Recent development of super-resolution fluorescence imaging technique such as stochastic optical reconstruction
microscopy (STORM) and photoactived localization microscope (PALM) has brought us beyond the diffraction limits. It
allows numerous opportunities in biology because vast amount of formerly obscured molecular structures, due to lack of
spatial resolution, now can be directly observed. A drawback of fluorescence imaging, however, is that it lacks complete
structural information. For this reason, we have developed a super-resolution multimodal imaging system based on
STORM and full-field optical coherence microscopy (FF-OCM). FF-OCM is a type of interferometry systems based on a
broadband light source and a bulk Michelson interferometer, which provides label-free and non-invasive visualization of
biological samples. The integration between the two systems is simple because both systems use a wide-field
illumination scheme and a conventional microscope. This combined imaging system gives us both functional
information at a molecular level (~20nm) and structural information at the sub-cellular level (~1μm). For thick samples
such as tissue slices, while FF-OCM is readily capable of imaging the 3D architecture, STORM suffer from aberrations
and high background fluorescence that substantially degrade the resolution. In order to correct the aberrations in thick
tissues, we employed an adaptive optics system in the detection path of the STORM microscope. We used our
multimodal system to obtain images on brain tissue samples with structural and functional information.
Raman spectroscopy has been a powerful tool in various fields of science and technology ranging from analytical
chemistry to biomedical imaging. In spite of unique features, Raman spectroscopy has also some limitations. Among
them are weak Raman signal compared to strong fluorescence and relatively complicated setup with expensive and bulky
spectrometer. In order to increase the sensitivity of Raman technique, many clever attempts have been made and some of
them were very successful including CARS, SRS, and so on. However, these still requires expensive and more
complicated setup. In this work, we have attempted to build a real-time compact Raman sensor without spectrometer.
Conventional spectrometer was replaced with a narrow-band optical filter and alternatively modulated two lasers with
slightly different wavelengths. At one laser, Raman signal from a target molecule was transmitted through the optical
filter. At the other laser, this signal was blocked by the optical filter and could not be detected by photon detector. The
alternative modulation of two lasers will modulate the Raman signal from a target molecule at the same modulation
frequency. This modulated weak Raman signal was amplified by a lock-in amplifier. The advantages of this setup
include compactness, low cost, real-time monitoring, and so on. We have tested the sensitivity of this setup and we found
that it doesn’t have enough sensitivity to detect single molecule-level, but it is still good enough to monitor the change of
major chemical composition in the sample.
High resolution optical imaging technologies, such as optical coherence tomography or multiphoton microscopy has given us an opportunity to do in vivo imaging noninvasively. However, due to the high laser scattering, these optical imaging techniques were prohibited from obtaining high resolution in the diffusive regime. Photoacoustic microscopy (PAM) can overcome this soft depth limit and maintain high resolution at the same time. In the past, PAM was limited to using an Nd:YAG laser, which requires an optical parametric oscillator (OPO) to obtain wavelengths selectively other than the second harmonic. However, OPO is unstable and cumbersome to control. We replaced the Nd:YAG laser and the OPO with a nanosecond pulsed Ti:Sapphire laser to give PAM more flexibility in the speed and the input wavelength while reducing the footprint of our system. This also increased our stability by removing OPO. Using a Ti:Sapphire laser allowed us to increase the pulse repetition rate to 100-500 kHz. Normally, micro-lasers with this pulse repetition rate will suffer from a significant decrease in pulse energy, but we were able to maintain stable pulses with a few hundreds nJ. Also, a well-known advantage of a Ti:Sapphire laser is its tunability from 650 to 1100 nm. For our PAM application, we used a range from 700 to 900 nm to obtain significant functional images. This added flexibility can help acquire functional images such as the angiogenesis process with better contrast. Here, we present a nanosecond Ti:Sapphire laser designated for PAM applications with increased contrast imaging.
Access to the requested content is limited to institutions that have purchased or subscribe to SPIE eBooks.
You are receiving this notice because your organization may not have SPIE eBooks access.*
*Shibboleth/Open Athens users─please
sign in
to access your institution's subscriptions.
To obtain this item, you may purchase the complete book in print or electronic format on
SPIE.org.
INSTITUTIONAL Select your institution to access the SPIE Digital Library.
PERSONAL Sign in with your SPIE account to access your personal subscriptions or to use specific features such as save to my library, sign up for alerts, save searches, etc.