Dual-wavelength differential confocal sensing technology with virtual pinhole detection

Aiming at the problem that when combining confocal microscopy and resonance Raman spectroscopy (RRS), the focusing accuracy of the system decreases due to the shift of the focusing plane of the beam caused by the change of the work wavelength, a laser dual-wavelength differential confocal sensing technology (DDCST) with virtual pinhole and CCD detection is proposed, the basic theory based on the wavelength and focusing plane position relationship has been established, which can provide a precise theoretical basis for the determination of the focusing accuracy of the Confocal microscopy (CM)-RRS, and greatly improve the system accuracy, and uses a CCD as detector. DDCST sets a microregion on the CCD imaging plane as a virtual pinhole, can freely adjust the position and size of the pinhole in the software to match with the sample reflectivity and detector response range in measurements. So, DDCST significantly simplifies the detection system and pinhole adjustment, eliminates the error attributed to the adjustment error of the physical pinhole. Theoretical analyses and preliminary experiments indicate that DDCST has an axial resolution of about 5nm and 6nm respectively, and a lateral resolution of about 1µm and 1.2µm respectively when the work wavelength is 532nm and 653nm respectively, can recognizes the influence of the change of the work wavelength on the focusing accuracy of the system, which can lay the foundation of the morphology imaging of the probing micro-region, and provide theoretical support and help for the development of the CM-RRS.