Using complementary optical microscopy techniques provides more detailed insight into biological samples. However, misinterpretation can occur by temporal discrepancies due to differences in temporal resolution and switching imaging modalities. Here, we demonstrated multimodal imaging of cryofixed cells using Raman and fluorescence structured illumination microscopy (SIM). Cryofixation preserves structures and chemical states of samples in their near-native states, allowing multimodal imaging without artifacts caused by temporal discrepancy. We demonstrated multimodal imaging of cryofixed HeLa cells stained with an actin probe, where Raman microscope visualized cytochromes, proteins and lipids, and SIM visualized fluorescence-labelled actin filaments.
Raman microscopy provides a variety of insights into molecular composition, chemical state, and environmental conditions in biological samples. However, biological imaging with Raman microscopy have faced challenges such a low signal-to-noise ratio, mainly due to the low scattering efficiency of Raman scattering. To overcome this limitation, we developed a cryo-Raman microscope integrated with a cryostat capable of rapid freezing of biological samples and low-temperature Raman imaging. The spatiotemporal cryofixation of biological samples allows long exposure measurements to accumulate signals without photodamage. We observed both reduction of photobleaching in resonant Raman scattering of cytochromes in cryofixed HeLa cells, and the preservation of redox states of cytochromes in rat heart tissue by cryofixation.
We developed a fast Raman spectroscopic discrimination system based on a slit-scanning confocal microscope and machine learning. The speed of discrimination was improved by reducing the number of measurements, without measuring all points in the field of view. During discrimination, the system continues to evaluate the spectra already obtained, which guarantees the accuracy of the discrimination and enables early detection of anomalies by optimizing the measurement positions. We performed discrimination using a mixture of polystyrene (PS) and polymethyl methacrylate (PMMA) microbeads as a sample to mimic cancer tissue and that of fatty liver tissue using mouse liver tissue samples. The results showed that the discrimination was about 2-11 times faster than that by slit scanning confocal microscopy.
Raman microscopy enables us to obtain molecular information in biological samples but has suffered from low signal-to-noise ratio (SNR) due to low of Raman scattering cross-section. Here we developed a cryo-stat equipped Raman microscope for low temperature measurement, allowing long time accumulation of Raman signals. We confirmed the SNR improvement in Raman imaging of cryofixed HeLa cells without photodamage under long time observation at low temperature. The reduction of photobleaching in resonant Raman scattering of carotenoid and cytochrome significantly increases the SNR, demonstrated by 7-color high SNR Raman imaging with multiple Raman tags, including EdU, MitoBADY, and alkyne-tagged Coenzyme Q (AltQ2). AltQ2 is a mobile small molecule that cannot be fixed by chemical fixation.
KEYWORDS: Raman spectroscopy, Light sources and illumination, Machine learning, Medical research, Random forests, Microscopy, Microscopes, Engineering, Diagnostics, Decision trees
We propose a method that combines high-speed Raman imaging with a machine learning technique, multi-armed bandit, to achieve rapid and accurate identification of samples under observation. First, our method dvides the field of view of the sample into small sections, and it returns either ’positive’ or ’negative’ based on whether the sections with high anomaly indices exceed a certain proportion. Moreover, the points to be measured are determined dynamically and automatically generating a series of optimal illumination patterns.
We developed spontaneous Raman microscopy using Bandit algorithm to realize fast diagnosis of the existence of anomalies or not with guaranteeing accuracy. The algorithm evaluates obtained Raman spectra during measurement to judge if the diagnosis is completed with ensuring an allowance error rate that users decided and also to generate optimal illumination patterns for the next irradiation which are optimized to accelerate the detection of anomaly. We present our simulation and experimental studies to show that our system can accelerate more than a few tens times faster than line-scanning Raman microscopy which requires full scanning over all pixels.
We used only a narrow range, 1397-1501 cm-1, for high-throughput analysis of cancerous and noncancerous human cell lines by spontaneous Raman microscopy. With baseline-corrected cellwise spectra in this range, two cell lines were discriminated at accuracy higher than 90%. This narrowband measurement allowed reduction of the signal readout time by 24-folds in comparison to a correspondent wideband measurement detecting 536-3132 cm-1, enabling cell analysis at 2.5 cells/min. To further improve the throughput, we employed detector binning, which allowed reduction not just of the readout time but also of the signal accumulation time with maintaining signal-to-background ratio and the accuracy. Improvement of the imaging speed by this approach reached at 4-folds, enabling a high-throughout analysis at 10 cells/min.
Photoacoustic microscopy (PAM) is a biological visualization technique that can provide high spatial resolution and high contrast images of deep structures in living tissues. In PAM, the lateral resolution is determined by the size of the focus spot. Generally, because the wavefront aberration, due to the difference of refractive index between samples and air (water) and the shape of samples, enlarges the focus spot, obtained deep images are blurred or distorted. In order to solve this problem, we corrected the wavefront aberration occurring in samples using a transmissive liquid-crystal adaptive optics (AO) element. Our AO element consists of three liquid-crystal layers which have different ITO (indium tin oxide) patterns and are controlled independently. Their patterns are designed to correct the wavefront aberration suitable for a 40X waterimmersion objective lens. The AO element with transmissive and thin structure is easily installed in the PAM system. Also, our AO element is inexpensive and has low power consumption. In this study, we compared photoacoustic images obtained without and with the AO element for a USAF test target, polystyrene beads diffused in glycerol and various tissue specimens. As a result, we found that the use of transmissive AO element improves the lateral resolution and signal-tonoise ratio in PAM.
Conventional one-photon photoacoustic microscopy (PAM) utilizes high-frequency components of generated photoacoustic waves to improve the depth resolution. However, to obtain optically-high resolution in PAM in the depth direction, the use of high-frequency ultrasonic waves is to be avoided. It is because that the propagation distance is shortened as the frequency of ultrasonic waves becomes high. To overcome this drawback, we have proposed and developed two-photon photoacoustic microscopy (TP-PAM). Two-photon absorption occurs only at the focus point. TPPAM does not need to use the high-frequency components of photoacoustic waves. Thus, TP-PAM can improve the penetration depth while preserving the spatial resolution. However, the image acquisition time of TP-PAM is longer than that of conventional PAM, because TP-PAM needs to scan the laser spot both in the depth and transverse directions to obtain cross-sectional images. In this paper, we have introduced a focus-tunable electrically-controlled liquid lens in TP-PAM. Instead of a mechanical stepping-motor stage, we employed electrically-controlled liquid lens so that the depth of the focus spot can be quickly changed. In our system, the imaging speed of TP-PAM using the liquid lens and one-axis stepping-motor stage was 10 times faster than that using a two-axis stepping-motor stage only. TP-PAM with focus-scanning head consisting of the liquid lens and stepping-motor stage will be a promising method to investigate the inside of living tissues.
Nerve-sparing surgery is essential to avoid functional deficits of the limbs and organs. Raman scattering, a label-free, minimally invasive, and accurate modality, is one of the best candidate technologies to detect nerves for nerve-sparing surgery. However, Raman scattering imaging is too time-consuming to be employed in surgery. Here we present a rapid and accurate nerve visualization method using a multipoint Raman imaging technique that has enabled simultaneous spectra measurement from different locations (n=32) of a sample. Five sec is sufficient for measuring n=32 spectra with good S/N from a given tissue. Principal component regression discriminant analysis discriminated spectra obtained from peripheral nerves (n=863 from n=161 myelinated nerves) and connective tissue (n=828 from n=121 tendons) with sensitivity and specificity of 88.3% and 94.8%, respectively. To compensate the spatial information of a multipoint-Raman-derived tissue discrimination image that is too sparse to visualize nerve arrangement, we used morphological information obtained from a bright-field image. When merged with the sparse tissue discrimination image, a morphological image of a sample shows what portion of Raman measurement points in arbitrary structure is determined as nerve. Setting a nerve detection criterion on the portion of “nerve” points in the structure as 40% or more, myelinated nerves (n=161) and tendons (n=121) were discriminated with sensitivity and specificity of 97.5%. The presented technique utilizing a sparse multipoint Raman image and a bright-field image has enabled rapid, safe, and accurate detection of peripheral nerves.
The peripheral nervous system plays an important role in motility, sensory, and autonomic functions of the human body. Preservation of peripheral nerves in surgery, namely nerve-sparing surgery, is now promising technique to avoid functional deficits of the limbs and organs following surgery as an aspect of the improvement of quality of life of patients. Detection of peripheral nerves including myelinated and unmyelinated nerves is required for the nerve-sparing surgery; however, conventional nerve identification scheme is sometimes difficult to identify peripheral nerves due to similarity of shape and color to non-nerve tissues or its limited application to only motor peripheral nerves. To overcome these issues, we proposed a label-free detection technique of peripheral nerves by means of Raman spectroscopy. We found several fingerprints of peripheral myelinated and unmyelinated nerves by employing a modified principal component analysis of typical spectra including myelinated nerve, unmyelinated nerve, and adjacent tissues. We finally realized the sensitivity of 94.2% and the selectivity of 92.0% for peripheral nerves including myelinated and unmyelinated nerves against adjacent tissues. Although further development of an intraoperative Raman spectroscopy system is required for clinical use, our proposed approach will serve as a unique and powerful tool for peripheral nerve detection for nerve-sparing surgery in the future.
Accurate and rapid evaluation of lymph node metastasis is required in tumor staging and the decision of treatment strategy.
General intraoperative pathological evaluation, however, takes at least a few tens of minutes or longer for metastasis
diagnosis. 5-aminolevulinic acid (5-ALA)-based fluorescence diagnosis is a solution for accurate and ultrarapid diagnosis
of malignant lesions. 5-ALA-based diagnosis evaluates fluorescence intensity of a fluorescent metabolite of 5-ALA,
protoporphyrin IX (PPIX); however, the fluorescence of PPIX is often affected by autofluorescence of tissue
chromophores, such as collagen and flavins. To enhance the accuracy of the diagnosis of malignant lesions based on the
PPIX fluorescence, elimination of the autofluroescence is required. In this study, we proposed and experimentally
demonstrated background-free PPIX fluorescence estimation method by simplified and optimized multispectral imaging.
To realize background-free PPIX fluorescence estimation, we computationally optimized observation wavelength regions
in terms of minimizing prediction error of PPIX fluorescence intensity in the presence of typical chromophores, collagen
and flavins. We verified the fundamental detection capability of our method by using known-chemical mixtures.
Furthermore, we applied our method to lymph node metastasis, and successfully realized background-free
histopathological evaluation of metastatic lesions of lymph node metastasis. Our results confirmed the potential of the
background-free estimation method of PPIX fluorescence for 5-ALA-based fluorescence diagnosis of malignant lesions,
and we expect this method to be beneficial for intraoperative and rapid cancer diagnosis.
To improve the penetration depth in photoacoustic microscopy while preserving high spatial resolution, we have proposed two-photon absorption-induced photoacoustic microscopy (TP-PAM). However, in tissue imaging, unwanted one-photon photoacoustic signals impair the image constructed from the two-photon photoacoustic signals, because the cross-section of two-photon absorption is smaller than that of one-photon absorption. To overcome this drawback, it is important to enhance (or extract) only the photoacoustic signals generated by two-photon absorption. In this study, to improve the detection selectivity and efficiency of two-photon photoacoustic signals, we investigated the dependence of TP-PAM signal intensity and image quality on the detection frequency range and excitation pulse duration in detail. The comparison among photoacoustic signals generated by optical pulses with various pulse durations (femtosecond to sub-nanosecond) enabled us to find that, the shorter the pulse duration is, the higher the generation efficiency of two-photon photoacoustic signals is. We also applied the confocal configuration between optical (excitation) and acoustic (detection) foci to TP-PAM. The optimization of the pulse duration, frequency filtering and confocal configuration improves the selectivity and efficiency of the TP-PAM signal. Such improvements can reduce the photon number required to obtain TP-PAM images and thus make the imaging speed faster and avoid tissue damage.
The peripheral nervous system plays an important role in motility, sensory, and autonomic functions of the human body. Preservation of peripheral nerves in surgery is essential for improving quality of life of patients. To preserve peripheral nerves, detection of ne peripheral nerves that cannot be identi ed by human eye or under white light imaging is necessary. In this study, we sought to provide a proof-of-principle demonstration of a label-free detection technique of peripheral nerve tissues against adjacent tissues that employs spontaneous Raman microspectroscopy. A line-illumination confocal Raman microscope was used for the experiment. A laser operating at the wavelength of 532 nm was used as an excitation laser light. We obtained Raman spectra of peripheral nerve, brous connective tissue, skeletal muscle, blood vessel, and adipose tissue of Wistar rats, and extracted speci c spectral features of peripheral nerves and adjacent tissues. By applying multivariate image analysis, peripheral nerves were clearly detected against adjacent tissues without any preprocessing neither xation nor staining. These results suggest the potential of the Raman spectroscopic observation for noninvasive and label-free nerve detection, and we expect this method could be a key technique for nerve-sparing surgery.
Recently autofluorescence imaging (AFI) endoscopy, visualizing tissue fluorescence in combination with reflected light,
has been adopted as a technique for detecting neoplasms in the colon and other organs. However, autofluorescence
colonoscopy is not infallible, and improvement of the detection method can be expected to enhance the performance.
Colonic mucosa contains metabolism-related fluorophores, such as reduced nicotinamide adenine dinucleotide, which
may be useful for visualizing neoplasia in autofluorescence endoscopy. We examined sliced cross-sections of
endoscopically resected tubular adenomas under a microscope. Fluorescence images acquired at 365-nm excitation
(F365ex) and 405-nm excitation (F405ex), and reflectance images acquired at 550 nm (R550) were obtained. Fluorescence
ratio (F365ex/F405ex) images and reflectance/fluorescence ratio (R550/F405ex) images were calculated from the acquired
images. The fluorescence ratio images could distinguish adenomatous mucosa from normal mucosa more clearly than the
reflectance/fluorescence ratio images. The results showed that the autofluorescence ratio imaging is a potential technique
for increasing the diagnostic power of autofluorescence endoscopy.
Estimating the distribution of myocardial fibrosis after myocardial infarct is important for appropriate therapeutic
planning. Here, we applied a Raman confocal microscope equipped with slit scanner for molecular tissue imaging of rat
infarcted hearts. Raman spectra of the cytoplasm of cardiomyocytes included the resonance Raman bands at 751, 1130
and 1582 cm-1 arising mainly from reduced b- and c- type cytochromes. Raman spectra of fibrotic tissues at the borderzone
of old myocardial infarct were highly consistent with that of collagen type I. Based on these findings, we
successfully obtained Raman tissue images of a cardiomyocyte and surrounding collagen at the cellular level.
Tumor invasion to the peritoneum is a poor prognostic factor in cancer patients. Accurate diagnosis of disseminated
peritoneal tumors is essential to accurate cancer staging. To date, peritoneal washing cytology during laparotomy has
been used for diagnosis of peritoneal dissemination of gastrointestinal cancer, but its sensitivity has not been satisfactory.
Thus, a more direct approach is indispensable to detect peritoneal dissemination in vivo. Fluorescein diacrylate (FDAcr)
is an esterase-sensitive fluorescent probe derived from fluorescein. In cancer cells, fluorescent fluorescein generated by
exogenous application of FDAcr selectively deposits owing to its stronger hydrolytic enzyme activity and its lower
leakage rate. We examined whether FDAcr can specifically detect disseminated peritoneal tumors in athymic nude
mouse models. Intraperitoneally administered FDAcr revealed disseminated peritoneal microscopic tumors not readily
recognized on white-light imaging. These results suggest that FDAcr is a useful probe for detecting disseminated
peritoneal tumors.
In recent years, various types of molecular imaging technologies have been developed, but many of them require probes
and may have some influence on the distribution of the target molecules. In contrast, Raman microscopic analysis is
effective for molecular identification of materials, and molecular imaging methods employing Raman scattering light can
be applied to living organisms without use of any exogenous probes. Unfortunately, Raman microscopic imaging is
rarely used in the biomedical field due to the weakness of Raman signals. When the conventional Raman microscopes
are used, the acquisition of an image of a cell usually takes several hours. Recently, a slit-scanning confocal Raman
microscope has been developed. It can acquire images of living cells and tissues with faster scanning speed. In this study,
we used the slit-scanning confocal Raman microscope (RAMAN-11) to image the distribution of a drug in living cells.
We could acquire images of the distribution of an anticancer reagent in living cells within several minutes. Since the
wavelength of Raman scattering light is determined by the frequency of molecular vibration, the in situ mapping of the
intracellular drugs without use of a probe is possible, suggesting that laser Raman imaging is a useful method for a
variety of pharmacokinetic studies.
Autofluorescence endoscopy is a promising modality for diagnosis of colonic tumors. This article discusses the
origin of autofluorescence of the normal colon. Excised normal colons were analyzed by using fluorescence
stereomicroscopy and a fluorescence-lifetime microscopy system. Fluorescence images showed that the mucosa had
stronger autofluorescence than the submucosa. The results of fluorescence-lifetime measurement showed that
nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FAD) might be responsible for the
autofluorescence of the colonic epithelia. Our results suggest that the mucosal autofluorescence generates by NADH and
FAD was an important source of the green autofluorescence.
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