Label-free and dynamic monitoring of cell evolutions using wavelength-multiplexing surface plasmon resonance holographic microscopy.

Dynamic characterizations of intracellular variations and cell-substrate interactions under different external environments are critical to study cell behaviors and exploring biological applications. However, techniques that are capable of dynamically and simultaneously measuring multiple parameters of living cells in a wide-field manner have rarely been reported. Here, we present a wavelength-multiplexing surface plasmon resonance holographic microscopy which allows wide-field, simultaneous, and dynamic measurements of cell parameters, including cell-substrate distance and cytoplasm refractive index (RI). We use two lasers of 632.8 nm and 690 nm as light sources. Two beam splitters are employed in the optical setup to separately adjust the incident angle of two light beams. Then, surface plasmon resonance (SPR) can be excited for each wavelength under SPR angles. We demonstrate the advances of the proposed apparatus by systematically studying the cell responses to osmotic pressure stimuli from the environmental medium at the cell-substrate interface. The SPR phase distributions of the cell are firstly mapped at two wavelengths, then the cell-substrate distance and cytoplasm RI are retrieved using a demodulation method. Based on phase response differences between two wavelengths and monotonic changes of SPR phase with cell parameters, cell-substrate distance, and cytoplasm RI can be determined simultaneously using an inverse algorithm. This work affords a new optical measurement technique to dynamically characterize cell evolutions and investigate cell properties in various cellular activities. It may become a useful tool in the bio-medical and bio-monitoring areas.