Optical sectioning in multifoci Raman hyperspectral imaging

Abstract

In this study, we compared the depth discrimination and speed performance of multifoci Raman hyperspectral imaging with the reference standard of a single laser point confocal Raman mapping. A liquid crystal spatial light modulator was employed for the generation of multifoci laser beams, and a digital micromirror device was used as a software-configurable reflective pinhole array. The patterns of the laser foci and pinhole array can be rapidly changed without requiring any hardware alterations. Confocal patterns with different distance-to-size ratios were tested and compared. After optimization of the laser-foci pattern, we demonstrated the feasibility of multifoci Raman hyperspectral microscopy for recording depth-resolved molecular maps of biological cells (Acanthamoeba castellanii trophozoites). Micrometric depth discrimination and short acquisition times (20 min for single plane confocal image) were achieved. KEYWORDSconfocal Raman imaging, cross-talk, multi-beam, optical sectioning, single cell imaging | INTRODUCTIONRaman microspectroscopy (RMS) is a powerful technique for highly specific molecular imaging of samples in three dimensions (3D).[1] RMS instruments typically utilize a single laser beam to excite Raman scattering at a sample, which is analyzed by a spectrometer, with hyperspectral images formed by raster-scanning the laser spot and obtaining a Raman spectrum at each point. A pinhole is often placed at the spectrometer entrance to ensure confocal measurement configuration for improved depth discrimination. Although RMS allows sensitive and chemically specific hyperspectral imaging, acquisition times for measurements in 3D are often lengthy, due to the weak spontaneous Raman scattering cross section of many materials. The addition of a pinhole also typically comes at the cost of optical throughput for RMS detection, further increasing the acquisition times. Several techniques have been proposed and demonstrated for achieving fast Raman spectral mapping, such as line-or slit-scanning [2,3] and wide-field Raman imaging. [4,5] Compared with confocal single-beam scanning Raman imaging, these power-sharing Raman imaging techniques effectively shorten the mapping time by measuring multiple sample locations simultaneously. However, both line-scanning and wide-field techniques tend to decrease the depth discrimination because of decreased or lack of confocality. Multifoci excitation is a promising strategy to improve Raman imaging speed while maintaining good depth discrimination. [6][7][8][9][10][11][12] Raman spectra from these ------------------------------------------This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

Raman Spectroscopy Study of Curvature-Mediated Lipid Packing and Sorting in Single Lipid Vesicles

“…A diagram of the HOT-Raman microscope is shown in Fig. 1 and has previously been described in ( 29 , 31 ). A Ti:Sapphire laser (Spectra-Physics, Santa Clara, CA) tuned to 785 nm was used to form the optical tweezers (beam TEM 00 ).…”

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“…We conclude that only for efficiently cooled 2D samples the claim of linear speed improvement in line-scan Raman microscopy [10] holds true. The same applies to multi confocal techniques [12]. For thick samples and long scan lines however, the line-confocal microscope can be left behind by the point confocal method in terms of working speed (cyan, blue in Fig 7 ).…”

Recent advances in linear and nonlinear Raman spectroscopy. Part XIII

“…After optimization of the laser foci pattern, they demonstrated the feasibility of multifoci Raman hyperspectral microscopy for recording depth‐resolved molecular maps of biological cells ( Acanthamoeba castellanii trophozoites). Micrometric depth discrimination and short acquisition times (20 min for single plane confocal image) were achieved . Moralejo et al described improving the frame rates of Raman image sequences recorded with integral field spectroscopy using windowing and binning methods.…”

“…Micrometric depth discrimination and short acquisition times (20 min for single plane confocal image) were achieved. [207] Moralejo et al described improving the frame rates of Raman image sequences recorded with integral field spectroscopy using windowing and binning methods. The potential of this approach was demonstrated with Raman image sequences of polystyrene beads in motion in a micro-fluid environment.…”