Label Free Linear and Non-Linear Excitation Nanoscopy

Abstract

Despite tremendous progress in super-resolution microscopy techniques, the imaging of non-fluorescent species with sub-diffraction resolution is a challenging task. To overcome this limit the subtraction microscopy approach has been proposed recently [1,2]. The concept is based on the acquisition and the successive mathematical subtraction of two images acquired with Gaussian and doughnut shaped focal spots, respectively.Here, we will show a novel rescaling method that avoids artifacts, maintaining the contrast and resolution enhancement. Nevertheless, Infrared absorption/saturation microscopy is also designed to be a label free super-resolution approach[3]. The main idea of InfraRed Nanoscopy (IRN) is an absorption/saturation effect similar to the conventional pump-probe technique[4], where the pump beam modifies the carrier density inside the sample, followed by the intensity changes in the transmitted probe beam. The introduction of an additional doughnut shaped pump beam can transiently saturate the induced transition in the periphery of the focal spot and enabling super-resolution. Finally we will show a novel implementation based on single wavelength 2PE-STED nanoscopy[5] by means of random access scanning, and a phasor approach for the analysis of polarization SHG images, that, providing quantitative information, can be used to extract a fingerprint of tissues.The LANIR research leading to these results has received funding from FP7/20012-2015 under grant agreement n°280804.1.Harold, D., Piche, M., De Koninck, Y.(2013) Optics express 21,13:15912-15925.2.Wang, N., Miyazaki, J., He, J., Seto, K., Kobayashi, T.(2015). Optics Communications 343,28-33.3.Silien, C., Liu, N., Hendaoui, N., Tofail, SAM., Peremans, A.(2012). Optics Express 20,29694-29704.4.Dong CY, So PT, French T, Gratton E.(1995) Biophysical Journal. 69(6):2234-2242.5. Bianchini, P., Peres, C., Oneto, M., Galiani, S., Vicidomini, G., Diaspro, A. (2015). Cell and Tissue Research 360,143-150.