A high-precision multi-dimensional microspectroscopic technique for morphological and properties analysis of cancer cell

Abstract

Raman and Brillouin scattering are sensitive approaches to detect chemical composition and mechanical elasticity pathology of cells in cancer development and their medical treatment researches. The application is, however, suffering from the lack of ability to synchronously acquire the scattering signals following three-dimensional (3D) cell morphology with reasonable spatial resolution and signal-to-noise ratio. Herein, we propose a divided-aperture laser differential confocal 3D Geometry-Raman-Brillouin microscopic detection technology, by which reflection, Raman, and Brillouin scattering signals are simultaneously in situ collected in real time with an axial focusing accuracy up to 1 nm, in the height range of 200 μm. The divided aperture improves the anti-noise capability of the system, and the noise influence depth of Raman detection reduces by 35.4%, and the Brillouin extinction ratio increases by 22 dB. A high-precision multichannel microspectroscopic system containing these functions is developed, which is utilized to study gastric cancer tissue. As a result, a 25% reduction of collagen concentration, 42% increase of DNA substances, 17% and 9% decrease in viscosity and elasticity are finely resolved from the 3D mappings. These findings indicate that our system can be a powerful tool to study cancer development new therapies at the sub-cell level.

Fig. 1. Schematic structure of the DDCGRBM system.

a Brillouin spectrum curve at focus. b Raman spectrum curve at focus. c The differential confocal axial light intensity curve. The characteristic that the zero-crossing point of the curve is the focal position with nanometer-focusing accuracy. d Utilizing the ultra-high focusing accuracy of the system, high stability and optimal resolution of spectral acquisition are guaranteed at each scanning point

Fig. 2. Anti-drift capability test results.

The Raman spectrum exposure time is 1 s and the Brillouin spectrum acquisition time is 10 s. a, d The schematic of axial focusing capability of DDCGRBM and CRM when the sample is tilted. b, e The normalized Raman spectrum intensity map with focus tracking measured by the DDCGRBM, the normalized defocused one by CRM. c, f The normalized Brillouin spectrum intensity map with focus tracking measured by the DDCGRBM, the normalized defocused one by CRM. g Variation curve of step edge width with defocus height of spectral intensity maps measured by DDCGRBM, CRM and CBM. h Variation curve of spectral intensity with defocus height of spectral intensity maps measured by DDCGRBM, CRM, and CBM

Fig. 3. Test of suppressing defocused stray light interference ability.

a Schematic diagram of the DDCGRBM system suppressing defocus-layer stray light interference experiment. PZT drives the objective lens to make the light spot enter the SiO₂ from the PMMA across the interface. b The intensity PSF diagram of DDCGRBM and CRM in the X and Z directions when the NA = 0.9 objective is divided into the excitation optical path pupil and the collection optical path pupil with equal diameters 1/2 of the diameter of the rear pupil of the objective lens, and the Iz plot at X = 0. c The Raman spectrum curve when the spot is located between PMMA and SiO₂. d The Raman spectral curves of each scanning position during the axial scanning of the DDCGRBM system. e The Raman intensity changes of PMMA (811 cm⁻¹) measured by the DDCGRBM and CRM systems

Fig. 4. Mapping results of gastric cancer tissue and normal tissue.

a Scanning area of gastric cancer tissue, adjacent normal tissue scanning area. Area ① is the gastric cancer cell area, area ② is the gastric cancer intercellular substance area, area ③ is the normal cell area, and area ④ is the normal intercellular substance area. b three-dimensional topographic map of gastric cancer tissue, adjacent normal tissue scanning area. c The average spectrum curve of the Raman spectrum of ten random points of gastric cancer tissue detected by the DDCGRBM system and the corresponding Raman peaks of various substances. d The fitting curve of the average spectrum of Brillouin spectrum of 10 random points detected by the DDCGRBM system in gastric cancer tissue. e The three-dimensional distribution map of various chemical components in the scanned area of gastric cancer tissue (upper left), the three-dimensional distribution map of various chemical components in the scanned area of normal tissue adjacent to cancer (lower left), The three-dimensional distribution map of loss modulus and storage modulus in the scanned area of gastric cancer tissue (upper right), and the three-dimensional distribution map of storage modulus and loss modulus in the scan area of normal tissue adjacent to cancer (lower right). f Concentration changes of various substances in gastric cancer cells and gastric cancer intercellular substance compared with normal cells and normal intercellular substance. g Changes in the storage modulus and loss modulus of gastric cancer cells and gastric cancer interstitial cells compared with normal cells and normal interstitial cells