We propose the acquisition of a two-photon/confocal microscope (Zeiss LSM 710) integrated with photoacoustic microscopy (PAM) to support NIH-funded research and establish imaging capabilities for the research community on the Danforth campus at Washington University. The integrated system will allow efficient imaging of the same sample or animal, eliminating the constraint of moving samples between imaging systems and allowing complementary phenotypic information to be co-registered automatically for a more holistic view of the sample. Technical innovations of LSM 710-which was released in 2008 and is not yet available at Washington University-provide new possibilities for research conducted with living, multi-labeled cells. LSM 710 can give new impetus to all areas of biological research by providing increased detection sensitivity, improved flexibility for new fluorescence dyes and multimodal experiments as well as new multiphoton detectors for deeper optical penetration into biological structures. Further hallmarks of the LSM 710 are its unique precision and reproducibility and markedly easier operation. The integrated PAM provides deeper penetration in scattering biological tissues as well as unique optical absorption contrast. Selective optical absorption is associated with molecules such as oxygenated and deoxygenated hemoglobin and melanin. Concentrations of multiple chromophores with different spectra of absorption coefficients can be qualified simultaneously by varying the wavelength of the irradiating laser. In this example, quantification of oxygenated and deoxygenated hemoglobin can provide functional imaging of the total concentration and oxygen saturation of hemoglobin. LSM 710 and PAM provide complementary contrasts. The former measures fluorescence signals from dyes or fluorophores, whereas the latter measures transient ultrasonic signal from optical absorption. PAM is one of the fastest growing biomedical imaging technologies with high-resolution sensing of rich optical absorption contrast in vivo at super-depths-depths beyond the optical transport mean free path (~1 mm in the skin). Previously available high- resolution three-dimensional optical microscopy modalities-including confocal microscopy, two-photon microscopy and optical coherence tomography-have fundamentally impacted biomedicine. Unfortunately, none can reach super- depths in scattering biological tissue. Taking advantage of low ultrasonic scattering, PAM equivalently improves tissue optical transparency by factor(s) of 100 to 1000 and consequently enables super-depth penetration at high resolution. Functional and molecular imaging based on optical contrast has been achieved. Further, PAM has multiscale in vivo imaging capabilities, allowing users to image subcellular organelles to organs with the same contrast origin. While PAM is expected to find broad applications, multiscale PAM will likely play a critical role in multiscale biology research.
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| Rao, Bin; Soto, Florentina; Kerschensteiner, Daniel et al. (2014) Integrated photoacoustic, confocal, and two-photon microscope. J Biomed Opt 19:36002 |
