Abstract
We describe an advanced DIVER (Deep Imaging Via Emission Recovery) detection system for two-photon fluorescence microscopy that allows imaging in multiple scattering media, including biological tissues, up to a depth of a few mm with micron resolution. This detection system is more sensitive to low level light signals than conventional epi-detection used in two-photon fluorescence microscopes. The DIVER detector efficiently collects scattered emission photons from a wide area of turbid samples at almost any entrance angle in a 2π spherical angle. Using an epi-detection scheme only photons coming from a relatively small area of a sample and at narrow acceptance angle can be detected. The transmission geometry of the DIVER imaging system makes it exceptionally suitable for Second and Third Harmonic Generation (SHG, THG) signal detection. It also has in-depth fluorescence lifetime imaging (FLIM) capability. Using special optical filters with sin-cos spectral response, hyperspectral analysis of images acquired in-depth in scattering media can be performed. The system was successfully employed in imaging of various biological tissues. The DIVER detector can be plugged into a standard microscope stage and used as an external detector with upright commercial two-photon microscopes.
Highlights
The ability to visualize features in deep layers of biological tissue with high resolution is a very sought-after feature of imaging systems employed in medical diagnostics, as well as in clinical and biological applications
The DIVER detector is coupled with the Fast-FLIMbox which allows acquisition of 3D fluorescence lifetime imaging (FLIM) images, where various sample features are colored according to their fluorescence lifetimes
We have shown that the DIVER detector, which is simple to construct, may significantly improve the imaging depth in turbid samples
Summary
The ability to visualize features in deep layers of biological tissue with high resolution is a very sought-after feature of imaging systems employed in medical diagnostics, as well as in clinical and biological applications. While transparent specimens can be imaged with a traditional microscope, biological tissue is an intrinsically turbid medium, which produces strong multiple scattering, and absorption and which exhibits inhomogeneity of the refractive index These features make traditional light and fluorescence microscopy inefficient at depths more than 100–200 μm from the surface of a sample [1], even with the aid of staining and the addition of fluorescent markers to improve the contrast. In order to access deeper layers of turbid samples, high power short pulsed lasers emitting in near infrared (NIR) are required This approach allows deeper light penetration into media, because of the reduced absorption and scattering at longer wavelengths. The application of spectral phasors to quantify dipolar
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
