The imaging of absorbing (i.e. nonfluorescent) chromophores in thick tissue poses a challenge for microscopists. Recently, a new technique called photothermal microscopy (PM) has been gaining attention, which involves the focusing two laser beams into a sample. One beam (the heating beam) is tuned to a chromophore absorption line while the other is set at wavelength outside any absorption bands (the probe beam). When the heating beam is absorbed, energy is deposited into the tissue, producing a local density fluctuation. This density change results in a small transient change in the refractive index about the chromophore, which is then monitored by the probe beam. In early applications, PM has been shown to track gold nanoparticles in cell cultures with unprecedented sensitivity. More recently, PM has also been shown to be remarkably effective at imaging endogenous chromophores in live cells. Examples include imaging of mitochondria and erythrocytes with 3D spatial resolution comparable to confocal microscopy. As promising as PM is, several open questions still remain. Specifically: is it possible to perform PM in thick tissue, and what exactly are the chromophore species responsible for PM contrast? To date, PM has only been performed with thin samples in a transmitted light configuration. Moreover, the chromophore species responsible for PM contrast are either unknown (in the case of mitochondrial imaging) or speculated (in the case of erythrocyte imaging). We propose to 1) develop a novel scanning PM that can perform photothermal imaging in thick tissue, for the first time, using on one- or two-photon absorption, and 2) unambiguously identify and characterize both existing and new endogenous contrast agents using a photothermal spectroscopy screening platform equipped with an ultra-wide bandwidth (UV to THz) laser. We will initially concentrate on the study of cytochrome in mitochondria, heme protein in blood cells, and channel rhodopsin. A completion of the above aims will be indispensible for PM to gain widespread acceptance in the biomedical imaging community, and will lay the groundwork for a new technology that provides high sensitivity, high resolution absorption contrast with molecular specificity.

Public Health Relevance

We propose to develop an optical microscopy technique that provides ultrahigh sensitivity 3D imaging of absorbing (i.e. non-fluorescent) proteins or molecules in tissue. This technology will be useful for in- vivo imaging research applications and rapid tissue diagnosis in the clinic.

National Institute of Health (NIH)
National Institute of Biomedical Imaging and Bioengineering (NIBIB)
Exploratory/Developmental Grants (R21)
Project #
Application #
Study Section
Special Emphasis Panel (ZRG1-IMST-L (90))
Program Officer
Conroy, Richard
Project Start
Project End
Budget Start
Budget End
Support Year
Fiscal Year
Total Cost
Indirect Cost
Boston University
Engineering (All Types)
Schools of Engineering
United States
Zip Code
Baritaux, J-C; Chan, C R; Li, J et al. (2014) View synthesis with a partitioned-aperture microscope. Opt Lett 39:685-8
Mërtiri, Alket; Jeys, Thomas; Liberman, Vladimir et al. (2012) Mid-infrared photothermal heterodyne spectroscopy in a liquid crystal using a quantum cascade laser. Appl Phys Lett 101:44101
Liberman, Vladimir; Adato, Ronen; Jeys, Thomas H et al. (2012) Rational design and optimization of plasmonic nanoarrays for surface enhanced infrared spectroscopy. Opt Express 20:11953-67
Parthasarathy, Ashwin B; Chu, Kengyeh K; Ford, Tim N et al. (2012) Quantitative phase imaging using a partitioned detection aperture. Opt Lett 37:4062-4