Ultrahigh-speed, phase-sensitive full-field interferometric confocal microscopy for quantitative microscale physiology

Abstract

We developed ultra-high-speed, phase-sensitive, full-field reflection interferometric confocal microscopy (FFICM) for the quantitative characterization of microscale biological motions and flows. We demonstrated 2D frame rates in excess of 1 kHz and pixel throughput rates up to 125 MHz. These fast FFICM frame rates were enabled by the use of a low spatial coherence, high-power laser source. Specifically, we used a dense vertical cavity surface emitting laser (VCSEL) array that synthesized low spatial coherence light through a large number of narrowband, mutually-incoherent emitters. Off-axis interferometry enabled single-shot acquisition of the complex-valued interferometric signal. We characterized the system performance (~2 μm lateral resolution, ~8 μm axial gating depth) with a well-known target. We also demonstrated the use of this highly parallelized confocal microscopy platform for visualization and quantification of cilia-driven surface flows and cilia beat frequency in an important animal model with >1 kHz frame rate. Such frame rates are needed to see large changes in local flow velocity over small distance (high shear flow), in this case, local flow around a single ciliated cell. More generally, our results are an important demonstration of low-spatial coherence, high-power lasers in high-performance, quantitative biomedical imaging.

Complex lasers with controllable coherence

“…The coherent noise from a highly scattering sample is strongly suppressed by the low spatial coherence illumination 88 . The ultrahigh-speed, full-field holographic confocal microscopy has been applied to in vivo quantitative studies of microscale physiology 89 .…”

Crosstalk-free volumetric in vivo imaging of a human retina with Fourier-domain full-field optical coherence tomography

“…The first attempt to use the spatially incoherent illumination for FD-FF-OCT was made back in 2006 [25], but due to the camera and laser technology limitations at the time it was too slow for any practical use. Benefits of spatially incoherent sources were also recognized in other types of interferometric imaging techniques, where, for example, a multimode fiber [26,27], a rotating diffuser [28,29], or narrowband VCSEL arrays [30,31] were used to reduce the crosstalk. In general, laser-based spatially incoherent sources are also used in non-interferometric imaging techniques due to their greater brightness compared to other light sources [32][33][34][35][36].…”

Interferometric Scattering (iSCAT) Microscopy and Related Techniques

“…In the decade that followed right up to the present day we see a broad array of research efforts, drawn from numerous communities, exploring interferometric detection of single nano-objects such as viruses, DNA, microtubules, exosomes, and proteins [75][76][77][78][79][80][81][82][83][84][85][86][87][88][89][90][91]. Interestingly, interferometric microscopies are also flourishing in the general context of label-free imaging of cells and membranes even if nanoparticles are not at the center of attention [75,76,90,[92][93][94][95][96][97][98][99][100][101][102][103][104][105][106]. The underlying physics of these methods remains the same although a plethora of acronyms such as interference reflectance imaging sensing (IRIS) [77], rotating coherent scattering (ROCS) [98], interference plasmonic imaging (iPM) [88], coherent bright-field imaging (COBRI) [107], stroboscopic interference scattering imaging (stroboSCAT) [108], interferometric scattering mass spectrometry (iSCAMS) [109] are on the rise.…”