Development of high-speed two-photon microscopy for biological and medical applications

Two-photon microscopy (TPM) is one of the most powerful microscopic technologies for in-vivo 3D tissue imaging up to a few hundred micrometers. It has been finding important applications in neuronal imaging, tumor physiology study, and optical biopsy. A practical limitation of TPM is its slow imaging speed (0.3 1 frames/s). We designed high-speed two-photon microscopes (HSTPMs) whose imaging speed is more than 10 times faster than traditional TPM, while their imaging depths, image contrast are comparable to those of TPM. The first high speed system is HSTPM based on polygonal mirror scanner. The scanning speed reaches 13 frames/s for typical tissues using a polygonal mirror scanner. This system is based on single-focus scanning and single-pixel signal collection. The usage of higher input power is required to compensate for the signal reduction due to higher scanning speed. However, since fluorescence signal is ultimately limited by the saturation of fluorophores due to their finite lifetimes, is the signal to noise ratio (SNR) of single focus scanning systems are also ultimately limited at high speed. This problem is circumvented in a second system based on parallelization by scanning specimens with multiple foci of excitation light and collecting signals with spatially resolved detectors. The imaging speed is increased proportional to the number of foci and similar excitation laser power per focus circumventing the problem of fluorophore saturation. However, it has been recognized that this method is severely limited for deep tissue imaging due to photon scattering.

Department dc:contributor.department

Massachusetts Institute of Technology. Department of Mechanical Engineering

Grantor dc:publisher

Massachusetts Institute of Technology

Year dc:date.issued

2005

Author dc:creator

• Kim, Ki Hean

Advisor dc:contributor.advisor

• George Barbastathis, Bevin P. Englward, Ian W. Hunter and Peter T.C. So.

• Mechanical Engineering.

Statement dc:rights

• M.I.T. theses are protected by copyright. They may be viewed from this source for any purpose, but reproduction or distribution in any format is prohibited without written permission. See provided URL for inquiries about permission.

Licence dc:rights.uri

• http://dspace.mit.edu/handle/1721.1/7582

Language dc:language.iso

eng

Handle dc:identifier.uri

http://hdl.handle.net/1721.1/32383

OAI identifier oai:identifier

oai:dspace.mit.edu:1721.1/32383