Microscopic Imaging and Spectroscopy with Scattered Light

Boustany, Nada N.; Boppart, Stephen A.; Backman, Vadim

doi:10.1146/annurev-bioeng-061008-124811

Cited by 120 publications

(111 citation statements)

References 143 publications

(165 reference statements)

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“…4 In the context of transmission microscopy, most of the developments aim at improving the sensitivity and speci¯city of the information retrieved from weakly absorbing specimens, by for instance retrieving quantitative values, or improve the image contrast. One approach in this direction consists in quantitative phase microscopy (QPM), for which contrast relies on refractive index values.…”

Section: Modern Linear Methodsmentioning

confidence: 99%

“…117 As OCT employs wide-band light sources to generate optical sectioning, an extension denoted as spectral OCT (S-OCT) consists in resolving spectrally the measurement to obtain both the 3D structure through OCT, as well as the absorption spectra. 4,118 This approach has been reported for both temporal 119 and Fourier domain 120,121 S-OCT, leading to di®erent performances, 122 which are also dependent on the employed compensation procedures. 123 The spectroscopic measurement can typically lead to various invaluable information such as blood oxygenation.…”

Section: Spectroscopic Multimodal Implementationsmentioning

confidence: 99%

“…The combination of label-free techniques with label-based ones are therefore brie°y explored in Sec. 4, with an emphasis on recent developments in this area.…”

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

Multimodal label-free microscopy

Pavillon

Fujita

Smith

2014

J. Innov. Opt. Health Sci.

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This paper reviews the di®erent multimodal applications based on a large extent of label-free imaging modalities, ranging from linear to nonlinear optics, while also including spectroscopic measurements. We put speci¯c emphasis on multimodal measurements going across the usual boundaries between imaging modalities, whereas most multimodal platforms combine techniques based on similar light interactions or similar hardware implementations. In this review, we limit the scope to focus on applications for biology such as live cells or tissues, since by their nature of being alive or fragile, we are often not free to take liberties with the image acquisition times and are forced to gather the maximum amount of information possible at one time. For such samples, imaging by a given label-free method usually presents a challenge in obtaining su±cient optical signal or is limited in terms of the types of observable targets. Multimodal imaging is then particularly attractive for these samples in order to maximize the amount of measured information. While multimodal imaging is always useful in the sense of acquiring additional information from additional modes, at times it is possible to attain information that could not be discovered using any single mode alone, which is the essence of the progress that is possible using a multimodal approach.

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Section: Modern Linear Methodsmentioning

confidence: 99%

Section: Spectroscopic Multimodal Implementationsmentioning

confidence: 99%

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Multimodal label-free microscopy

Pavillon

Fujita

Smith

2014

J. Innov. Opt. Health Sci.

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“…Biomedical applications of light scattering phenomena have been actively investigated, yielding useful insights for tissue diagnosis, imaging and characterization [1]. Coherent optical techniques, such as optical coherence tomography (OCT), were developed to acquire high-resolution, depth-resolved images even in the presence of severe scattering / turbidity, enabling micron-scale 3D visualization of biological tissue microstructure [2,3].…”

Section: Introductionmentioning

confidence: 99%

Analysis of scattering statistics and governing distribution functions in optical coherence tomography

Sugita

Weatherbee

Bizheva

et al. 2016

Biomed. Opt. Express

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Abstract:The probability density function (PDF) of light scattering intensity can be used to characterize the scattering medium. We have recently shown that in optical coherence tomography (OCT), a PDF formalism can be sensitive to the number of scatterers in the probed scattering volume and can be represented by the K-distribution, a functional descriptor for non-Gaussian scattering statistics. Expanding on this initial finding, here we examine polystyrene microsphere phantoms with different sphere sizes and concentrations, and also human skin and fingernail in vivo. It is demonstrated that the K-distribution offers an accurate representation for the measured OCT PDFs. The behavior of the shape parameter of Kdistribution that best fits the OCT scattering results is investigated in detail, and the applicability of this methodology for biological tissue characterization is demonstrated and discussed.

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“…Meanwhile, the refractive index change demonstrated here suggests an alternative use of dyes as contrast agents in the field of light scattering to investigate subdiffractional structures such as specific organelles, chromatin fibers, proteins, etc. In clinic, this effect can advance cancer diagnosis when utilized by scattering-based techniques such as optical coherence microscopy (OCM), light scattering spectroscopy (LSS), confocal light absorption and scattering spectroscopy (CLASS), and partial wave spectroscopic (PWS) microscopy [18,19]. [a] shows the measured cell height and absorptance at 600nm; [b] illustrates the n' and n" for 3 areas (black corresponds to unstained), letters H and E point to the absorption peaks of hematoxylin and eosin correspondingly; [c] illustrates the spatial dis tribution of n' and n" at 600nm within the cell; [d] size-dependent absorption efficiency (Q abs ) and amplification of scattering efficiency (Q sca ) (refractive index of surrounding medium assumed to be 1.52).…”

mentioning

confidence: 99%

Targeted Alteration of Real and Imaginary Refractive Index of Biological Cells by Histological Staining

Cherkezyan

Subramanian

Yang

et al. 2012

Biomedical Optics and 3-D Imaging

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Various staining techniques are commonly used in biomedical research to investigate cellular morphology. By inducing absorption of light, staining dyes change the intracellular refractive index due to the Kramers-Kronig relationship. We present a method for creating 2-D maps of real and imaginary refractive indices of stained biological cells using their thickness and absorptance. We validate our technique on dyed polystyrene microspheres and quantify the alteration in refractive index of stained biological cells. We reveal that specific staining of individual organelles can increase their scattering cross-section by orders of magnitudes implying a major impact in the field of biophotonics.Staining provides a tool for visualization of macromolecules, biological cells, and tissues in applications that extend from basic biology to clinical diagnostics [1,2]. Within the field of optics, the change in light scattering properties of stained organelles caused by the absorption of specific dyes is used to determine their refractive indices [3]. However, while most of the attention has been focused on the absorption (i.e. the imaginary part of refractive index) introduced by staining dyes, the subsequent alteration in the real part of refractive index according to the Kramers-Kronig relation has often been overlooked [4,5]. A recently proposed linear relationship between the real and imaginary parts of refractive index does not treat the effects of spectral dispersion in either quantity [6]. In this Letter, we demonstrate a method to quantify the wavelength dependent change in both real and imaginary parts of the refractive index of epithelial cells caused by histological stains such as hematoxylin and eosin-containing cytostain.We construct 2-D maps for real and imaginary refractive indices (n' and n") of stained cells as a function of wavelength λ as follows: first, we calculate the n" after measuring the transmission intensity I and the sample thickness L using: (1) where I 0 (x,y,λ 0 ) is the transmission intensity at a wavelength λ 0 where absorption is absent (to isolate n" from other causes of attenuation). For dyes used in this study λ 0 = 700nm. Then, we extract the n' (λ):* Corresponding author: v-backman@northwestern.edu. where χ' dye +jχ" dye is the electric susceptibility of dye molecules embedded in a nonabsorbing material with a refractive index n 0 ; χ' dye is determined from χ" dye using Kramers-Kronig relations [4,5,7]: (3) where P denotes Cauchy principal value and k is the wavenumber k=2π/λ. Our instrumentation includes an atomic force microscope (AFM, Bruker Bioscope II) with 39 nm resolution to measure the thickness L(x,y) and a custom built spectroscopic microscope (SM) to image the sample, as well as measure the wavelength dependent transmission through it I(x,y,λ). The sample of interest is mounted between 2 glass slides using Permaslip (Alban Scientific) to remove specular reflections at the specimen-air interface. The bottom glass slide is coated with a 100nm layer of Aluminum (Deposit...

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Microscopic Imaging and Spectroscopy with Scattered Light

Cited by 120 publications

References 143 publications

Multimodal label-free microscopy

Multimodal label-free microscopy

Analysis of scattering statistics and governing distribution functions in optical coherence tomography

Targeted Alteration of Real and Imaginary Refractive Index of Biological Cells by Histological Staining

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