Fourier phase microscopy for investigation of biological structures and dynamics Gabriel Popescu, Lauren P. Def lores, and Joshua C. Vaughan
George R. Harrison Spectroscopy Laboratory, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139
Harvard Medical School and Massachusetts General Hospital, Boston, Massachusetts 02114
Photonics K.K., Hamamatsu, Shizuoka 430-8587, Japan
Ramachandra R. Dasari and Michael S. Feld
George R. Harrison Spectroscopy Laboratory, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139 Received May 26, 2004 By use of the Fourier decomposition of a low-coherence optical image field into two spatial components that can be controllably shifted in phase with respect to each other, a new high-transverse-resolution quantitative-phase microscope has been developed. The technique transforms a typical optical microscope into a quantitative-phase microscope, with high accuracy and a path-length sensitivity of l 5500, which is stable over several hours. The results obtained on epithelial and red blood cells demonstrate the potential of this instrument for quantitative investigation of the structure and dynamics associated with biological systems without sample preparation. © 2004 Optical Society of America OCIS codes: 180.0180, 170.1530.
Phase-contrast and differential interference-contrast microscopy provide high-contrast intensity images of transparent biological structures without sample preparation.1,2 However, although these techniques reveal the structure of the sample, the phase information provided is qualitative. Phase-shifting interferometry (PSI) is commonly used for quantitative metrology,3 and interferometric techniques based on this principle have been demonstrated for fast imaging.4 Digital holography has also been developed for phase-contrast imaging5 and integrated with PSI.6 Recently, by using two harmonically related wavelengths, our group developed an interferometric technique for single-point phase measurements and demonstrated its potential for cell biology studies.7 A phase-imaging instrument that combines a stereo microscope with PSI was used for biological applications.8 However, this experimental arrangement does not take any precautions to suppress phase noise, which may affect the long-term stability of the instrument. A noninterferometric technique based on the irradiance transport equation was also developed for extracting optical phase images.9 This method requires displacement of the sample through the focus and requires extensive computations, which may limit its applicability to dynamic biological studies. The description of an optical image as an interference phenomenon was noted by Abbe more than 125 years ago10 and represents the underlying principle of phase-contrast microscopy.1 In this spirit, Kadono et al.11 developed a phase-shifting interferometer 0146-9592/04/212503-03$15.00/0
that is based on the interference of the scattered and unscattered light from transparent samples. In that setup the sample was illuminated by a laser and stability was achieved due to the common optical path for interfering beams. In addition to collecting the typical PSI four-frame interferograms, the phase image reconstruction required the measurement of the unscattered field amplitude, which imposed drastic restrictions on the system alignment. Recently, a similar system was combined with oblique illumination to enhance optical resolution.12 In this Letter we present a highly sensitive phase-imaging instrument, referred to as the Fourier phase microscope (FPM), which relies on the similar principle of decomposing a given field into its average and a spatially varying field. In our setup the two interfering fields are derived by Fourier transforming the output image of an existing transmission microscope, which provides excellent transverse...