Optical Path Design for Laser Confocal Inverted Microscope Raman
Spectrometer
HUANG Bao-kun1*, SONG Xin-ze2, CHENG Jing3, LI Yu-meng1, HUANG Tian-yun-zi1, SHEN Tian-yang1, KONG Xin-lan1, TAO Sha1*, ZHANG Yun-hong4*
1. School of Science, Jiangsu Ocean University，Lianyungang 222005, China
2. School of Mechanical Engineering, Jiangsu Ocean University，Lianyungang 222005, China
3. Jiangsu Tanggou Liangxianghe Liquor Co., Ltd.，Lianyungang 222535, China
4. Beijing Institute of Technology，Beijing 100081, China
Abstract：Micro-Raman spectrometers with a confocal design exhibit advantages such as high sensitivity, high spectral resolution, and high spatial resolution—owing to their highefficiencyin utilizing excitation light energy and in collecting and transmitting Raman scattering signals. Thus, they have become one of the primary analytical tools in laboratories. Inverted microscopes, by orienting their light exit ports upward, offer advantages including minimal restrictions on sample volume, rapid sample replacement, and upward beam propagation (which is suitable for detecting samples in containers like petri dishes). As a result, they hold promising applications in fields such as biomaterials and optical tweezers-based Raman spectroscopy.In this study, a laser confocal inverted micro-Raman spectrometer was developed by independently designing the optical path (comprising a laser optical system, an inverted microscope, a Raman signal optical system, and a spectrometer) and integrating these components with commercially procured lasers and charge-coupled devices (CCDs) via reserved interfaces. This developed spectrometer features a stable structure, low stray light levels, minimal installation requirements, and convenient maintenance. In accordance with the General Specification for Raman Spectrometers (GB_T 40219—2021), the sensitivity and spectral resolution of the laser confocal inverted micro-Raman spectrometer were tested. The results showed:When the spectrometer's entrance slit widths were 50 and 20 μm, the spectral resolution at the 1 710 cm-1 peak of a neon lamp was 2.5 and 1.5 cm-1, respectively—meeting the Class II index for “spectral resolution” specified in the General Specification; When a laser (wavelength: 532 nm, output power: 50 mW) was used as the excitation source with an exposure time of 300 s, the signal-to-noise ratios (SNRs) of the third-order Raman characteristic peak of monocrystalline silicon reached 11∶1 and 20∶1 when using open-electrode CCDs and backscattering CCDs, respectively. Additionally, the fourth-order peak was observable—meeting the Class I index for “signal-to-noise ratio” in the General Specification. Furthermore, the diameter of the laser-focused spot was calculated using the Rayleigh criterion. When a Leica objective lens (50× magnification, numerical aperture [NA]=0.75, focal length=0.5 mm) was employed, the spot size after the laser was focused through the objective lens was approximately 0.433 μm. Using the optical imaging magnification formula, the magnification of the Raman signal optical system was calculated to be 200, resulting in a spot diameter of ~86.6 μm at the slit. Given the spectrometer's magnification of 1, the spot diameter reaching the CCD remained ~86.6 μm. With the CCD having a pixel size of 16×16 μm, the spot diameter occupied approximately 5.4 pixels. When the slit width was 50 μm, the image of the slit (formed by the spectrometer) occupied ~3.1 pixels on the CCD. These calculation results were verified through actual spot size measurements. Finally, details of the instrument design were discussed, including the design philosophy and opto-mechanical design method for the laser beam expander, the application of long-pass filters and associated optical path design, the numerical aperture matching between the spectrometer and the pre-slit lens, the spectrometer's structure, the selection of collimating mirrors and focusing mirrors, and the choice of incident angle for grating diffraction.
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