial Diffraction Limit: A Guide to Axial Super‐Resolution Fluorescence Microscopy

“…Similar to TIRFM, SAFM can also provide good optical sectioning capacity and high SBR, which has been applied to highly sensitive biosensing, cell adhesion and motility quantification, plasma membrane association and protein diffusion measurement, and near‐membrane refractometry implementation . In addition, SAFM can be combined with existing SMLM methods to perform 3D super‐resolution imaging .…”

“…In summary, SAFM has superior performance, but greater system complexity than TIRFM. Combining these two methods, i.e., using TIRFM for excitation and SAFM for detection, allows further improvement of the optical sectioning capacity …”

Supercritical Angle Fluorescence Microscopy and Spectroscopy

“…However, although the lateral resolution and axial optical sectioning can be of the order of, respectively, 220 and 100 nm (18), the stage-scanning requirement makes image acquisition too slow for imaging near-membrane dynamics in live cells. 2) as a full-field technique, either with a resolutioncompromising aperture disk (30) or else in the resolution-preserving vSAF variant (13,31,40), SAF microscopy is a simple, cheap, and less alignmentdemanding whole-field alternative to TIRF. Additionally, no laser source is needed.…”

“…We advocated the use of SAF in conjunction with TIRF (13), particularly when using TIRF in the prismless, objective-type configuration. Here, SAF can help to remove impurities, imperfections, and unknowns associated with the passage of the laser beam close to the limiting NA of the objective and those resulting from conjugate image planes inside the microscope body (4,40). Also, contrary to the earlier discussed case of SAF with EPI illumination, the combined excitation and emission optical sectioning result in a better surface selectivity of fluorescence detection.…”