Pixel super-resolution in serial time-encoded amplified microscopy (STEAM)

Paper no. CTu3J.4We propose pixel super-resolution serial time-encoded amplified microscopy (STEAM) for achieves high speed and high-resolution imaging - relaxing the stringent requirement on the digitizer bandwidth while preserving the ultrahigh frame-rate (>MHz). © 2012 OSA.published_or_final_versio

Speed-dependent resolution analysis of ultrafast laser-scanning fluorescence microscopy

published_or_final_versio

Coherent laser source for high frame-rate optical time-stretch microscopy at 1.0 μm

We demonstrate a coherent picosecond pulsed fiber laser for the high frame-rate optical time-stretch microscopy at 1.0 μm. The spectrum of a picosecond pulsed laser is commonly broadened before the time-stretch imaging, which however will degrade its stability and coherence. As a result, it is required to enhance the degraded signal-to-noise ratio by averaging, which would compromise the frame rate on the other hand. Instead of pursuing such kind of spectrum-broadened picosecond pulsed laser sources, we propose a pulse train extracted directly from an all-normal dispersion mode-locked fiber laser with a rectangle-shaped optical spectrum. It delivers stable and coherent performance for the serial time-encoded amplified microscopy at 1.0 μm. With this robust picosecond pulsed laser, real-time stain-free flow imaging with a frame rate of 26.25 MHz and a spatial resolution of <; 2 μm is demonstrated. Featured with the compact configuration and good coherence property, it is a promising picosecond pulsed fiber laser source for the ultrafast interferometric time-stretch microscopy at 1.0 μm.published_or_final_versio

Revisit laser scanning fluorescence microscopy performance under fluorescence-lifetime-limited regime

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. © 2014 SPIE

Interferometric time-stretch microscopy for ultrafast quantitative cellular and tissue imaging at 1 μm

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-mum 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 ms). 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).published_or_final_versio

All-passive pixel super-resolution of time-stretch imaging

Based on image encoding in a serial-temporal format, optical time-stretch imaging entails a stringent requirement of state-of-the- art fast data acquisition unit in order to preserve high image resolution at an ultrahigh frame rate --- hampering the widespread utilities of such technology. Here, we propose a pixel super-resolution (pixel-SR) technique tailored for time-stretch imaging that preserves pixel resolution at a relaxed sampling rate. It harnesses the subpixel shifts between image frames inherently introduced by asynchronous digital sampling of the continuous time-stretch imaging process. Precise pixel registration is thus accomplished without any active opto-mechanical subpixel-shift control or other additional hardware. Here, we present the experimental pixel-SR image reconstruction pipeline that restores high-resolution time-stretch images of microparticles and biological cells (phytoplankton) at a relaxed sampling rate (approx. 2--5 GSa/s) --- more than four times lower than the originally required readout rate (20 GSa/s) --- is thus effective for high-throughput label-free, morphology-based cellular classification down to single-cell precision. Upon integration with the high-throughput image processing technology, this pixel-SR time- stretch imaging technique represents a cost-effective and practical solution for large scale cell-based phenotypic screening in biomedical diagnosis and machine vision for quality control in manufacturing.Comment: 17 pages, 8 figure

Intra-operative spectroscopic assessment of surgical margins during breast conserving surgery

Background: In over 20% of breast conserving operations, postoperative pathological assessment of the excised tissue reveals positive margins, requiring additional surgery. Current techniques for intra-operative assessment of tumor margins are insufficient in accuracy or resolution to reliably detect small tumors. There is a distinct need for a fast technique to accurately identify tumors smaller than 1 mm2 in large tissue surfaces within 30 min. Methods: Multi-modal spectral histopathology (MSH), a multimodal imaging technique combining tissue auto-fluorescence and Raman spectroscopy was used to detect microscopic residual tumor at the surface of the excised breast tissue. New algorithms were developed to optimally utilize auto-fluorescence images to guide Raman measurements and achieve the required detection accuracy over large tissue surfaces (up to 4 × 6.5 cm2). Algorithms were trained on 91 breast tissue samples from 65 patients. Results: Independent tests on 121 samples from 107 patients - including 51 fresh, whole excision specimens - detected breast carcinoma on the tissue surface with 95% sensitivity and 82% specificity. One surface of each uncut excision specimen was measured in 12–24 min. The combination of high spatial-resolution auto-fluorescence with specific diagnosis by Raman spectroscopy allows reliable detection even for invasive carcinoma or ductal carcinoma in situ smaller than 1 mm2. Conclusions: This study provides evidence that this multimodal approach could provide an objective tool for intra-operative assessment of breast conserving surgery margins, reducing the risk for unnecessary second operations

Optical time-stretch microscopy for ultrafast optofluidic imaging

Session - Biomedical and Life Sciences: (IN)BLS007 (Invited

Cellular imaging by time-stretch confocal microscopy in the 1 µm window

Frontier in Optics (FiO) 2012/Laser Science (LS) XXVIIIWe demonstrate, for the first time, optical time-stretch confocal microscopy in the 1 µm spectral window, which is the optimal diagnostic window for biophotonics, for single-shot high-speed (10 MHz) and high-resolution (< 2 µm) cellular imaging.link_to_OA_fulltex

Ultrafast Microfluidic Cellular Imaging by Optical Time Stretch

There is an unmet need in biomedicine for measuring a multitude of parameters of individual cells (i.e., high content) in a large population ef ciently (i.e., high throughput). This is particularly driven by the emerging interest in bringing Big-Data analysis into this arena, encompassing pathology, drug discovery, rare cancer cell detection, emulsion microdroplet assays, to name a few. This momentum is particularly evident in recent advancements in ow cytometry. They include scaling of the number of measurable colors from the labeled cells and incorporation of imaging capability to access the morphological information of the cells. However, an unspoken predicament appears in the current technologies: higher content comes at the expense of lower throughput, and vice versa. For example, accessing additional spatial information of individual cells, imaging ow cytometers only achieve an imaging throughput ~1000 cells/s, orders of magnitude slower than the non- imaging ow cytometers. In this chapter, we introduce an entirely new imaging platform, namely optical time-stretch microscopy, for ultrahigh speed and high contrast label-free single-cell (in a ultrafast micro uidic ow up to 10 m/s) imaging and analysis with an ultra-fast imaging line-scan rate as high as tens of MHz. Based on this technique, not only morphological information of the individual cells can be obtained in an ultrafast manner, quantitative evaluation of cellular information (e.g., cell volume, mass, refractive index, stiffness, membrane tension) at nanometer scale based on the optical phase is also possible. The technology can also be integrated with conventional uorescence measurements widely adopted in the non-imaging ow cytometers. Therefore, these two combinatorial and complementary measurement capabilities in long run is an attractive platform for addressing the pressing need for expanding the “parameter space” in high-throughput single-cell analysis. This chapter provides the general guidelines of constructing the optical system for time stretch imaging, fabrication and design on the micro uidic chip for ultrafast uidic ow, as well as the image acquisition and processing