15 Feb 2008
A new holographic technique that achieves dynamic high-contrast imaging offers a unique solution for studying biological processes at the molecular level.
A team of US, Swiss and German scientists has found that second harmonic signals generated by a femtosecond laser can be used to produce high-contrast images with a temporal resolution of about 150 fs. The researchers believe that the new imaging technique, which they have called harmonic holography, will enable the dynamics of complex biological processes to be studied in three dimensions (Appl. Opt. 47 A103).
"To the best of my knowledge, this is the first time that second harmonic signals have been used to record holograms," Ye Pu of the California Institute of Technology told
Although a number of imaging techniques are currently used to probe biological samples, none are able to combine the high-contrast imaging needed to identify individual molecules with the ability to track fast-moving biological processes. Fluorescence microscopy, for example, has become popular for picking out specific molecules or nanostructures, but is largely constrained to two dimensions.
"Fluorescence is an incoherent process, and it is generally difficult to infer 3D information from such signals," Pu explained. "Although confocal and two-photon laser scanning microscopy do provide 3D capability, the point-scanning is too time consuming to capture dynamic events."
In contrast, holography is well known for its 3D imaging capability, and taking a number of images in quick succession enables dynamic processes to be followed in time as well as space. The problem is that conventional holography lacks contrast between what is of interest (the signal) and what is not (the background), which has so far limited its use in biological imaging.
The solution adopted by Pu, working in collaboration with Martin Centurion of the Max Planck Institute of Quantum Physics in Garching, Germany, and Demetri Psaltis of the Ecole Polytechnique Fédérale de Lausanne in Switzerland, is to produce a holographic image from second harmonic signals that are generated when pulses from a femtosecond laser pass through a special type of nanocrystal.
"Second harmonic generation provides a strong contrast mechanism in the coherent domain," said Pu. "Because most liquids and biological structures are isotropic, they are incapable of frequency doubling. Second harmonic generating nanocrystals can be picked up easily in the double-frequency band with a dark background."
Only nanocrystals with a noncentrosymmetric structures are capable of producing such second harmonic signals. The Caltech group used 100 nm nanocrystals of barium titanate (BaTiO3), a low-cost, nontoxic material that has the right crystal structure to produce strong second harmonic signals.
In the experiment, 810 nm femtosecond laser pulses with an energy of 2 mJ are split into a pump and a reference beam. Firing the pump beam at the nanocrystals produces second harmonic signals, which then interfere with the frequency-doubled reference beam to produce the holographic image.
According to Pu, the temporal resolution of the current set-up is about 150 fs, limited only by the laser source. The spatial resolution is around 1.8 µm in the lateral direction and 4 µm axially at a numerical aperture of 0.5, although these figures could be improved by increasing the numerical aperture of the system. "When the image is formed with doubled frequency while illuminated with the fundamental frequency, the spatial resolution doubles accordingly," said Pu.
One critical issue is the signal-to-noise ratio, since second harmonic emissions from nanocrystals are usually very weak. If a normal imaging device were used at such low photon counts, the overall signal noise would be dominated by the device noise – which is usually relatively high for an ultrafast imaging system. In contrast, the best imaging performance is obtained when there are enough photons for the device noise to be much smaller than the so-called shot noise, which is caused by intrinsic fluctuations in the number of photons.
In the case of harmonic holography, however, the overall noise is dominated by the shot noise of the reference beam, and the device noise is negligible. "With a coherent reference serving as a bias, the holographic fringes swing around the bias level with an amplified amplitude," explained Pu. "In the holographic reconstruction, the shot noise of the reference cancels the amplification in the holographic fringe amplitude, and the final signal-to-noise ratio remains limited by the shot noise of the signal, not the reference, beam." The end result is shot-noise limited performance at low photon counts.
To demonstrate the technique, the Caltech team produced images of bare nanocrystals, but for imaging biological samples the idea is to tag the nanocrystals to the molecules or structures being investigated. "Together with Scott Fraser's group in the Biology Division here at Caltech, we are working to push the second harmonic generating nanocrystals into the critical 10 nm regime for real biological applications," commented Pu.
Pu is also confident that the harmonic holography technique can be realized in a practical instrument. "Besides the investment of an amplified femtosecond laser system, a practical harmonic holography microscope only requires minimal changes to a modern microscope," he said. "We are working hard to make it happen."