Variable-Temperature Stage for Raman Microprobing Spectroscopy

Abstract

Although Raman microprobing spectroscopy (RMS) has been becoming increasingly applicable in the area of structural study of biological samples, such as DNA in a single living species or chromosome, and protein chromophores in single cells, potential impairment of sample integrity by an excitation photon could be one of the toughest obstacles to its wide utilization. In RMS, the excitation laser beam is tightly focused at the sample—in the case of high magnification and numeric aperture objectives, down to a diffraction limit of sub-microns laterally, with a depth of focus on the order of 1 to 10 microns, producing high irradiant levels. Consequently, photo-damage or integrity degradation of samples can occur, especially in the case of resonance Raman measurements where a sample absorbs excitation photons, resulting in heat generation. A device that can cool the sample far below ambient temperature and at the same time fit properly between the microscope objectives and sample stage is highly desirable. Application of a liquid nitrogen-cooled sample holder for resonance Raman spectroscopic studies of visual cone cells with a microscope has recently been reported. However, the capabilities of variable temperature, precise control, and convenient operation are also very important for the study of thermally dependent structural changes in biological samples. Here we report the use of a commercial Joule-Thompson cooling device in a Raman microprobing spectroscopic system designed for resonance Raman study of biological molecules.