Speckle-field reflection phase microscopy

Using a broadband source is a straightforward way to achieve depth-sectioning in a reflection phase microscope. Another approach to achieve depth-sectioning is to utilize dynamic speckle illumination, which is also offers high lateral and axial resolution [1]. By introducing twin copies of an identical time-varying speckle-field into the two arms of an interferometer, depth-resolved amplitude and phase images of an object can be acquired through the automatic correlation process offered by the interferometric measurement. The quick decorrelation nature of 3-D speckle-fields allows us to achieve confocal equivalent depth selectivity.

Dynamic speckle phase microscopy

Figure 1. (a) Schematic diagram of the experimental setup. D: diffuser, M: Mirror, PBS: polarizing beam splitter, HWP: half wave plate, QWP: quarter wave plate, OLR and OLS: objective lenses for reference and sample arms, G: grating, Pα>: polarizer with α-degree rotation. Multiple diffraction orders from the grating other than ±1 are omitted. (b), (c) Intensity distribution of the speckle field from reference and sample arms, respectively, with no path length difference. (d), (e) Interference pattern at the camera with stationary and rotating diffuser, respectively.

Figure 1(a) shows the schematic of dynamic speckle phase microscope. A mode-locked Ti:Sapphire laser (λ0 = 800nm, Δλ = 17nm) is used as a broadband light source. The collimated laser beam illuminates a rotating ground glass diffuser, which generates a dynamic speckle field. Next, the speckle field is delivered to a Linnik-type interferometer where a polarization beam splitter (PBS) is used to split the incoming speckle field. A quarter wave plate is placed in each arm of the interferometer such that the polarization of back-scattered light from a reference mirror and a sample arm is rotated by 90o. This enables the combine the two fields at the port of the PBS and launched into a commonpath interferometer for detection. A diffraction grating is placed at the first conjugate image plane of the sample. In the Fourier plane of the grating, we place a cross-polarizers (P0, and P90) such that 1st (-1st) diffraction order contains only reference (sample) signal. Finally, in the second conjugate image plane, we place a camera after passing through a 45-degree polarizer (P45) to record the interference signal.

Due to the short temporal and spatial coherence lengths of the broadband speckle field, the proposed imaging system features superior lateral resolution, 520 nm, as well as high depth selectivity, 1.03 µm. The off-axis holography is implemented for single-shot, wide-field imaging suitable for high-speed phase measurements. In addition, the phase sensitivity for measuring axial motion, such as membrane dynamics, was up 21 mrad/nm which is more than 40 times better than that offered by transmission-mode quantitative phase microscope. Combined with the high-depth selectivity and the phase sensitivity, we have successfully identified the motion of the top membrane surface from that of the bottom in red blood cells as shown in Fig. 1(b-d). These features make this technique an ideal system for studying the dynamics of plasma and nuclear membrane and related biomechanical characteristics of complex eukaryotic cells [2].


  1. "Dynamic speckle illumination wide-field reflection phase microscopy," Optics Letters, 39(20), pp. 6062-5, (2009). [ Pubmed ]
  2. "Reflection phase microscopy using spatio-temporal coherence of light," Optica, 5(11), pp. 1468-1473, (2018). [ Pubmed ]