Chemical imaging offers critical information to understand the fundamentals in biology and to assist clinical diagnostics. Label-free chemical imaging piques a general interest since it avoids the use of bio-perturbing molecular labels and holds promises to characterize human tissue in vivo. Coherent Raman scattering (CRS), which utilizes lasers to excite the vibrations of molecules, renders new modalities to map chemicals in living samples without the need of labeling and provides significantly improved speed, resolution, and sensitivity compared to spontaneous Raman scattering. Although microscopy systems based on CRS have seen rapid development in the past two decades, remaining challenges, which emerge in diverse aspects, start to impede the continuous advancement of the field. In this perspective, we review the history of CRS microscopy, scrutinize the pros and cons of different modalities, and discuss the current challenges and possible future directions of the field. Infiltration of conceptual and technological ideals from other fields will promote CRS microscopy towards a versatile tool for basic science and medical research.

Perspective: Coherent Raman scattering microscopy, the future is bright

• Over 70 new Data Analysis Problems, with answers and explanations FIND OUT • New online videos, referenced throughout the book MORE INSIDE... , “I have enjoyed using this book because it cuts through the mountain of details flooding into this topic and presents in a simple way the techniques currently in use and some cutting-edge research. When I chose this book, it was after looking at several other books on the market —this one thoroughly covered the topics, but in a simple, easy-to-understand way for the student.”

The Cell: A Molecular Approach, Seventh Edition.

We experimentally demonstrated Bessel-like beams utilizing digital micromirror device (DMD). DMD with images imitating the equivalent axicon can shape the collimated Gaussian beam into Bessel beam. We reconstructed the 3D spatial field of the generated beam through a stack of measured cross-sectional images. The output beams have the profile of Bessel function after intensity modulation, and the beams extend at least 50 mm while the lateral dimension of the spot remains nearly invariant. Furthermore, the self-healing property has also been investigated, and all the experimental results agree well with simulated results numerically calculated through beam propagation method. Our observations demonstrate that the DMD offers a simple and efficient method to generate Bessel beams with distinct nondiffracting and self-reconstruction behaviors. The generated Bessel beams will potentially expand the applications to the optical manipulation and high-resolution fluorescence imaging owing to the unique nondiffracting property.

Generation of nondiffracting Bessel beam using digital micromirror device

An ideal direct imaging system entails a method to illuminate on command a single diffraction-limited region in a generally thick and turbid volume. The best approximation to this is the use of large-aperture lenses that focus light into a spot. This strategy fails for regions that are embedded deep into the sample, where diffraction and scattering prevail. Airy beams and Bessel beams are solutions of the Helmholtz Equation that are both non-diffracting and self-healing, features that make them naturally able to outdo the effects of distance into the volume but intrinsically do not allow resolution along the propagation axis. Here, we demonstrate diffraction-free self-healing three-dimensional monochromatic light spots able to penetrate deep into the volume of a sample, resist against deflection in turbid environments, and offer axial resolution comparable to that of Gaussian beams. The fields, formed from coherent mixtures of Bessel beams, manifest a more than ten-fold increase in their undistorted penetration, even in turbid milk solutions, compared to diffraction-limited beams. In a fluorescence imaging scheme, we find a ten-fold increase in image contrast compared to diffraction-limited illuminations, and a constant axial resolution even after four Rayleigh lengths. Results pave the way to new opportunities in three-dimensional microscopy.

Diffraction-free light droplets for axially-resolved volume imaging.

Light scattered by turbid tissue is known to degrade optical coherence tomography (OCT) image contrast progressively with depth. Bessel beams have been proposed as an alternative to Gaussian beams to image deeper into turbid tissue. However, studies of turbid tissue comparing the image quality for different beam types are lacking. We present such a study, using numerically simulated beams and experimental OCT images formed by Bessel or Gaussian beams illuminating phantoms with optical properties spanning a range typical of soft tissue. We demonstrate that, for a given scattering parameter, the higher the scattering anisotropy the lower the OCT contrast, regardless of the beam type. When focusing both beams at the same depth in the sample, we show that, at focus and for equal input power and resolution, imaging with the Gaussian beam suffers less reduction of contrast. This suggests that, whilst Bessel beams offer extended depth of field in a single depth scan, for low numerical aperture (NA   0.95), superior contrast (by up to ~40%) may be obtained over an extended depth range by a Gaussian beam combined with dynamic focusing.

/pdf/quantifying-the-influence-of-bessel-beams-on-image-quality-4jeetjszff.pdf

Quantifying the influence of Bessel beams on image quality in optical coherence tomography.

Monitoring and visualizing specimens at a large penetration depth is a challenge. At depths of hundreds of microns, several physical effects (such as, scattering, PSF distortion and noise) deteriorate the image quality and prohibit a detailed study of key biological phenomena. In this study, we use a Bessel-like beam in-conjugation with an orthogonal detection system to achieve depth imaging. A Bessel-like penetrating diffractionless beam is generated by engineering the back-aperture of the excitation objective. The proposed excitation scheme allows continuous scanning by simply translating the detection PSF. This type of imaging system is beneficial for obtaining depth information from any desired specimen layer, including nano-particle tracking in thick tissue. As demonstrated by imaging the fluorescent polymer-tagged-CaCO3 particles and yeast cells in a tissue-like gel-matrix, the system offers a penetration depth that extends up to 650 mu m. This achievement will advance the field of fluorescence imaging and deep nano-particle tracking.

/pdf/spatial-filter-based-bessel-like-beam-for-improved-1jpblpsyyp.pdf

Sampa Bera

Papers

Spatial Filter Based Bessel-Like Beam for Improved Penetration Depth Imaging in Fluorescence Microscopy

Studying the Variations of Complex Electrical Bio-Impedance of Plant Tissues During Boiling

Electrical Impedance Spectroscopy (EIS) based fruit characterization : A technical review

Multiple inhomogeneity phantom imaging with a LabVIEW-based Electrical Impedance Tomography (LV-EIT) System