Spectral focusing-based stimulated Raman scattering microscopy using compact glass blocks for adjustable dispersion

Abstract

Spectral focusing is a well-established technique for increasing spectral resolution in coherent Raman scattering microscopy. However, current methods for tuning optical chirp in setups using spectral focusing, such as glass rods, gratings, and prisms, are very cumbersome, time-consuming to use, and difficult to align, all of which limit more widespread use of the spectral focusing technique. Here, we report a stimulated Raman scattering (SRS) configuration which can rapidly tune optical chirp by utilizing compact adjustable-dispersion TIH53 glass blocks. By varying the height of the blocks, the number of bounces in the blocks and therefore path length of the pulses through the glass can be quickly modulated, allowing for a convenient method of adjusting chirp with almost no necessary realignment. To demonstrate the flexibility of this configuration, we characterize our system's signal-to-noise ratio and spectral resolution at different chirp values and perform imaging in both the carbon-hydrogen stretching region (MCF-7 cells) and fingerprint region (prostate cores). Our findings show that adjustable-dispersion glass blocks allow the user to effortlessly modify their optical system to suit their imaging requirements. These blocks can be used to significantly simplify and miniaturize experimental configurations utilizing spectral focusing.

Fig. 1.

Spectral focusing SRS microscopy using compact TIH53 glass blocks. (a) Schematic of setup. HWP: Half-wave plate; POL: Polarizer; AOM: Acousto-optic modulator; DL: Delay line; T: Telescope; GB: Glass block; DM: Dichroic mirror; GM: Galvanometer mirrors; OBJ: Objective; S: Sample; CON: Condenser; F: Filter; PD: Photodetector. (b) Schematic (top) and photo (bottom left) of the adjustable-dispersion TIH53 glass block. The beam enters the block, bounces between the two silver mirrors, and exits from an unsilvered ramp. (c) Experimentally determined pulse durations (circle) versus theoretical predictions (square).

Fig. 2.

Characterization measurements for SRS microscope in the FP region. (a) SRS spectral profile of the benzonitrile Raman band at 1001 cm^-1 for 6–6 (6 for the pump and Stokes each), 7–7, and 8–8 bounces in the FP region alongside the corresponding spontaneous Raman spectrum. (b) SRS image of 0.6 µm polystyrene beads at the 1004 cm^-1 Raman band. (c) Zoom-in of the highlighted region in the SRS image shown in (a). (d) Measured and fitted (solid line) transverse SRS intensity profile associated with a single 0.6 µm polystyrene bead. A FWHM value of 0.7 µm is obtained for the transverse resolution. The pixels analyzed in this curve are shown in (c) as a yellow line.

Fig. 3.

SRS imaging of PC tissue. Images were collected at 512 × 512 pixels with an integration time of 20 µs for a total acquisition time of 5.2 seconds per image. (a) SRS spectra of benzonitrile, oleic acid, paraffin, and acetaminophen. SRS images of PC tissue when using 6 bounces for pump and Stokes at the Raman bands of (b) 1004 cm^-1, (c) 1250 cm^-1, (d) 1477 cm^-1, and (e) 1644 cm^-1. (f) Co-registered image of SRS PC tissue at the Raman bands corresponding to 1004 cm^-1 (red), 1477 cm^-1 (green), and 1644 cm^-1 (blue).

Fig. 4.

SRS imaging of MCF-7 cells at 24 hours-post exposure to X-ray radiation with 2 bounces each in the TIH53 glass blocks for the pump and Stokes beams. Images were collected at 512 × 512 pixels with an integration time of 20 µs for a total acquisition time of 5.2 seconds per image. Images of unirradiated (0 Gy) MCF-7 cells at the (a) 2850 cm^-1 and (b) 2926 cm^-1 bands. (c) A co-registered image of the 0 Gy images, with the 2850 cm^-1 image in green and the 2926 cm^-1 image in purple. Images of MCF-7 cells irradiated with 30 Gy at (d) 2850 cm^-1 and (e) 2926 cm^-1. (f) A co-registered image of the 30 Gy images, with the same color scheme as in (c).