Abstract
We analyze the influence of the anisotropy of molecular and biological samples on polarization resolved nonlinear microscopy imaging. We show in particular the detrimental influence of birefringence on Second Harmonic Generation (SHG) and Two-Photon Excited Fluorescence (TPEF) polarization resolved microscopy imaging, which, if not accounted for, can lead to an erroneous determination of the sample properties and thus to a misinterpretation of the read-out information. We propose a method to measure this birefringence and account for this effect in nonlinear polarization resolved experiments.
Highlights
Since its first developments [1,2,3] and its introduction in bio-imaging [4,5,6], second harmonic generation (SHG) microscopy is widely used to image ordered biomolecular assemblies in complex samples at depths reaching a few hundreds of micrometers
Coherent SHG occurring naturally in non-centrosymmetric structures such as collagen [2], skeletal muscles [5] and microtubules [6], is today exploited as a functional contrast [7, 8], possibly in conjunction with Two-Photon Excited Fluorescence (TPEF) [9, 10], with the ultimate goal of developing diagnostics of pathological effects related to tissues and cell architecture
Note that still accounting for the variations of φ0 seen on the SHG images does not notably change these results. These results show that particular care has to be brought on the local properties of the sample, which can be very distinct from averaged birefringence since the optical axis of the system can typically rotate progressively through the whole sample thickness
Summary
Since its first developments [1,2,3] and its introduction in bio-imaging [4,5,6], second harmonic generation (SHG) microscopy is widely used to image ordered biomolecular assemblies in complex samples at depths reaching a few hundreds of micrometers. Coherent SHG occurring naturally in non-centrosymmetric structures such as collagen [2], skeletal muscles [5] and microtubules [6], is today exploited as a functional contrast [7, 8], possibly in conjunction with Two-Photon Excited Fluorescence (TPEF) [9, 10], with the ultimate goal of developing diagnostics of pathological effects related to tissues and cell architecture In addition to their unique imaging capabilities, these contrasts are dependent on the incident light polarization state, providing an interesting way to probe molecular orientation and disorder. These contrasts are chosen for the possibility to combine them on the same microscope, the present study can be adapted to one-photon fluorescence or other nonlinear processes
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