Bio-microrheology is the quantitative study of mechanical properties of live cells [1
]. Variations in mechanical properties are intrinsic indicators of ongoing cellular processes such as increase in elasticity of certain cancer cells [2
], change of membrane stiffness in malaria-infected red blood cells [3
], changes in cellular adhesion [4
], and so forth. The measurement of rheological properties of cell membranes is advantageous since it may also indirectly provide information on the internal structures of cell [1
]. A number of different techniques exist to assess membrane rheological properties of live cells. These include atomic force microscopy (AFM) [5
], optical and magnetic tweezers [6
], pipette aspiration [8
], electric field deformation [11
], and full-field transmission phase microscopy [12
]. Many of these methods are invasive and use large deformations that may lead to cell damage or cell’s response to mechanical strain rather than its intrinsic response. For point-measurement techniques such as AFM, the time scales to probe large surface areas of cell membrane are in minutes, preventing the study of high-speed cell membrane dynamics over a wider surface area. Full-field transmission phase microscopy has been successfully utilized to measure membrane flcutuations in red blood cells over a broad range of temporal and spatial frequencies [12
]. Furthermore, the measured membrane fluctuations can be used in appropriate mathematical models to calculate rheological properties of red blood cells [13
]. However, for most type of cells, which have complicated 3-D internal cellular structures, transmission-type optical techniques will not be suitable as they will probe a combination of membrane as well as bulk properties of cells that are difficult to decouple. In this context, properly designed reflection-based phase microscopy with depth-sectioning capability can play vital role to exclusively access the membrane dynamics of nucleated cells. Moreover, transmission mode techniques measure relative phase shift induced by the sample with respect to that by the medium. Thus, the measured phase shift is proportional to the refractive index difference, Δn
, between the sample and the medium. In contrast, reflection phase microscopy techniques yield phase measurement proportional to the index of refraction, n
, of the medium rather than the relative index, Δn
. Thus, reflection-based optical methods promise a 2n
advantage in measurement sensitivity over the transmission-based optical techniques.
Low-coherence interferometry is necessary to exclusively sample the reflection signal from the depth of interest. In the past, both spectral domain as well as time domain optical coherence tomography (OCT) based implementations of reflection phase microscopy have been reported [15
]. Joo et al.
and Choma et al.
have independently developed similar setups using self phase-referenced spectral domain phase microscopy setups using point illumination [15
]. Ellerbee et al.
used the phase sensitive OCT based configuration in [15
] to visualize the motion of intracellular structures [20
]. In recent past, we have designed and developed a quantitative phase microscope based on spectral domain OCT and line-field illumination [19
]. The line-field reflection phase microscope exploited low-coherent illumination and confocal gating to successfully obtain the surface profile of cell membrane with sub-nanometer axial resolution. Using the line-field approach, we demonstrated 1 kHz frame rate with more than hundred data points along the line illumination. The first full-field phase sensitive OCT was reported using swept-source OCT configuration, which required 1024 wavelength encoded images to generate a volume phase image [17
]. Moreover, the acquisition rate (25 ms integration time per wavelength) was not sufficient to observe cellular dynamics. In order to observe intrinsic membrane motion of living cells, Yamauchi et al.
developed a full-field time-domain reflection phase microscope based on phase shifting interferometry and captured sectional surface profile of living cells. But the time resolution was limited to 1.25 sec due to the need for taking multiple images [18
]. There was an attempt to use an off-axis digital holography with low-coherence source to take a full-field phase image in a single shot [21
]. But the tilting of reference mirror caused uneven interference contrast and thereby impeded full-field imaging.
In this paper, we present the first single-shot full-field reflection phase microscope based on low-coherence interferometry and off-axis interferometry. Its unique design provides the wavefront tilt in the reference beam such that it interferes with the sample beam across the whole field-of-view. The single-shot interferograms are processed to determine the optical phase of the beam reflected back from the sample under investigation, providing its surface profile without the need for raster or 1-D scanning. Since single-shot interferograms are required to retrieve sample phase, the amount of light returning from the cell and camera frame rate will define the speed of the surface imaging. We have demonstrated 1 kHz full-field imaging (2400 times faster than the work reported in Ref [18
].) to observe the membrane motion related to the thermal fluctuations in HeLa cells. The measured membrane fluctuations, which are typically on the order of a nanometer or less, can in turn be used to estimate mechanical properties of plasma membrane in nucleated cells using methods similar to that for erythrocytes [13