Low-cost,chromatic confocal endomicroscope for cellular imaging in vivo

We have developed a low-cost, chromatic confocal endomicroscope (CCE) that can image a cross-section of the tissue at cellular resolution. In CCE, a custom miniature objective lens was used to focus different wavelengths into different tissue depths. Therefore, each tissue depth was encoded with the wavelength. A custom miniature spectrometer was used to spectrally-disperse light reflected from the tissue and generate cross-sectional confocal images. The CCE prototype had a diameter of 9.5 mm and a length of 68 mm. Measured resolution was high, 2 µm and 4 µm for lateral and axial directions, respectively. Effective field size was 468 µm. Preliminary results showed that CCE can visualize cellular details from cross-sections of the tissue in vivo down to the tissue depth of 100 µm.     

Reflectance confocal endomicroscope with optical axial scanning for in vivo imaging of the oral mucosa

This paper presents the design and evaluation of a reflectance confocal laser endomicroscope using a miniature objective lens within a rigid probe in conjunction with an electrically tunable lens for axial scanning. The miniature lens was characterized alone as well as in the endoscope across a 200 µm axial scan range using the tunable lens. The ability of the confocal endoscope to probe the human oral cavity is demonstrated by imaging of the oral mucosa in vivo. The results indicate that reflectance confocal endomicroscopy has the potential to be used in a clinical setting and guide diagnostic evaluation of biological tissue.OCIS codes: (220.3620) Lens system design, (350.3950) Micro-optics, (170.1790) Confocal microscopy, (170.2150) Endoscopic imaging, (120.3890) Medical optics instrumentation, (170.3880) Medical and biological imaging     

Achromatized endomicroscope objective for optical biopsy

Currently, researchers and clinicians lack achromatized endomicroscope objectives that are as narrow as biopsy needles. We present a proof-of-concept prototype that validates the optical design of an NA0.4 objective. The objective, built with plastic lenses, has a 0.9 mm clear aperture and is achromatized from 452 nm to 623 nm. The objective’s measured Strehl ratio is 0.74 ± 0.05 across a 250 μm FOV. We perform optical sectioning via structured illumination through the objective while capturing fluorescence images of breast carcinoma cells stained with proflavine and cresyl violet. This technology has the potential to improve optical biopsies and provide the next step forward in cancer diagnostics.OCIS codes: (080.3620) Lens system design, (220.0220) Optical design and fabrication, (170.2520) Fluorescence microscopy, (170.3880) Medical and biological imaging, (220.1920) Diamond machining     

Dynamic real-time subtraction of stray-light and background for multiphoton imaging

We introduce a new approach to reduce uncorrelated background signals from fluorescence imaging data, using real-time subtraction of background light. This approach takes advantage of the short fluorescence lifetime of most popular fluorescent activity reporters, and the low duty-cycle of ultrafast lasers. By synchronizing excitation and recording, laser-induced multiphoton fluorescence can be discriminated from background light levels with each laser pulse. We demonstrate the ability of our method to – in real-time – remove image artifacts that in a conventional imaging setup lead to clipping of the signal. In other words, our method enables imaging under conditions that in a conventional setup would yield corrupted data from which no accurate information can be extracted. This is advantageous in experimental setups requiring additional light sources for applications such as optogenetic stimulation.     

A high speed multifocal multiphoton fluorescence lifetime imaging microscope for live-cell FRET imaging

We demonstrate diffraction limited multiphoton imaging in a massively parallel, fully addressable time-resolved multi-beam multiphoton microscope capable of producing fluorescence lifetime images with sub-50ps temporal resolution. This imaging platform offers a significant improvement in acquisition speed over single-beam laser scanning FLIM by a factor of 64 without compromising in either the temporal or spatial resolutions of the system. We demonstrate FLIM acquisition at 500 ms with live cells expressing green fluorescent protein. The applicability of the technique to imaging protein-protein interactions in live cells is exemplified by observation of time-dependent FRET between the epidermal growth factor receptor (EGFR) and the adapter protein Grb2 following stimulation with the receptor ligand. Furthermore, ligand-dependent association of HER2-HER3 receptor tyrosine kinases was observed on a similar timescale and involved the internalisation and accumulation or receptor heterodimers within endosomes. These data demonstrate the broad applicability of this novel FLIM technique to the spatio-temporal dynamics of protein-protein interaction.OCIS codes: (180.0180) Microscopy, (170.2520) Fluorescence microscopy, (180.4315) Nonlinear microscopy, (040.1240) Arrays, (090.1970) Diffractive optics     

Fluorescence-guided optical coherence tomography imaging for colon cancer screening: a preliminary mouse study

A new concept for cancer screening has been preliminarily investigated. A cancer targeting agent loaded with a near-infrared (NIR) dye was topically applied on the tissue to highlight cancer-suspect locations and guide optical coherence tomography (OCT) imaging, which was used to further investigate tissue morphology at the micron scale. A pilot study on ApcMin mice has been performed to preliminarily test this new cancer screening approach. As a cancer-targeting agent, poly(epsilon-caprolactone) microparticles (PCLMPs), labeled with a NIR dye and functionalized with an RGD (argenine-glycine-aspartic acid) peptide, were used. This agent recognizes the α(ν)β(3) integrin receptor (ABIR), which is over-expressed by epithelial cancer cells. The contrast agent was administered topically in vivo in mouse colon. After incubation, the animals were sacrificed and fluorescence-guided high resolution optical coherence tomography (OCT) imaging was used to visualize colon morphology. The preliminary results show preferential staining of the abnormal tissue, as indicated by both microscopy and laser-induced fluorescence imaging, and OCT's capability to differentiate between normal mucosal areas, early dysplasia, and adenocarcinoma. Although very preliminary, the results of this study suggest that fluorescence-guided OCT imaging might be a suitable approach for cancer screening. If successful, this approach could be used by clinicians to more reliably diagnose early stage cancers in vivo.     

Design of an endomicroscope including a resonant fiber-based microprobe dedicated to endoscopic polarimetric imaging for medical diagnosis

We report on a novel endomicroscope, to the best of our knowledge, designed for achieving full 4×4 Mueller polarimetric images of biological tissues through a fiber endoscope for medical diagnosis. The polarimetric technique is based on a previously published two-wavelength differential method (TWDM). A key component of the endomicroscope is a resonant fiber-based microprobe including a highly-selective fiber Bragg grating (FBG), free of detrimental polarimetric effects, photo written in the core of the fiber, near the output face. By means of the TWDM, and using the specially designed microprobe (diameter 2.9 mm, length 30 mm), full Mueller images of 250×250 pixels were produced at the rate of 1 image/2 s through a 2 m single mode fiber, paving the way to in vivo applications in polarimetric endomicroscopy.     

Confocal foveated endomicroscope for the detection of esophageal carcinoma

By mimicking the variable resolution of the human eye, a newly designed foveated endomicroscopic objective shows the potential to improve current endoscopic based techniques of identifying abnormal tissue in the esophagus and colon. The prototype miniature foveated objective is imaged with a confocal microscope to provide large field of view images combined with a high resolution central region to rapidly observe morphological structures associated with cancer development in a mouse model.OCIS codes: (170.2150) Endoscopic imaging, (350.3950) Micro-optics, (170.3880) Medical and biological imaging, (170.3890) Medical optics instrumentation, (170.1790) Confocal microscopy     

Intravital imaging of the kidney using multiparameter multiphoton microscopy

Intravital optical microscopy provides a powerful means of studying the cell biology in the most physiologically relevant setting. The ability of multiphoton microscopy to collect optical sections deep into biological tissues has opened up the field of intravital microscopy to high-resolution studies of multiple organs. Presented here are examples of how two-photon microscopy can be applied to intravital studies of kidney physiology and the study of disease processes. These include studies of cell vitality and apoptosis, fluid transport, receptor-mediated endocytosis, blood flow, and leukocyte trafficking. Efficient two-photon excitation of multiple fluorophores permits comparison of multiple probes and simultaneous characterization of multiple parameters. Two-photon microscopy can now provide a level of investigation previously unattainable in intravital microscopy, enabling kinetic analyses and physiological studies of the organs of living animals with subcellular resolution. Therefore, application of this technology will provide direct visualization of organ-specific and cell-specific responses to an array of stimuli and therapeutic approaches, enhancing our understanding and treatment of disease processes.     

Measurements of multiphoton action cross sections for multiphoton microscopy

We report quantitative measurements of two-, three-, and four-photon excitation action cross sections of several commonly used fluorophores and fluorescent proteins at three different excitation wavelengths of 800 nm, 1300 nm, and 1680 nm. The measured cross section values are consistent with simple quantum mechanic estimations. These values indicate that the optimum repetition rate for deep tissue 3-photon microscopy is approximately 1 to 2 MHz. We further demonstrate that it is feasible to perform 4-photon fluorescence microscopy of GFP labeled microglia in mouse brain in vivo at 1700 nm. 4-photon excitation increases the accessibility of fluorophores at the long wavelength spectral window of 1700 nm.OCIS codes: (190.0190) Nonlinear optics, (190.4180) Multiphoton processes     

Shot noise limits on binary detection in multiphoton imaging

Much of fluorescence-based microscopy involves detection of if an object is present or absent (i.e., binary detection). The imaging depth of three-dimensionally resolved imaging, such as multiphoton imaging, is fundamentally limited by out-of-focus background fluorescence, which when compared to the in-focus fluorescence makes detecting objects in the presence of noise difficult. Here, we use detection theory to present a statistical framework and metric to quantify the quality of an image when binary detection is of interest. Our treatment does not require acquired or reference images, and thus allows for a theoretical comparison of different imaging modalities and systems.     

Deep learning-based high-speed,large-field,and high-resolution multiphoton imaging

Multiphoton microscopy is a formidable tool for the pathological analysis of tumors. The physical limitations of imaging systems and the low efficiencies inherent in nonlinear processes have prevented the simultaneous achievement of high imaging speed and high resolution. We demonstrate a self-alignment dual-attention-guided residual-in-residual generative adversarial network trained with various multiphoton images. The network enhances image contrast and spatial resolution, suppresses noise, and scanning fringe artifacts, and eliminates the mutual exclusion between field of view, image quality, and imaging speed. The network may be integrated into commercial microscopes for large-scale, high-resolution, and low photobleaching studies of tumor environments.     

Imaging carious dental tissues with multiphoton fluorescence lifetime imaging microscopy

In this study, multiphoton excitation was utilized to image normal and carious dental tissues noninvasively. Unique structures in dental tissues were identified using the available multimodality (second harmonic, autofluorescence, and fluorescence lifetime analysis) without labeling. The collagen in dentin exhibits a strong second harmonic response. Both dentin and enamel emit strong autofluorescence that reveals in detail morphological features (such as dentinal tubules and enamel rods) and, despite their very similar spectral profiles, can be differentiated by lifetime analysis. Specifically, the carious dental tissue exhibits a greatly reduced autofluorescence lifetime, which result is consistent with the degree of demineralization, determined by micro-computed tomography. Our findings suggest that two-photon excited fluorescence lifetime imaging may be a promising tool for diagnosing and monitoring dental caries.     

Imaging carious dental tissues with multiphoton fluorescence lifetime imaging microscopy

In this study, multiphoton excitation was utilized to image normal and carious dental tissues noninvasively. Unique structures in dental tissues were identified using the available multimodality (second harmonic, autofluorescence, and fluorescence lifetime analysis) without labeling. The collagen in dentin exhibits a strong second harmonic response. Both dentin and enamel emit strong autofluorescence that reveals in detail morphological features (such as dentinal tubules and enamel rods) and, despite their very similar spectral profiles, can be differentiated by lifetime analysis. Specifically, the carious dental tissue exhibits a greatly reduced autofluorescence lifetime, which result is consistent with the degree of demineralization, determined by micro-computed tomography. Our findings suggest that two-photon excited fluorescence lifetime imaging may be a promising tool for diagnosing and monitoring dental caries.OCIS codes: (170.1850) Dentistry, (170.3880) Medical and biological imaging, (170.6935) Tissue characterization, (170.6920) Time-resolved imaging     

Retinal cell imaging in myopic chickens using adaptive optics multiphoton microscopy

Abnormal eye growth induced by visual deprivation can modify the structure and density of the retinal cells. We have used an adaptive optics multiphoton microscope to image photoreceptors (PRs) and ganglion cells (GCs) at different retinal locations in unstained retinas of chicken eyes with about 10D of myopia and their normal-sighted fellow eyes. In all samples, the local averaged inter-PR distance increased with eccentricity. No significant differences in PR density were found between control and myopic eyes. GC density declined in myopic eyes compared to control eyes and the inter-cell distance increased. In normal eyes, the size of the GC cell bodies increased approximately two-fold between the area centralis and the peripheral retina. In myopic eyes, this trend was preserved but the GC bodies were larger at each retinal location, compared to control eyes. Obviously, GC morphology is changing when the retinal area is enlarged in myopic eyes.OCIS codes: (170.3880) Medical and biological imaging, (180.4315) Nonlinear microscopy, (110.1080) Active or adaptive optics, (170.4470) Ophthalmology     

Signal enhancement in multiphoton imaging by the use of coated glass substrates

In nonlinear optical imaging of biological specimens, more than half of the generated luminescence signal is lost, when signal collection is performed in the epi-illuminated geometry. In this study, we enhanced the collected luminescence signal by the use of alternating multiply-coated layers of tantalum pentoxide (Ta₂O₅) and silicon dioxide (SiO₂) on standard microscope cover glasses that has high transmission in the near-infrared wavelength region and high reflection of the visible, luminescence signal. Our coating is biocompatible, allows visual examination of the specimens and optimize collection of the luminescence signal. We demonstrated this approach on a number of specimens including sulforhodamine solution, fluorescence microspheres, and labeled 3T3 cells. In all cases, the use of coated cover glass enhanced signal, optimally by a factor of about 2. Image analysis of labeled 3T3 cells also shows signal enhancement did not contribute to additional photobleaching. Our results show that properly designed coated cover glass can enhance detected signal in multiphoton microscopy and result in improved image quality.OCIS codes: (300.6410) Spectroscopy, multiphoton; (000.2700) General science     

In-vivo imaging of psoriatic lesions with polarization multispectral dermoscopy and multiphoton microscopy

Psoriasis is a skin autoimmune disease characterized by hyperkeratosis, hyperproliferation of the epidermis and dilatation of dermal papillary blood vessels. Healthy skin (5 volunteers) and psoriatic lesions (3 patients) were visualized in vivo, with high contrast and resolution, with a Polarization Multispectral Dermoscope and a Multiphoton Microscope. Psoriatic features were identified and quantified. The effective diameter of the superficial blood vessels was measured at 35.2 ± 7.2 μm and the elongated dermal papillae had an effective diameter of 64.2 ± 22.6 μm. The methodologies developed could be employed for quantitative diagnostic purposes and furthermore serve as a monitoring method of the effect of personalized treatments.OCIS codes: (180.4315) Nonlinear microscopy, (170.1870) Dermatology, (100.2980) Image enhancement, (170.6900) Three-dimensional microscopy, (170.6510) Spectroscopy, tissue diagnostics     

Real-time histology in liver disease using multiphoton microscopy with fluorescence lifetime imaging

Conventional histology with light microscopy is essential in the diagnosis of most liver diseases. Recently, a concept of real-time histology with optical biopsy has been advocated. In this study, live mice livers (normal, with fibrosis, steatosis, hepatocellular carcinoma and ischemia-reperfusion injury) were imaged by MPM-FLIM for stain-free real-time histology. The acquired MPM-FLIM images were compared with conventional histological images. MPM-FLIM imaged subsurface cellular and subcellular histopathological hallmarks of live liver in mice models at high resolution. Additional information such as distribution of stellate cell associated autofluorescence and fluorescence lifetime changes was also gathered by MPM-FLIM simultaneously, which cannot be obtained from conventional histology. MPM-FLIM could simultaneously image and quantify the cellular morphology and microenvironment of live livers without conventional biopsy or fluorescent dyes. We anticipate that in the near future MPM-FLIM will be evaluated from bench to bedside, leading to real-time histology and dynamic monitoring of human liver diseases.OCIS codes: (180.4315) Nonlinear microscopy, (170.3880) Medical and biological imaging, (170.6510) Spectroscopy, tissue diagnostics