1 March 2010 Signal improvement in multiphoton microscopy by reflection with simple mirrors near the sample
Author Affiliations +
J. of Biomedical Optics, 15(2), 026017 (2010). doi:10.1117/1.3374337
In conventional fluorescence or confocal microscopy, emitted light is generated not only in the focal plane but also above and below. The situation is different in multiphoton-induced fluorescence and multiphoton-induced higher harmonic generation. Here, restriction of signal generation to a single focal point permits that all emitted photons can contribute to image formation if collected, regardless of their path through the specimen. Often, the intensity of the emitted light is rather low in biological specimens. We present a method to significantly increase the fraction of photons collected by an epi (backward) detector by placing a simple mirror, an aluminum-coated coverslip, directly under the sample. Samples investigated include fluorescent test slides, collagen gels, and thin-layered, intact mouse skeletal muscles. Quantitative analysis revealed an intensity increase of second- and third-harmonic generated signal in skeletal muscle of nine- and sevenfold respectively, and of fluorescent signal in test slides of up to twofold. Our approach thus allows significant signal improvement also for situations were a forward detection is impossible, e.g., due to the anatomy of animals in intravital microscopy.
Markus Rehberg, Fritz Krombach, Ulrich Pohl, Steffen Dietzel, "Signal improvement in multiphoton microscopy by reflection with simple mirrors near the sample," Journal of Biomedical Optics 15(2), 026017 (1 March 2010). http://dx.doi.org/10.1117/1.3374337
Submission: Received ; Accepted


Second-harmonic generation

Signal detection





Back to Top