Due to the chromatic dispersion properties inherent in all optical materials, even the best designed multi-spectral objective will exhibit residual chromatic aberration effect. Here we show that the aberration correction ability of Fourier Ptychographic Microscopy (FPM) is well matched and well suited for post-image acquisition correction of these effects to render in-focus images. We show that an objective with significant spectral focal shift (up to 0.02 μm/nm) and spectral field curvature (up to 0.05 μm/nm drift at off-axis position of 800μm) can be computationally corrected to render images with effectively null spectral defocus and field curvature. This approach of combining optical objective design and computational microscopy provides a good strategy for high quality multi-spectral imaging over a broad spectral range, and eliminating the need for mechanical actuation solutions.
Demands of multi-well plate readers has been on the rise for drug discovery and cell line development applications, as it can obtain fluorescence, absorbance, and morphology information from cell cultures grown in tens to hundreds of conditions. Existing systems typically only house one camera, requiring slow mechanical actuation to cover a large area on the multi-well plate, or to sacrifice speed for area, forgoing the precious spatial information. We mitigate the time-resolution trade off with the Fourier ptychographic microscopy (FPM) technology by simultaneously capturing 96 high-resolution phase images (>20,000 cells per plate) with CMOS-based cameras with custom-designed microscope objectives. By illuminating the samples with a permutation of lighting conditions, we achieve synthetic numerical aperture (NA) of 0.3 at an extended depth-of-field of 20 micrometer for at most 96 conditions at one time. Unlike our previous invention of the 6-well plate reader (EmSight), the same illumination condition can be shared among adjacent cameras. Therefore, image acquisition and data transfer can be performed in a massively parallel manner. Along with computational acceleration with graphical processing units (GPUs), all these approaches reduces the plate-to-image turnover from hours to minutes – an eight-fold reduction in time over existing mechanical-scanning plate readers. In addition to providing phase imaging, the system is also capable of fluorescence imaging at the native resolution of the objectives. We anticipate that our high-throughout 96-camera imaging system will help advance the high content analysis of cell cultures beyond hundreds of test conditions, thus facilitates more in-depth characterization of biological screens and drug testing.
Optical time-stretch microscopy enables cellular images captured at tens of MHz line-scan rate and becomes a potential tool for ultrafast dynamics monitoring and high throughput screening in scientific and biomedical applications. In time-stretch microscopy, to achieve the fast line-scan rate, optical fibers are used as the pulse-stretching device that maps the spectrum of a light pulse to a temporal waveform for fast digitization. Consequently, existing time-stretch microscopy is limited to work at telecom windows (e.g. 1550 nm) where optical fiber has significant pulse-stretching and small loss. This limitation circumscribes the potential application of time-stretch microscopy.
Here we present a new optical time-stretch imaging modality by exploiting a novel pulse-stretching technique, free-space angular-chirp-enhanced delay (FACED), which has three benefits: (1) Pulse-stretching in FACED generates substantial, reconfigurable temporal dispersion in free-space with low intrinsic loss at visible wavelengths; (2) Pulse-stretching in FACED inherently provides an ultrafast all-optical laser-beam scanning mechanism for time-stretch imaging. (3) Pulse-stretching in FACED can be wavelength-invariant, which enables time-stretch microscopy implemented without spectral-encoding.
Using FACED, we demonstrate optical time-stretch microscopy with visible light (~700 nm). Compared to the prior work, bright-field time-stretch images captured show superior contrast and resolution, and can be effectively colorized to generate color time-stretch images. More prominently, accessing the visible spectrum regime, we demonstrate that FACED enables ultrafast fluorescence time-stretch microscopy. Our results suggest FACED could unleash a wider scope of applications that were once forbidden with the fiber based time-stretch imaging techniques.
Based on the interferometric or holographic approaches, recent QPM techniques provide quantitative-phase information, e.g cell volume, dry mass and optical scattering properties for label-free cellular physical phenotyping. These approaches generally rely on iterative phase-retrieval algorithms to obtain quantitative-phase information, which are computationally intensive. Moreover, current QPM techniques can only offer limited image acquisition rate by using CMOS/CCD image sensors, these two limitations hinder QPM for high-throughput quantitative image-based single-cell analysis in real-time. To this end, we demonstrate an interferometry-free quantitative phase microscopy developed on a new generation of time-stretch microscopy, asymmetric-detection time-stretch optical microscopy (ATOM), which is coined quantitative ATOM (Q-ATOM) - featuring an unprecedented cell measurement throughput together with the assorted intrinsic optical phenotypes (e.g. angular light scattering profile) and the derived physical properties of the cells (e.g. cell size, dry mass density etc.). Based on a similar concept to Schlieren imaging, Q-ATOM retrieves quantitative-phase information through multiple off-axis light-beam detection at a line-scan rate of <10 MHz - a speed unachievable by any existing QPM techniques. Phase retrieval in Q-ATOM relies on a non-iterative method, significantly reducing the computational complexity of the technique. It is a particularly important feature which facilitates real-time continuous label-free single-cell analysis in Q-ATOM. With the use of a non-interferometric configuration, we demonstrate ultrafast Q-ATOM of mouse chondrocytes and hypertrophic chondrocytes in ultrafast microfluidic flow with sub-cellular resolution at an imaging throughput equivalent to ~100,000 cells/sec without image blur. This technique shows a great potential for ultrahigh throughput label-free image-based single-cell biophysical phentotyping.
Optical time-stretch imaging entails a stringent requirement of state-of-the-art high-speed data acquisition unit in order to preserve high image resolution at an ultrahigh frame rate — hampering the widespread application of such technology. We here propose a pixel super-resolution (pixel SR) technique tailored for time-stretch imaging that can relax the sampling rate requirement. It harnesses a concept of equivalent-time sampling, which effectively introduces sub-pixel shifts between frames. It involves no active opto-mechanical subpixel-shift control and any additional hardware. We present the system design rules and a proof-of-principle experiment which restores high-resolution images at a relaxed sampling rate of 5 GSa=s.
Fluorescence imaging using radio frequency-multiplexed excitation (FIRE) has emerged to enable an order-of-magnitude higher frame rate than the current technologies. Similar to all high-speed realtime imaging modalities, FIRE inherently generates massive image data continuously. While this technology entails high-throughput data sampling, processing, and storage in real-time, strategies in data compression on the fly is also beneficial. We here report that it is feasible to exploit the radio frequency-multiplexed excitation scheme in FIRE for implementing compressed sensing (CS) without any modification of the FIRE system. We numerically demonstrate that CS-FIRE can reduce the effective data rate by 95% without severe degradation of image quality.
Quantitative phase imaging (QPI) has been proven to be a powerful tool for label-free characterization of biological specimens. However, the imaging speed, largely limited by the image sensor technology, impedes its utility in applications where high-throughput screening and efficient big-data analysis are mandated. We here demonstrate interferometric time-stretch (iTS) microscopy for delivering ultrafast quantitative phase cellular and tissue imaging at an imaging line-scan rate >20 MHz—orders-of-magnitude faster than conventional QPI. Enabling an efficient time-stretch operation in the 1-μm wavelength window, we present an iTS microscope system for practical ultrafast QPI of fixed cells and tissue sections, as well as ultrafast flowing cells (at a flow speed of up to 8 m/s). To the best of our knowledge, this is the first time that time-stretch imaging could reveal quantitative morphological information of cells and tissues with nanometer precision. As many parameters can be further extracted from the phase and can serve as the intrinsic biomarkers for disease diagnosis, iTS microscopy could find its niche in high-throughput and high-content cellular assays (e.g., imaging flow cytometry) as well as tissue refractometric imaging (e.g., whole-slide imaging for digital pathology).
Continuing desire for higher-speed laser scanning fluorescence microscopy (LSFM) and progressive advancement in ultrafast and sensitive photodetectors might imply that our conventional understanding of LSFM is not adequate when approaching to the intrinsic speed limit — fluorescence lifetime. In this regard, we here revisit the theoretical framework of LSFM and evaluate its general performance in lifetime-limited and noise-limited regimes. Our model suggests that there still exists an order-of-magnitude gap between the current LSFM speed and the intrinsic limit. An imaging frame rate of > 100 kHz could be viable with the emerging laser-scanning techniques using ultrafast wavelength-swept sources, or optical time-stretch.
Optical imaging based on time-stretch process has recently been proven as a powerful tool for delivering ultra-high
frame rate (< 1MHz) which is not achievable by the conventional image sensors. Together with the capability of optical
image amplification for overcoming the trade-off between detection sensitivity and speed, this new imaging modality is
particularly valuable in high-throughput biomedical diagnostic practice, e.g. imaging flow cytometry. The ultra-high
frame rate in time-stretch imaging is attained by two key enabling elements: dispersive fiber providing the time-stretch
process via group-velocity-dispersion (GVD), and electronic digitizer. It is well-known that many biophotonic
applications favor the spectral window of ~1μm. However, reasonably high GVD (< 0.1 ns/nm) in this range can only be
achieved by using specialty single-mode fiber (SMF) at 1μm. Moreover, the ultrafast detection has to rely on the state-of-
the-art digitizer with significantly wide-bandwidth and high sampling rate (e.g. <10 GHz, <40 GS/s). These stringent
requirements imply the prohibitively high-cost of the system and hinder its practical use in biomedical diagnostics. We
here demonstrate two cost-effective approaches for realizing time-stretch confocal microscopy at 1μm: (i) using the
standard telecommunication SMF (e.g. SMF28) to act as a few-mode fiber (FMF) at 1μm for the time-stretch process,
and (ii) implementing the pixel super-resolution (SR) algorithm to restore the high-resolution (HR) image when using a
lower-bandwidth digitizer. By using a FMF (with a GVD of ~ 0.15ns/nm) and a modified pixel-SR algorithm, we can
achieve time-stretch confocal microscopy at 1μm with cellular resolution (~ 3μm) at a frame rate 1 MHz.
Access to the requested content is limited to institutions that have purchased or subscribe to SPIE eBooks.
You are receiving this notice because your organization may not have SPIE eBooks access.*
*Shibboleth/Open Athens users─please
sign in
to access your institution's subscriptions.
To obtain this item, you may purchase the complete book in print or electronic format on
SPIE.org.
INSTITUTIONAL Select your institution to access the SPIE Digital Library.
PERSONAL Sign in with your SPIE account to access your personal subscriptions or to use specific features such as save to my library, sign up for alerts, save searches, etc.