SignificanceStimulated emission depletion (STED) is a powerful super-resolution microscopy technique that can be used for imaging live cells. However, the high STED laser powers can cause significant photobleaching and sample damage in sensitive biological samples. The dynamic intensity minimum (DyMIN) technique turns on the STED laser only in regions of the sample where there is fluorescence signal, thus saving significant sample photobleaching. The reduction in photobleaching allows higher resolution images to be obtained and longer time-lapse imaging of live samples. A stand-alone module to perform DyMIN is not available commercially.AimIn this work, we developed an open-source design to implement three-step DyMIN on a STED microscope and demonstrated reduced photobleaching for timelapse imaging of beads, cells, and tissue.ApproachThe DyMIN system uses a fast multiplexer circuit and inexpensive field-programmable gate array controlled by Labview software that operates as a stand-alone module for a STED microscope. All software and circuit diagrams are freely available.ResultsWe compared time-lapse images of bead samples using our custom DyMIN system to conventional STED and recorded a ∼46% higher signal when using DyMIN after a 50-image sequence. We further demonstrated the DyMIN system for time-lapse STED imaging of live cells and brain tissue slices.ConclusionsOur open-source DyMIN system is an inexpensive add-on to a conventional STED microscope that can reduce photobleaching. The system can significantly improve signal to noise for dynamic time-lapse STED imaging of live samples.
SignificanceIn vivo imaging and electrophysiology are powerful tools to explore neuronal function that each offer unique complementary information with advantages and limitations. Capturing both data types from the same neural population in the freely moving animal would allow researchers to take advantage of the capabilities of both modalities and further understand how they relate to each other.AimHere, we present a head-mounted neural implant suitable for in vivo two-photon imaging of neuronal activity with simultaneous extracellular electrical recording in head-fixed or fiber-coupled freely moving animals.ApproachA gradient refractive index (GRIN) lens-based head-mounted neural implant with extracellular electrical recording provided by tetrodes on the periphery of the GRIN lens was chronically implanted. The design of the neural implant allows for recording from head-fixed animals, as well as freely moving animals by coupling the imaging system to a coherent imaging fiber bundle.ResultsWe demonstrate simultaneous two-photon imaging of GCaMP and extracellular electrophysiology of neural activity in awake head-fixed and freely moving mice. Using the collected information, we perform correlation analysis to reveal positive correlation between optical and local field potential recordings.ConclusionSimultaneously recording neural activity using both optical and electrical methods provides complementary information from each modality. Designs that can provide such bi-modal recording in freely moving animals allow for the investigation of neural activity underlying a broader range of behavioral paradigms.
Significance: Three-photon (3P) microscopy significantly increases the depth and resolution of in vivo imaging due to decreased scattering and nonlinear optical sectioning. Simultaneous excitation of multiple fluorescent proteins is essential to studying multicellular interactions and dynamics in the intact brain.
Aim: We characterized the excitation laser pulses at a range of wavelengths for 3P microscopy, and then explored the application of tdTomato or mScarlet and EGFP for dual-color single-excitation structural 3P imaging deep in the living mouse brain.
Approach: We used frequency-resolved optical gating to measure the spectral intensity, phase, and retrieved pulse widths at a range of wavelengths. Then, we performed in vivo single wavelength-excitation 3P imaging in the 1225- to 1360-nm range deep in the mouse cerebral cortex to evaluate the performance of tdTomato or mScarlet in combination with EGFP.
Results: We find that tdTomato and mScarlet, expressed in oligodendrocytes and neurons respectively, have a high signal-to-background ratio in the 1300- to 1360-nm range, consistent with enhanced 3P cross-sections.
Conclusions: These results suggest that a single excitation wavelength source is advantageous for multiple applications of dual-color brain imaging and highlight the importance of empirical characterization of individual fluorophores for 3P microscopy.
Detection of circulating tumor cells with image cytometry is limited by the sensitivity and specificity of the biomarker panel. We collected confocal images of ~100,000 cells labeled for DNA, lipids, CD45, and Cytokeratin on a model system of MCF7 and WBCs representing disease positive, D+ and disease negative, D- populations. We computed spatial image metrics and performed multivariable regression and feature selection, increasing the separation of the D+ and D- populations to 7 standard deviations with detection limit of ~1 in 480. In conclusion, simple regression analysis holds promise to improve the separation of rare cells in cytometry applications.
Vagus nerve interfacing is of interest due to its central role in parasympathetic regulation of the visceral organs, as well as its modulatory effects on the brain, which have been shown to influence epilepsy, depression and migraines. Electrical vagus nerve stimulation (VNS) has shown therapeutic effect in humans, yet it lacks the specificity for controlling and studying targeted pathways. In contrast, optical techniques may enable axon-specific neuromodulation using genetically targeted opsin expression and spatial patterning of the photo-stimulus. In addition to light-activated stimulation, calcium-sensitive fluorescent reporters such as GCaMP6 present a pathway for axon-specific optical recording of activity. We demonstrate in vivo photo-stimulation and two-photon GCaMP6 fluorescence imaging in the vagus nerve using a novel GRIN lens-coupled nerve cuff in the anesthetized mouse. A pulsed near-IR laser (1040 nm, 300 fs) was modified by a spatial light modulator (SLM) in the Fourier plane and focused by the microscope objective through a GRIN relay lens to the cervical vagus nerve. By actuating the SLM, spatially selected regions of axons could be differentially stimulated within the nerve. Mouse vitals were monitored with a MouseOx suite and used to detect physiological changes in response to photo-stimulation. We were able to induce differential modulations in heart rate, respiratory rate, and blood-oxygen saturation upon photo-stimulation of selective spatial regions of the nerve. Additionally, we recorded two-photon GCaMP6 Ca2+ transients in vagal axons in response to both photo-stimulation and electrical stimulation.
We report a miniature head mounted two-photon fiber-coupled microscope (TP-FCM) for neuronal imaging with active axial focusing enabled using a miniature electrowetting lens. Full three-dimensional two-photon imaging of GCaMP6s showing individual neuron activity in multiple focal planes was achieved in a freely-moving mouse. Two-color simultaneous imaging of GFP and tdTomato fluorescence is demonstrated. Additionally, the axial scanning of the electrowetting lens allows dynamic control of tilt to the focal plane allowing rapid scanning of different regions of interest in three dimensions. Two-photon imaging allows increased penetration depth in tissue with a field-of-view of 240 μm diameter and 200 μm variable axial focus. The TP-FCM has a light-weight design (~4 g) and excellent image stability. TP-FCM with dynamic axial scanning provides a new capability to record from functionally distinct neuronal layers, opening up unique opportunities in neuroscience research.
Neural-machine interfaces using optogenetics are of interest due to their minimal invasiveness and potential for parallel read in and read out of activity. One possible biological target for such an interface is the peripheral nerve, where axonlevel imaging or stimulation could greatly improve interfacing with artificial limbs or enable neuron/fascicle level neuromodulation in the vagus nerve. Two-photon imaging has been successful in imaging brain activity using genetically encoded calcium or voltage indicators, but in the peripheral nerve, this is severely limited by scattering and aberrations from myelin. We employ a Shack-Hartman wavefront sensor and two-photon excitation guidestar to quantify optical scattering and aberrations in peripheral nerves and cortex. The sciatic and vagus nerves, and cortex from a ChAT-Cre ChR-eYFP transgenic mouse were excised and imaged directly. In peripheral nerves, defocus was the strongest aberration followed by astigmatism and coma. Peripheral nerve had orders of magnitude higher aberration compared with cortex. These results point to the potential of adaptive optics for increasing the depth of two-photon access into peripheral nerves.
Two-photon microscopy is a powerful tool of current scientific research, allowing optical visualization of structures below the surface of tissues. This is of particular value in neuroscience, where optically accessing regions within the brain is critical for the continued advancement in understanding of neural circuits. However, two-photon imaging at significant depths have typically used Ti:Sapphire based amplifiers that are prohibitively expensive and bulky. In this study, we demonstrate deep tissue two-photon imaging using a compact, inexpensive, turnkey operated Ytterbium fiber laser (Y-Fi, KM Labs). The laser is based on all-normal dispersion (ANDi) that provides short pulse durations and high pulse energies. Depth measurements obtained in ex vivo mouse cortex exceed those obtainable with standard two-photon microscopes using Ti:Sapphire lasers. In addition to demonstrating the capability of deep-tissue imaging in the brain, we investigated imaging depth in highly-scattering white matter with measurements in sciatic nerve showing limited optical penetration of heavily myelinated nerve tissue relative to grey matter.
We performed stimulated emission depletion (STED) imaging of isolated olfactory sensory neurons (OSNs) using a custom-built microscope. The STED microscope uses a single pulsed laser to excite two separate fluorophores, Atto 590 and Atto 647N. A gated timing circuit combined with temporal interleaving of the different color excitation/STED laser pulses filters the two channel detection and greatly minimizes crosstalk. We quantified the instrument resolution to be ∼81 and ∼44 nm, for the Atto 590 and Atto 647N channels. The spatial separation between the two channels was measured to be under 10 nm, well below the resolution limit. The custom-STED microscope is incorporated onto a commercial research microscope allowing brightfield, differential interference contrast, and epifluorescence imaging on the same field of view. We performed immunolabeling of OSNs in mice to image localization of ciliary membrane proteins involved in olfactory transduction. We imaged Ca2+-permeable cyclic nucleotide gated (CNG) channel (Atto 594) and adenylyl cyclase type III (ACIII) (Atto 647N) in distinct cilia. STED imaging resolved well-separated subdiffraction limited clusters for each protein. We quantified the size of each cluster to have a mean value of 88±48 nm and 124±43 nm, for CNG and ACIII, respectively. STED imaging showed separated clusters that were not resolvable in confocal images.
Circulating tumor cell (CTC) identification has applications in both early detection and monitoring of solid cancers. The rarity of CTCs, expected at ~1-50 CTCs per million nucleated blood cells (WBCs), requires identifying methods based on biomarkers with high sensitivity and specificity for accurate identification. Discovery of biomarkers with ever higher sensitivity and specificity to CTCs is always desirable to potentially find more CTCs in cancer patients thus increasing their clinical utility. Here, we investigate quantitative image cytometry measurements of lipids with the biomarker panel of DNA, Cytokeratin (CK), and CD45 commonly used to identify CTCs. We engineered a device for labeling suspended cell samples with fluorescent antibodies and dyes. We used it to prepare samples for 4 channel confocal laser scanning microscopy. The total data acquired at high resolution from one sample is ~ 1.3 GB. We developed software to perform the automated segmentation of these images into regions of interest (ROIs) containing individual cells. We quantified image features of total signal, spatial second moment, spatial frequency second moment, and their product for each ROI. We performed measurements on pure WBCs, cancer cell line MCF7 and mixed samples. Multivariable regressions and feature selection were used to determine combination features that are more sensitive and specific than any individual feature separately. We also demonstrate that computation of spatial characteristics provides higher sensitivity and specificity than intensity alone. Statistical models allowed quantification of the required sensitivity and specificity for detecting small levels of CTCs in a human blood sample.
Increasing interest in the role of lipids in cancer cell proliferation and resistance to drug therapies has motivated the need to develop better tools for cellular lipid analysis. Quantification of lipids in cells is typically done by destructive chromatography protocols that do not provide spatial information on lipid distribution and prevent dynamic live cell studies. Methods that allow the analysis of lipid content in live cells are therefore of great importance. Using micro-Raman spectroscopy and coherent anti-Stokes Raman scattering (CARS) microscopy, we generated a lipid profile for breast (T47D, MDA-MB-231) and prostate (LNCaP, PC3) cancer cells upon exposure to medroxyprogesterone acetate (MPA) and synthetic androgen R1881. Combining Raman spectra with CARS imaging, we can study the process of hormone-mediated lipogenesis. Our results show that hormone-treated cancer cells T47D and LNCaP have an increased number and size of intracellular lipid droplets and higher degree of saturation than untreated cells. MDA-MB-231 and PC3 cancer cells showed no significant changes upon treatment. Principal component analysis with linear discriminant analysis of the Raman spectra was able to differentiate between cancer cells that were treated with MPA, R1881, and untreated.
We performed super-resolution imaging of isolated olfactory sensory neurons (OSNs) using a custom-built Stimulated Emission Depletion (STED) microscope. The design for the STED microscope is based on the system developed in the laboratory of Dr. Stefan Hell1. Our system is capable of imaging with sub-diffraction limited resolution simultaneously in two color channels (at Atto 590/Atto 647N wavelengths). A single, pulsed laser source (ALP; Fianium, Inc.) generates all four laser beams, two excitation and two STED. The two STED beams are coupled into one polarization maintaining (PM) fiber and the two excitation beams into another. They are then collimated and both STED beams pass through a vortex phase plate (RPC Photonics) to allow shaping into a donut at the focus of the objective lens. The beams are then combined and sent into an inverted research microscope (IX-71; Olympus Inc.) allowing widefield epifluorescence, brightfield and DIC imaging on the same field of view as STED imaging. A fast piezo stage scans the sample during STED and confocal imaging. The fluorescent signals from the two color channels are detected with two avalanche photodiodes (APD) after appropriate spectral filtering. The resolution of the system was characterized by imaging 40 nm fluorescent beads as ~60 nm (Atto 590) and ~50 nm (Atto 647N). We performed STED imaging on immunolabeled isolated OSNs tagged at the CNGA2 and ANO2 proteins. The STED microscope allows us to resolve ciliary CNGA2 microdomains of ~54 nm that were blurred in confocal.
Increasing interest in the role of lipids in cancer cell proliferation or resistance to drug therapies has motivated the need
to develop better tools for cellular lipid analysis. Quantification of lipids in cells is typically done by destructive
chromatography protocols that do not provide spatial information on lipid distribution and prevent dynamic live cell
studies. Methods that allow the analysis of lipid content in live cells is therefore of great importance for research. Using
Raman micro-spectroscopy we investigated whether the female hormone medroxyprogesterone acetate (MPA) and the
synthetic androgen R1881 affect the lipid expression in breast (T47D) and prostate (LNCaP) cancer cells. Differences
were noted in the spectral regions at 830-1800 cm-1 and 2800-3000 cm-1 when comparing different drug treatments.
Significant changes were noticed for saturated (1063 - 1125 cm-1, 1295 cm-1 and 1439 cm-1), unsaturated (1262 cm-1 and
1656 cm-1, and 1720 - 1748 cm-1) chemical bonds, suggesting that the composition of the lipid droplets was changed by
the hormone treatments. Also, significant differences were observed in the high frequency regions of lipids and proteins
at 2851 cm-1 and around 2890 cm-1. Principal component analysis with Linear Discriminant Analysis (PCA-LDA) of the
Raman spectra was able to differentiate between cancer cells that were treated with MPA, R1881 or vehicle (P < 0.05).
Future work includes analysis to determine exact lipid composition and concentrations as well as development of clinical
techniques to characterize differences in patient tumor lipid profiles to determine response to drug treatment and
prognosis.
The aqueous outflow system (AOS), including the trabecular meshwork (TM), the collector channels (CC) and the
Schlemm’s canal (SC), regulates intraocular pressure (IOP) through the drainage of the aqueous humor (AH). Abnormal
IOP elevation leads to increased pressure stress to retinal ganglion cells, resulting in cell loss that can ultimately lead to complete loss of eyesight. Therefore, development of imaging tools to detect abnormal structural and functional changes of the AOS is important in early diagnosis and prevention of glaucoma. Multiphoton microscopy (MPM), including twophoton autofluorescence (TPAF) and second harmonic generation (SHG), is a label-free microscopic technique that allows molecular specific imaging of biological tissues like the TM. Since the TM and other AOS structures are located behind the highly scattering scleral tissue, transscleral imaging of the TM does not provide enough optical resolution. In this work, a gonioscopic lens is used to allow direct optical access of the TM through the cornea for MPM imaging. Compared to transscleral imaging, the acquired MPM images show improved resolution as individual collagen fiber bundles of the TM can be observed. MPM gonioscopy may have the potential to be developed as a future clinical imaging tool for glaucoma diagnostics.
The development of technologies to characterize the ocular aqueous outflow system (AOS) is important for the understanding of the pathophysiology of glaucoma. Multiphoton microscopy (MPM) offers the advantage of high-resolution, label-free imaging with intrinsic image contrast because the emitted signals result from the specific biomolecular content of the tissue. Previous attempts to use MPM to image the murine irido-corneal region directly through the sclera have suffered from degradation in image resolution due to scattering of the focused laser light. As a result, transscleral MPM has limited ability to observe fine structures in the AOS. In this work, the porcine irido-corneal angle was successfully imaged through the transparent cornea using a gonioscopic lens to circumvent the highly scattering scleral tissue. The resulting high-resolution images allowed the detailed structures in the trabecular meshwork (TM) to be observed. Multimodal imaging by two-photon autofluorescence and second harmonic generation allowed visualization of different features in the TM without labels and without disruption of the TM or surrounding tissues. MPM gonioscopy is a promising noninvasive imaging tool for high-resolution studies of the AOS, and research continues to explore the potential for future clinical applications in humans.
High harmonic generation (HHG) is a useful source of coherent light in the extreme ultraviolet (EUV) region of the spectrum. However, both the conversion efficiency and the highest achievable photon energy have in the past been limited in the past by the inability to phase-match the frequency conversion process. In this paper, we summarize recent results on the development of new techniques for phase-matching the high-harmonic conversion process. We also summarize finding from three series of experiments that make use of the coherent EUV light generated using HHG: 1) probing of acoustic dynamics in materials; 2) monitoring of chemical dynamics at surfaces using photoelectron spectroscopy; and 3) time-resolved plasma imaging.
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