Fluorescence microscopy is an essential technique in biomedical research because it can reveal morphological details of complex organisms. Over the past few decades, the spatial resolution of fluorescence microscopy has dramatically improved with super-resolution techniques, contributing significantly to the evolution of life science. On the other hand, temporal resolution (imaging speed) has been overlooked, and its applications have been limited. Therefore, we are developing high-speed fluorescence microscopy techniques to open up new frontiers in life science. Here, we will introduce our recently developed high-speed fluorescence microscopy techniques and their applications to imaging flow cytometry and 3D imaging. We will also discuss the future of high-speed fluorescence imaging that will lead to integrating photonics, informatics, and life science.
We demonstrate ultrafast fluorescence lifetime imaging microscopy (FLIM) based on frequency-division multiplexing. As a proof-of-concept demonstration, we obtained images with fluorescence intensity and lifetime contrasts of MCF-7 breast cancer cells stained by SYTO16 at a record high frame rate of 16,000 fps, which is 100 times higher than that of previously reported FLIM techniques. Our method is expected to expand the utility of FLIM to quantitative analysis of rapid intracellular dynamics and high-throughput cell screening based on fluorescence lifetime images.
Frequency-division-multiplexed laser-scanning fluorescence microscopy is a powerful imaging method for biological tissues that enables an imaging speed of >10,000 frames/s. Despite its unprecedented high speed, its large-scale implementation that includes a bulky and unstable Mach-Zehnder interferometer has hampered its practical applications, especially in biomedical studies. Here we present a compact implementation of frequency-division-multiplexed microscopy to overcome this issue. The compactness is enabled by introducing an inline interferometer for generating an excitation beam array. In this setup, the laser beam is separated and recombined with small beam separation angles (<2°) by optical components such as acousto-optic deflectors or Wollaston prisms, thus implementing an interferometer with a relay lens system and drastically downsizing the setup. Compared with our previous setup with a Mach-Zehnder interferometer, the footprint of the optical setup for the excitation beam generation was downsized from ~20 cm x 70 cm to ~130 cm x 2.54 cm (defined by one-inch optical components used in the setup). Furthermore, our design concept allows for an ultra-compact implementation (~10 cm x 1 cm) by using custom optical components and omitting the relay lens systems. As a proof-of-concept demonstration, we obtained two-color (fluorescence and brightfield) images of Euglena gracilis cells (autofluorescent) and MCF-7 cells (fluorescence from nuclei stained by SYTO16) at a scanning speed of 0.84 m/s, which corresponds to a frame rate of 15,300 frames/s at a 55-μm field of view in the scanning direction. By virtue of the wide modulation bandwidth of the excitation beam (200 MHz), it is also possible to measure fluorescence lifetimes of target fluorophores, leading to potential applications for fluorescence lifetime imaging (FLIM).
While fluorescence imaging flow cytometry is a promising method for high-throughput single-cell analysis, it has not been suitable for analysis of large populations of cells (e.g., blood samples) due to its low imaging sensitivity at a high cell throughput. Here we present fluorescence imaging flow cytometry with an ultrahigh imaging sensitivity, which is enabled by virtual motion freezing. In this method, we prepare a wide-field imaging system with a CMOS camera and scan images of flowing cells by a scanning device, such as a polygon scanner, equipped in the imaging system so that the motion of the cells is canceled in the imaging plane, thus significantly extending the exposure time of the camera without suffering from motion blur. Additionally, we scan a loosely focused excitation beam during the exposure time of the camera in the direction opposite to the cell flow using a beam scanner such as an acousto-optic deflector, which significantly reduces motion cancellation errors caused by the image distortion of the imaging system and hence allows further extension of the exposure time. Consequently, our method improves imaging sensitivity by a factor of ~1,000 compared with a conventional wide-field excitation method, enabling acquisition of microscopy-grade images of fast flowing cells. As a proof-of-concept, we obtained fluorescence images of nuclei of murine white blood cells stained by SYTO16 at a flow speed of 1 m/s (corresponding to a cell throughput of 10,000 cells/s assuming the 100-μm cell spacing) and determined the population of nuclear lobulation from the high-signal-to-noise-ratio images obtained.
Fluorescence imaging flow cytometry offers highly accurate analysis of a large number of cells compared with conventional flow cytometry by virtue of its imaging capability. Unfortunately, the throughput of conventional fluorescence imaging flow cytometers is limited to ~1,000 cells/sec, which is one order of magnitude lower than that of conventional non-imaging flow cytometers. This is due to the low data transfer rate of a CCD image sensor with a time-delay integration technique employed to achieve sufficient sensitivity for fluorescence imaging of fast flowing cells. Replacing the CCD image sensor with a CMOS image sensor can potentially overcome the throughput limitation by virtue of its high data transfer rate, but critically sacrifice the imaging sensitivity because the time-delay integration cannot be employed to current CMOS image sensors. Here we present a fluorescence imaging flow cytometer that achieves comparable throughput and sensitivity with non-imaging flow cytometers. It is enabled by high-energy-density light-sheet excitation of flowing cells on a mirror-embedded PDMS-based microfluidic chip and by fluorescence image acquisition with a CMOS image sensor. The light-sheet excitation allows us obtain fluorescence images of flowing cells at a speed of >1 m/s, corresponding to a high throughput of >10,000 cells/sec. To show its biomedical utility, we use it combined with machine learning to demonstrate accurate screening of white blood cells and real-time identification of cancer cells in blood.
Fluorescence imaging flow cytometry is an emerging technique for analyzing a large number of cells with high accuracy over conventional flow cytometry by virtue of its imaging capability. Unfortunately, the cell throughput of conventional fluorescence imaging flow cytometers (~1,000 cells/sec) is much lower than that of standard non-imaging flow cytometers due to the use of a CCD image sensor having a limited data transfer rate, making it difficult to analyze a large population of cells. Here we report our experimental demonstration of highly accurate classification of microalgae with a frequency-division-multiplexed confocal imaging flow cytometer (IFC) that enables imaging of every single microalgal cell with an unprecedentedly high throughput of 20,000 cells/sec. The high-speed imaging performance of the IFC is enabled by employing frequency-division-multiplexed confocal microscopy, which uses a sensitive single-pixel photodetector such as an avalanche photodetector or a photomultiplier tube to obtain images of flowing cells. We stained three species of microalgae (Chlamydomonas reinhardtii, Haematococcus lacustris, and Euglena gracilis) with SYTO16 and obtained three-color images of the cells (bright-field, fluorescence staining of nuclei, and autofluorescence of chlorophyll). We extracted 243-dimensional features from each three-color image and employed a support vector machine to classify the cells with the obtained multi-dimensional data. As a result, the cells were successfully classified with an accuracy of 99.7%. Due to the IFC’s multi-color imaging capability with an unprecedentedly high throughput, our technique has a wide variety of potential applications other than microalga classification, such as accurate blood screening and liquid biopsy.
We present methods of fluorescence confocal microscopy that enable unprecedentedly high frame rate of > 10,000 fps. The methods are based on a frequency-division multiplexing technique, which was originally developed in the field of communication engineering. Specifically, we achieved a broad bandwidth (~400 MHz) of detection signals using a dual- AOD method and overcame limitations in frame rate, due to a scanning device, by using a multi-line focusing method, resulting in a significant increase in frame rate. The methods have potential biomedical applications such as observation of sub-millisecond dynamics in biological tissues, in-vivo three-dimensional imaging, and fluorescence imaging flow cytometry.
Clinical examination crucially relies on the ability to quickly examine large tissue areas and rapidly zoom in to regions of interest. Skin lesions often show irregularity in color and appearance in general, especially when they start to progress towards malignancy. Large field of view (FOV) and automatic translation of the imaging area are critical in the assessment of the entire lesion. Imaging of limited FOVs of the lesion can easily result in false negative diagnosis. We present a multiphoton microscope based on two-photon excited fluorescence and second-harmonic generation that images FOVs of about 0.8 mm2 (without stitching adjacent FOVs) at speeds of 10 frames/second (800 x 800 pixels) with lateral and axial resolutions of 0.5 μm and 2.5 μm, respectively. The main novelty of this instrument is the design of the scan head, which includes a fast galvanometric scanner, relay optics, a beam expander and a high NA objective lens. We optimized the system based on the Olympus 25x, 1.05NA water immersion lens, that features a long working distance of 1 mm. Proper tailoring of the beam expander, which consists of the scan and tube lens elements, enables scaling of the FOV. The design criteria include a flat wavefront of the beam, minimum field curvature, and suppressed spherical aberrations. All aberrations in focus are below the Marechal criterion of 0.07λ rms for diffraction-limited performance. We demonstrate the practical utility of this microscope by ex-vivo imaging of wide FOVs in normal human skin.
Fluorescence imaging using radiofrequency-tagged emission (FIRE) is an emerging technique that enables higher imaging speed (namely, temporal resolution) in fluorescence microscopy compared to conventional fluorescence imaging techniques such as confocal microscopy and wide-field microscopy. It works based on the principle that it uses multiple intensity-modulated fields in an interferometric setup as excitation fields and applies frequency-division multiplexing to fluorescence signals. Unfortunately, despite its high potential, FIRE has limited imaging speed due to two practical limitations: signal bandwidth and signal detection efficiency. The signal bandwidth is limited by that of an acousto-optic deflector (AOD) employed in the setup, which is typically 100-200 MHz for the spectral range of fluorescence excitation (400-600 nm). The signal detection efficiency is limited by poor spatial mode-matching between two interfering fields to produce a modulated excitation field. Here we present a method to overcome these limitations and thus to achieve higher imaging speed than the prior version of FIRE. Our method achieves an increase in signal bandwidth by a factor of two and nearly optimal mode matching, which enables the imaging speed limited by the lifetime of the target fluorophore rather than the imaging system itself. The higher bandwidth and better signal detection efficiency work synergistically because higher bandwidth requires higher signal levels to avoid the contribution of shot noise and amplifier noise to the fluorescence signal. Due to its unprecedentedly high-speed performance, our method has a wide variety of applications in cancer detection, drug discovery, and regenerative medicine.
A novel multi-level scheme using optical phase is proposed. It overcomes drawbacks in conventional multi-level
schemes and greatly enhances capacity and transfer rate of microholographic optical discs. We demonstrate its feature of
high signal-to-noise ratio.
A system using homodyne detection to read out signals from optical discs was simplified by using a high-coherence
laser light source. The quality of the readout signal with the simplified system was substantially greater than with
conventional detection.
We experimentally demonstrated improvement in optical disc readout signal quality by homodyne detection. We
introduced an optical phase servo system as an alternative to the phase-diversity detection scheme for stabilization of the
interference signal in homodyne detection, which resulted in further enhancement of readout signal quality.
Experimental results indicate that at least a 16-layer recordable disc can be reliably read out with a jitter of less than 8%
at a 1-mW read power by homodyne detection. The estimated amplification of signal amplitude was 13 times.
Optical signal amplification by a homodyne detection scheme is proposed and experimentally
demonstrated. We estimated that this scheme improved the signal-to-noise ratio of an 8-layer 3x
read-speed Blu-ray Disc (BD) by more than 20 dB.
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