Time-stretch microscopy had mostly been operated in bright-field (BF) imaging mode in the longer wavelength range (~1500 nm), and is thus incapable of revealing high-contrast and high-resolution morphology of the transparent cells—hindering accurate cell recognition and screening. As a result, effective use of time-stretch imaging had been limited to microparticle or cell screening in high-speed flow with trivial size and shape differences, especially when the targeted cells are labeled with contrast agents [6, 7]. Similar to the classical optical imaging modalities, the time-stretch image quality, which is typically characterized by image resolution and image contrast, is still compromised by the higher imaging speed. To enlarge the scope of time-stretch imaging applications, particularly in biomedical diagnostics, we have recently introduced a new generation of time-stretch imaging, dubbed ATOM, for obtaining label-free, high-contrast images of the transparent cells at ultrahigh speed, and with sub-cellular resolution.

ATOM relies on the same spectral-encoding strategy as its BF time-stretch imaging counterpart. The key difference is that ATOM generates the enhanced phase-gradient contrast in the time-stretch image based on a simple asymmetric detection scheme after the spectral-encoding step (see Fig. 4). The phase-gradient contrast results in three-dimensional (3D) appearance in the image, resembling the contrast-enhancement effect in Schlieren imaging [8]. Moreover, by time-multiplexing two ATOM images with opposite phase-gradient contrasts, we further obtain two different contrasts from the same specimen: one with differential (enhanced) phase-gradient contrast and another with absorption contrast, simultaneously (Fig. 2). This method decouples the phase-gradient information from absorption, resulting in further enhancement of the image contrast. Together with operating in the 1 μm wavelength regime, a more favorable spectral window for biophotonic applications as opposed to the telecommunications wavelength band (~1500 nm) used by most of the previously reported time-stretch imaging systems, the ATOM system is able to achieve higher diffraction-limited resolution and high-contrast cellular time-stretch imaging. Similar to the original time-stretch imaging, the detection sensitivity in ATOM is not compromised by the high-speed operation because of the in-line optical image amplification—a feature rarely implemented in typical optical microscopy. We have demonstrated the unique capability of ATOM in visualizing the detailed cellular morphology (e.g. normal human blood cells from fresh blood and human leukemic monocytes) without contrast agent in ultrafast microfluidic flow (up to ~10 m/s), which is yet to be demonstrated in the existing time-stretch imaging modality (Fig. 2) [8]. The achieved flow speed here is equivalent to an imaging throughput of ~ 100,000 cells/s. ATOM thus represents a significant advancement in bringing the essential imaging metrics—high resolution and high contrast—to high-speed time-stretch imaging, making it a genuinely appealing platform for realizing high-throughput image-based cellular assays.

The functional and structural information of the biological cells and tissues can commonly be inferred by the correlated image contrasts. While exogenous fluorescent labels are the well-acclaimed and thus the prevalent contrast agent used in many applications, they are however not always ideal in view of the complication introduced by cytotoxicity and photobleaching. In contrast, endogenous image contrast, e.g., absorption and scattering, could sometimes serve as the effective intrinsic biomarker without the need for label or stain and the associated laborious sample preparation procedures. Among different label-free optical imaging modalities, quantitative phase-contrast imaging (QPI) is proven to be an effective approach to scavenge different useful intrinsic information about the cells and tissues. By mapping the optical phase shift across the transparent cells or tissues, QPI can provide not only high image contrast non-invasively, but also quantitative evaluation of cellular information (e.g., cell volume, mass, refractive index, stiffness, membrane tension) at nanometer scale based on the optical phase [14, 15]. The scattering properties of the cells/tissues can also be extracted from the optical phase [16–19]. All information derived from QPI can then be used as the intrinsic biomarkers for cellular identification and understanding the corresponding physiological information, such as disease state of cells/tissues. Notably, refractive index of cell nuclei serves a distinct indicator for label-free detection of cancer cells with high sensitivity [20, 21].

Motivated by that time-stretch imaging is intrinsically compatible with label-free phase-contrast imaging, it has been demonstrated iTS microscopy for quantitative phase contrast tissue and cellular imaging at an ultrahigh imaging scan rate as high as tens of MHz, with nanometer precision [13, 15]. Again, iTS microscopy employs the same spectral-encoding imaging scheme as ordinary BF time-stretch imaging. The key difference is that an additional interferometer (e.g., Michelson interferometer) is required in iTS microscopy such that the spectrally-encoded pulsed beam from the sample arm is interfered with its uncoded replica from the reference arm. The interfered pulses then undergo optical time-stretch which generates the temporal interferograms. The quantitative phase information can then be extracted from the interferograms by the phase-retrieval and unwrapping algorithms routinely used in classical QPI (e.g., Goldstein’s algorithm) (Fig. 3). It should be emphasized again that the quantitative phase information has long been known as valuable biomarkers of label-free cellular bioassay [13]. However, the use of these intrinsic biomarkers for large-scale screening has almost been impossible because of the limited imaging throughput of the existing technologies.