Nanoscale refractive index fluctuations detected via sparse spectral microscopy

Abstract

Partial Wave Spectroscopic (PWS) Microscopy has proven effective at detecting nanoscale hallmarks of carcinogenesis in histologically normal-appearing cells. The current method of data analysis requires acquisition of a three-dimensional data cube, consisting of multiple images taken at different illumination wavelengths, limiting the technique to data acquisition on ~30 individual cells per slide. To enable high throughput data acquisition and whole-slide imaging, new analysis procedures were developed that require fewer wavelengths in the same 500-700nm range for spectral analysis. The nanoscale sensitivity of the new analysis techniques was validated (i) theoretically, using finite-difference time-domain solutions of Maxwell's equations, as well as (ii) experimentally, by measuring nanostructural alterations associated with carcinogenesis in biological cells.

Label-free imaging of the native, living cellular nanoarchitecture using partial-wave spectroscopic microscopy

“…Acquisition of the full cell reference interference spectrum (500-700 nm) for spectral analysis takes under 30 s, with each wavelength collection produced from <100-ms exposures. Using a reduced wavelength approach to subsample the interference spectrum, this can be further reduced to under 2 s per acquisition (27). Even with full spectral collection, as demonstrated in Indeed, one of the most critical features of this rapid acquisition is the capacity to directly study the underlying heterogeneity of both chromatin structure and its temporal evolution within the cell population over time.…”

Review of interferometric spectroscopy of scattered light for the quantification of subdiffractional structure of biomaterials

“…16 At the same time, an optical parameter equivalent toΣ can be calculated when the standard deviation of reflectance (at a single wavelength of light) f Σ p ðλÞ is taken across pixels of the registered bright-field microscope image, instead of evaluating it across different wavelengths as reviewed herein. 48 That is, the standard deviation of reflectance across different realizations of media with same statistical properties (i.e., standard deviation across pixels) is identical to standard deviation of reflectance from the same medium (registered in a single pixel) measured at different wavelengths of light. In general, measuring the spectral standard deviation Σðx; yÞ has the advantage of quantifying the sample subvolumes imaged by individual pixels independently and thus not making the assumption of different pixels imaging statistically equivalent samples with similar physical thicknesses.…”

“…In general, measuring the spectral standard deviation Σðx; yÞ has the advantage of quantifying the sample subvolumes imaged by individual pixels independently and thus not making the assumption of different pixels imaging statistically equivalent samples with similar physical thicknesses. Nevertheless, in cases when samples can be assumed to be statistically homogeneous, evaluation of a sample's internal properties using few (≈30) wavelengths of light instead of 200 has the advantage of system speed improvement by orders of magnitudes along with the possibility of obtaining measurements of whole microscopy slides, 48 both of which have great promise for the translational applications of the technique. The applicability of reduced-wavelengthΣ p ðλÞ to a given type of samples can be tested by performing an initial set of full-wavelength Σðx; yÞ measurements after which the spatial homogeneity of structural properties within those sample can be evaluated whenΣ is compared toΣ p .…”

Multimodal interferometric imaging of nanoscale structure and macromolecular motion uncovers UV induced cellular paroxysm

“…Structural measurements are collected by imaging the backscattered light across a range of wavelengths (500-700nm) to produce a spectral data cube, I(λ, x, y), where λ is the wavelength and (x, y) correspond to pixel positions. Each frame within a spectral data cube is acquired with a 35ms exposure, and the total cube can be obtained in under two seconds depending on the number of wavelengths collected 39 . For this study, temporal measurements consisted of at least 201 frames acquired with 1.2ms, 32ms, or 35ms exposures each for a total time of ranging between 240ms to 7 seconds.…”