The key reasons for developing reflection mode phase microscopy systems includes depth-sectioning and higher (by a factor of 2n/Δn) phase measurement sensitivity, where n is the refractive of the medium and Δn is the refractive index contrast. One approach to achieve depth-sectioning is to use a broadband source with low coherence length. LBRC has developed both line-illumination as well as wide-field low coherence reflection phase microscopes. The following briefly describes the working principle and characteristics of a line-field reflection phase microscope.
Figure 1 shows the schematic of light illumination low coherence phase microscopy setup [1]. The setup is a classical spectral-domain implementation, which employs a separate reference arm (using identical optics) for interference. By measuring the relative phase of the reflections from the specimen and the coverslip surface (outside the sample), the common-mode noise can be effectively rejected which delivers superior phase stability ideally suited for high-sensitivity phase measurements. Compared to point illumination systems [2,3], the system shown in Fig. 1 allows simultaneous depth-resolved phase measurement of multiple lateral points, thus enabling the study of spatial and temporal coherence of multiple locations within a B-scan image. Light from a modelocked Ti:Sapphire laser (center wavelength, λc = 800 nm) is coupled into a single-mode fiber for delivery as well as for spectrum broadening. The full-width-half-maximum spectral width, Δλ, at the fiber output measures 50 nm, which corresponds to coherence length of 4 μm in medium with refractive index n = 1.37. A cylindrical lens (f = 300 mm) is used in the path of the collimated beam (1/e2 diameter = 6 mm) along with achromatic lenses L2, L3 and a water immersion 60x (NA = 1.2) microscope objective to yield line focused illumination beam (~60 µm x 0.5 µm) in the object plane.
The returning light beams from sample and reference arms combine at the beam splitter and reach a two-dimensional (2-D) spectrometer. A vertical slit S is also introduced along the way to reduce the light coming from out-of-confocal region in the object plane and arriving at the spectrometer. The 2-D spectrometer consists of a reflection grating (600 lines/mm), a focusing lens L7, and a high-speed CMOS camera (Photron 1024PCI). The collinear reference and sample beams are dispersed by the grating before reaching the camera where the spatial and spectral components are measured along the two axis of the detector. Furthermore, for each lateral position, the spectral data is resampled evenly in wavenumber space, numerically compensated for dispersion, and Fourier transformed to get the depth-resolved phase and amplitude information of the sample.
Figure 2 shows a 2-D surface profile of a HeLa cell measured using the line-field quantitative phase microscope. For imaging, the center of the line focus beam was focused on the cell surface and the sample was displaced using a motorized linear translational stage (step size ~100 nm) in a direction orthogonal to the line focus beam. A 2-D interferogram was acquired at each step of the linear stage. The measured interferograms were processed to calculate the 2-D surface profile of the HeLa cell as shown in Fig. 2. A total phase of more than 100 radians was measured with respect to the glass coverslip, illustrating 5 µm total cell height assuming that the average index of the cell was 1.37 [4].