Nonlinear optical (NLO) microscopy is emerging as a powerful technique for the study of biological samples. By combining several different imaging modalities such as multiphoton (MP) fluorescence, second-harmonic and thirdharmonic generation (SHG and THG), and coherent Raman scattering techniques such as coherent anti-Stokes Raman scattering (CARS) and stimulated Raman scattering (SRS), it is possible to combine the best practices of label and label-free imaging into a single platform capable of imaging structures within single cells and elucidating the health of biological tissue samples, even at the submicron level. Single-substrate, ion-beam-sputtered (IBS) thinfilm interference filters are a key enabling technology in laser-based optical microscopy and play a critical role in multimodal NLO imaging. In microscopy applications, optical filters are used to select and discriminate exactly which wavelengths of light are to be transmitted, reflected and suppressed. In this paper we discuss various important characteristics of hard-coated thin-film interference filters, such as high light throughput, steep edges, and high out-of-band blocking, all of which require careful consideration when designing and manufacturing optical filters for NLO imaging applications. To understand the true performance of hard-coated IBS filters, a simple CARS imaging experiment was performed. We found a 2.6 times increase in signal enhancement and 70% improvement in image contrast when compared to a commercially available filter commonly used in CARS microscopy applications.
[1]
W. Webb,et al.
Live tissue intrinsic emission microscopy using multiphoton-excited native fluorescence and second harmonic generation
,
2003,
Proceedings of the National Academy of Sciences of the United States of America.
[2]
F. Ganikhanov,et al.
Multimodal nonlinear optical imaging of collagen arrays.
,
2008,
Journal of structural biology.
[3]
W. Webb,et al.
Nonlinear magic: multiphoton microscopy in the biosciences
,
2003,
Nature Biotechnology.
[4]
Mortazavi,et al.
Supporting Online Material Materials and Methods Figs. S1 to S13 Tables S1 to S3 References Label-free Biomedical Imaging with High Sensitivity by Stimulated Raman Scattering Microscopy
,
2022
.
[5]
Chris B Schaffer,et al.
In vivo imaging of myelin in the vertebrate central nervous system using third harmonic generation microscopy.
,
2011,
Biophysical journal.
[6]
Jane A Dickerson,et al.
Current Applications of Liquid Chromatography / Mass Spectrometry in Pharmaceutical Discovery After a Decade of Innovation
,
2008
.