Full-field Interferometric Confocal Microscopy using a VCSEL Array

Abstract

We present an interferometric confocal microscope using an array of 1200 VCSELs coupled to a multimode fiber. Spatial coherence gating provides ~18,000 continuous virtual pinholes allowing an entire en face plane to be imaged in a snapshot. This approach maintains the same optical sectioning as a scanning confocal microscope without moving parts, while the high power of the VCSEL array (~5 mW per laser) enables high-speed image acquisition with integration times as short as 100 µs. Interferometric detection also recovers the phase of the image, enabling quantitative phase measurements and improving the contrast when imaging phase objects.Confocal microscopy combines high-resolution with improved contrast and optical sectioning, making it an invaluable tool in developmental biology, clinical medicine, and optical metrology [1,2]. However, traditional confocal microscopes rely on raster scanning, which limits image acquisition speed and increases system complexity. With typical frame rates of a few Hz for 1000×1000 pixel frames, scanning confocal systems are susceptible to motion artifacts and poorly suited for the study of dynamic samples or use in vivo. While video-rate confocal microscopes have been demonstrated using very high speed scanning [3,4], the complexity required to achieve such high scan rates has limited their adoption. Given a fixed lateral scan rate, image acquisition speed can also be improved through parallelization. The most common approach to parallelization is through the use of an array of spatially separated pinholes (i.e. a Nipkow disk) [5]; however, this approach has obvious limitations, since the pinholes must be sufficiently separated to prevent cross-talk [6,7]. Researchers have also proposed sacrificing confocality in one-dimension (i.e. line-scan confocal microscopy [8][9][10]) to improve imaging speed, but cross-talk limits this approach to weakly scattering samples [11]. Spectral encoding can provide parallelization in one dimension without cross-talk by using a grating to map different wavelengths to a line on the sample; however, scanning in the second dimension is still required to form an image [12].An alternative approach to completely parallelize confocal image acquisition is to combine interferometric detection with spatial coherence gating [13][14][15][16][17] However, the main advantage of parallelization-faster image acquisition-has thus far been mitigated by the lack of an appropriate light source. Traditional low-spatial coherence sources (e.g. thermal sources or LEDs) lack sufficient power per mode for high-speed imaging, and methods to reduce the spatial coherence of lasers (e.g. rotating diffusers) require relatively long integration times to achieve sufficiently low spatial coherence.In this work, we use a recently developed vertical cavity surface emitting laser (VCSEL) array [18] which combines high power per mode with low spatial coherence to demonstrate full-field confocal image acquisition with integration times as short as 100 µsec. The VCSEL array con...

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

“…In this framing, the low spatial coherence is a consequence of using spatial multiplexing to perform highly parallelized confocal microscopy. We also note that the evidence suggests that the one-to-one relationship between source spatial mode and images resolution element can be relaxed at low to moderate levels of scattering [19,25].…”

“…In a prior work, we used a multimode fiber to deliver light from the VCSEL array to the sample [25]. In this work, we used bulk-optic Köhler illumination.…”

“…Although the general concept of spatial coherence gating has been described for some time in both narrowband and broadband interferometric imaging, recent advances in low spatial coherence sources make the concept attractive for high-speed (kHz regime) imaging. These advances are yielding light sources that are highly spatially multimode (low spatial coherence) while maintaining high-power per spatial mode [24][25][26][27][28].…”

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.…”

Modeling the depth-sectioning effect in reflection-mode dynamic speckle-field interferometric microscopy

“…Similarly, the spatial-coherence gating effect has also been utilized in interferometric microscopy to obtain depth-resolved measurements [23]. B. Redding et al reported a fullfield interferometric confocal imaging method, where the spatial coherence was manipulated by using a multimode fiber [24]. The measured spatial resolution however was limited to a few microns.…”