Cell mechanics plays an important role in a number of complex biological processes including but not limited to metabolism, cell signaling, growth, and wound healing. The most common approaches to assess cellular rheology including atomic force microscopy (AFM), optical and magnetic tweezers, pipette aspiration, and electric field deformation are invasive and thus are not suitable especially for nuclear biomechanical studies in intact cells. To this end, interferometric microscopy is an appealing approach to quantify cell biomechanics by measuring nanometer scale axial motions of the cell membrane. The LBRC has successfully used transmission-type interferometric microscopy techniques to study biomechanical properties of RBCs in different pathophysiological conditions. To extend this approach to complex eukaryotic cells, LBRC aims to develop next-generation interferometric biomechanical assays based on 3D-resolved measurement of sub-nanometer-scale membrane motions to quantify plasma/nuclear membrane mechanics. Figure 1: Schematic of the confocal reflectance interferometric microscope. P: Polarizer, L1-L5: Convex Lenses, PBS: Polarizing Beam Splitter, DMD: Digital Micro-mirror Device, TL: Tube Lens, OBJ: Microscope Objective, A: Analyzer, M: Mirror and G: grating.
Scanning confocal reflection phase microscopy
Over the years, a variety of depth-resolved wide-field reflection phase microscopy systems have been developed at LBRC. These include systems based on temporally low-coherent source such as mode-locked Ti:Sapphire laser or a superluminescent diode [1], temporally and spatially low-coherent source such as white light [2], and dynamic speckle illumination [3]. These systems have not been utilized to study biomechanics of nucleic envelope either due to lack of desired depth sectioning [1], lower imaging speed [2], or limited phase measurement sensitivity [3]. To address this challenge, LBRC has recently developed a confocal reflectance interferometric microscope [4]. The microscope utilizes a high-speed digital micro-mirror device (DMD-1) to generate a scanning confocal spots grid on the sample, as shown in Fig. 1. The back-scattered optical field from the specimen is projected back to DMD-1 for confocal detection. Wide field-of-view imaging is achieved by raster scanning the confocal pinhole pattern. A near common-path interferometer, similar to diffraction phase microscopy, is implemented for optical phase detection with high stability. Specifically, a second DMD placed at the Fourier plane of L4 allows generating sample and reference beams for off-axis holography. A CMOS camera is used to acquire single-shot full-field interferogram at 70 Hz. Typically, the measured single-shot off-axis interferograms can be analyzed to compute the phase maps φ(x,y;t) of the specimen using Hilbert transform. To improve the measurement accuracy, LBRC has also developed a method to characterize the effect of strong reflector in vicinity of the cell to obtain correct phase reconstruction [4].
To demonstrate the utility of the system, nucleic envelope and plasma membrane fluctuations in mouse embryonic stem cells have been measured. Figure 2(a) shows an interferogram recorded at the cell-dish interface, where the optical signal is primarily dominated by the contribution from the dish. Measurements were also performed for the background signal (outside cell region) as well as at the nucleic envelope and plasma membrane interfaces. Figure 2(b) shows plots of instantaneous fluctuation amplitudes at single spatial location of cell-dish interface as well as for the nucleic envelope and plasma membrane interfaces. The root-mean-square (rms) fluctuation amplitudes were also computed for 10 different spatial locations of corresponding interfaces and plotted in Fig. 2(c). The nucleic membrane median rms fluctuation amplitude (~2.59 nm) was found to be smaller than that of the plasma membrane (~9.93 nm), indicating higher stiffness of nuclear membrane.
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