Two-photon Scanning Microscopy Research Articles

Multibeam continuous axial scanning two-photon microscopy for in vivo volumetric imaging in mouse brain.

This study presents an alternative approach for two-photon volumetric imaging that combines multibeam lateral scanning with continuous axial scanning using a confocal spinning-disk scanner and an electrically focus tunable lens. Using this proposed system, the brain of a living mouse could be imaged at a penetration depth of over 450 μm from the surface. In vivo volumetric Ca2+ imaging at a volume rate of 1.5 Hz within a depth range of 130-200 μm, was segmented with an axial pitch of approximately 5-µm and revealed spontaneous activity of neurons with their 3D positions. This study offers a practical microscope design equipped with compact scanners, a simple control system, and readily adjustable imaging parameters, which is crucial for the widespread adoption of two-photon volumetric imaging.

Optical coherence tomography-based design for a real-time motion corrected scanning microscope.

While two-photon fluorescence microscopy is a powerful platform for the study of functional dynamics in living cells and tissues, the bulk motion inherent to these applications causes distortions. We have designed a motion tracking module based on spectral domain optical coherence tomography which compliments a laser scanning two-photon microscope with real-time corrective feedback. The module can be added to fluorescent imaging microscopes using a single dichroic and without additional contrast agents. We demonstrate that the system can track lateral displacements as large as 10 μm at 5 Hz with latency under 14 ms and propose a scheme to extend the system to 3D correction with the addition of a remote focusing module. We also propose several ways to improve the module's performance by reducing the feedback latency. We anticipate that this design can be adapted to other imaging modalities, enabling the study of samples subject to motion artifacts at higher resolution.

Background Rejection in Two-Photon Fluorescence Image Scanning Microscopy

We discuss the properties of signal strength and integrated intensity in two-photon excitation confocal microscopy and image scanning microscopy. The resolution, optical sectioning and background rejection are all improved over nonconfocal two-photon microscopy. Replacing the pinhole of confocal two-photon microscopy with a detector array increases the peak intensity of the point spread function. The outer pixels of a detector array give signals from defocused regions, and thus the processing of these, such as through subtraction, can further improve optical sectioning and background rejection.

Ten-kilohertz two-photon microscopy imaging of single-cell dendritic activity and hemodynamics in vivo.

The studying of rapid neuronal signaling across large spatial scales in intact, living brains requires both high temporal resolution and versatility of the measurement device. We introduce a high-speed two-photon microscope based on a custom-built acousto-optic deflector (AOD). This microscope has a maximum line scan frequency of 400kHz and a maximum frame rate of 10,000 frames per second (fps) at . For stepwise magnification from population view to subcellular view with high spatial and temporal resolution, we combined the AOD with resonance-galvo (RS) scanning. With this combinatorial device that supports both large-view navigation and small-view high-speed imaging, we measured dendritic calcium propagation velocity and the velocity of single red blood cells (RBCs). We measured dendritic calcium propagation velocity ( ) in OGB-1-labeled single cortical neurons in mice in vivo. To benchmark the spatial precision and detection sensitivity of measurement in vivo, we also visualized the trajectories of single RBCs and found that their movement speed follows Poiseuille's law of laminar flow. This proof-of-concept methodological development shows that the combination of AOD and RS scanning two-photon microscopy provides both versatility and precision for quantitative analysis of single neuronal activities and hemodynamics in vivo.

Natural Solar Irradiation Produces Fluorescent and Biodegradable Nanoplastics.

Nanoplastics (NPs) have raised global concern owing to their potential health effects. Herein, after simulated and natural solar irradiation, polyethylene, polypropylene, polystyrene, and poly(vinyl chloride) nanoplastics (PVC NPs) were observed to exhibit enhanced fluorescence, particularly PVC NPs. Furthermore, the role of photoaged NPs as a potential fluorescence indicator was evaluated by exposing a model aquatic organism Daphnia magna to these NPs. Our results revealed that photoaged NPs exhibited strong fluorescence owing to the generation of conjugated π bonds, which can achieve π-π* electron transition with low energy consumption. Photogenerated fluorescence also enabled the photoaged NPs to act as efficient fluorescent tracers, which can help track NP migration in various organisms. The results of two-photon laser confocal scanning microscopy revealed that the photoaged NPs could translocate across biological barriers and accumulate in extraintestinal tissues in addition to being ingested and excreted. Moreover, compared with pristine NPs, the photoaged NPs underwent biodegradation more easily, probably because of increased hydrophilicity due to photogenerated oxygen-containing moieties. Therefore, in addition to producing fluorescent NPs without the attachment of external fluorescent dyes, the natural photoaging process can promote the migration and degradation of photoaged NPs in food chains.

Differentiable model-based adaptive optics for two-photon microscopy.

Aberrations limit scanning fluorescence microscopy when imaging in scattering materials such as biological tissue. Model-based approaches for adaptive optics take advantage of a computational model of the optical setup. Such models can be combined with the optimization techniques of machine learning frameworks to find aberration corrections, as was demonstrated for focusing a laser beam through aberrations onto a camera [Opt. Express2826436 (26436)10.1364/OE.403487]. Here, we extend this approach to two-photon scanning microscopy. The developed sensorless technique finds corrections for aberrations in scattering samples and will be useful for a range of imaging application, for example in brain tissue.

The functional organization of excitatory synaptic input to place cells

Hippocampal place cells contribute to mammalian spatial navigation and memory formation. Numerous models have been proposed to explain the location-specific firing of this cognitive representation, but the pattern of excitatory synaptic input leading to place firing is unknown, leaving no synaptic-scale explanation of place coding. Here we used resonant scanning two-photon microscopy to establish the pattern of synaptic glutamate input received by CA1 place cells in behaving mice. During traversals of the somatic place field, we found increased excitatory dendritic input, mainly arising from inputs with spatial tuning overlapping the somatic field, and functional clustering of this input along the dendrites over ~10 µm. These results implicate increases in total excitatory input and co-activation of anatomically clustered synaptic input in place firing. Since they largely inherit their fields from upstream synaptic partners with similar fields, many CA1 place cells appear to be part of multi-brain-region cell assemblies forming representations of specific locations.

Amplitude-modulation-type multi-ring mask for two-photon excitation scanning microscopy

A smaller point spread function (PSF) is desirable for high-resolution image acquisition. In two-photon excitation microscopy, the focus size of the excitation light is directly related to the two-photon fluorescence PSF. To achieve a small PSF, we designed amplitude-modulation-type multi-ring pupil masks using two design methods. When the intensity distribution of the excitation light has a top-hat form, the volume of the PSF obtained with the first designed multi-ring mask is 1.46 times smaller than that obtained without the mask. Furthermore, the second multi-ring mask is less influenced by spherical aberration occurring during observation inside the sample. Our method, which is robust against spherical aberration, reduces the size of the PSF in the observation of fluorescent beads and biological samples.

Ultrahigh-speed point scanning two-photon microscopy using high dynamic range silicon photomultipliers

Conventional two-photon microscopes use photomultiplier tubes, which enable high sensitivity but can detect relatively few photons per second, forcing longer pixel integration times and limiting maximum imaging rates. We introduce novel detection electronics using silicon photomultipliers that greatly extend dynamic range, enabling more than an order of magnitude increased photon detection rate as compared to state-of-the-art photomultiplier tubes. We demonstrate that this capability can dramatically improve both imaging rates and signal-to-noise ratio (SNR) in two-photon microscopy using human surgical specimens. Finally, to enable wider use of more advanced detection technology, we have formed the OpenSiPM project, which aims to provide open source detector designs for high-speed two-photon and confocal microscopy.

Image scanning microscopy with multiphoton excitation or Bessel beam illumination.

Image scanning microscopy is a technique of confocal microscopy in which the confocal pinhole is replaced by a detector array, and the image is reconstructed most straightforwardly by pixel reassignment. In the fluorescence mode, the detector array collects most of the fluorescent light, so the signal-to-noise ratio is much improved compared with confocal microscopy with a small pinhole, while the resolution is improved compared with conventional fluorescence microscopy. Here we consider two cases in which the illumination and detection point spread functions are dissimilar: illumination with a Bessel beam and multiphoton microscopy. It has been shown previously that for Bessel beam illumination in image scanning microscopy with a large array, the imaging performance is degraded. On the other hand, it is also known that the resolution of confocal microscopy is improved by Bessel beam illumination. Here we analyze image scanning microscopy with Bessel beam illumination together with a small array and show that an improvement in transverse resolution (width of the point spread function) by a factor of 1.78 compared with a conventional fluorescence microscope can be obtained. We also examine the behavior of image scanning microscopy in two- or three-photon fluorescence and for two-photon excitation also with Bessel beam illumination. The combination of the optical sectioning effect of image scanning microscopy with multiphoton microscopy reduces background from the sample surface, which can increase penetration depth. For a detector array size of two Airy units, the resolution of two-photon image scanning microscopy is a factor 1.85 better and the peak of the point spread function 2.84 times higher than in nonconfocal two-photon fluorescence. The resolution of three-photon image scanning microscopy is a factor 2.10 better, and the peak of the point spread function is 3.77 times higher than in nonconfocal three-photon fluorescence. The resolution of two-photon image scanning microscopy with Bessel beam illumination is a factor 2.13 better than in standard two-photon fluorescence. Axial resolution and optical sectioning in two-photon or three-photon fluorescence are also improved by using the image scanning modality.

Quantitative High-Resolution Imaging of Live Microbial Cells at High Hydrostatic Pressure

The majority of the Earth’s microbial biomass exists in the deep biosphere, in the deep ocean, and within the Earth’s crust. Although other physical parameters in these environments, such as temperature or pH, can differ substantially, they are all under high pressures. Beyond emerging genomic information, little is known about the molecular mechanisms underlying the ability of these organisms to survive and grow at pressures that can reach over 1000-fold the pressure on the Earth’s surface. The mechanisms of pressure adaptation are also important in food safety, with the increasing use of high-pressure food processing. Advanced imaging represents an important tool for exploring microbial adaptation and response to environmental changes. Here, we describe implementation of a high-pressure sample chamber with a two-photon scanning microscope system, allowing for the first time, to our knowledge, quantitative high-resolution two-photon imaging at 100 MPa of living microbes from all three kingdoms of life. We adapted this setup for fluorescence lifetime imaging microscopy with phasor analysis (FLIM/Phasor) and investigated metabolic responses to pressure of live cells from mesophilic yeast and bacterial strains, as well as the piezophilic archaeon Archaeoglobus fulgidus. We also monitored by fluorescence intensity fluctuation-based methods (scanning number and brightness and raster scanning imaging correlation spectroscopy) the effect of pressure on the chromosome-associated protein HU and on the ParB partition protein in Escherichia coli, revealing partially reversible dissociation of ParB foci and concomitant nucleoid condensation. These results provide a proof of principle that quantitative, high-resolution imaging of live microbial cells can be carried out at pressures equivalent to those in the deepest ocean trenches.

Volumetric Ca2+ Imaging in the Mouse Brain Using Hybrid Multiplexed Sculpted Light Microscopy

Calcium imaging using two-photon scanning microscopy has become an essential tool in neuroscience. However, in its typical implementation, the tradeoffs between fields of view, acquisition speeds, and depth restrictions in scattering brain tissue pose severe limitations. Here, using an integrated systems-wide optimization approach combined with multiple technical innovations, we introduce a new design paradigm for optical microscopy based on maximizing biological information while maintaining the fidelity of obtained neuron signals. Our modular design utilizes hybrid multi-photon acquisition and allows volumetric recording of neuroactivity at single-cell resolution within up to 1× 1× 1.22mm volumes at up to 17Hz in awake behaving mice. We establish the capabilities and potential of the different configurations of our imaging system at depth and across brain regions by applying it to invivo recording of up to 12,000 neurons in mouse auditory cortex, posterior parietal cortex, and hippocampus.

Cell Imaging Using Two-Photon Excited CdS Fluorescent Quantum Dots Working within the Biological Window.

In recent years, two-photon excited semiconductor quantum dots (QDs) have been the subject of intense investigation due to their long excitation wavelength which helps to achieve deeper penetration and higher image resolution in optical bioimaging. In this paper, water-soluble CdS QDs were synthesized using a hydrothermal method and applied to human liver hepatocellular carcinoma (HepG2) cells. The first-principles calculation suggested that the S-rich defected structure contributes to a narrower band gap compared to the pristine structure. The resulting fluorescence wavelength was significantly red shifted, which was attributed to the deep defect states emission. The large Stokes shifts (> 200 nm) of the QDs can eliminate the possible cross-talk between the excitation light and the emission light. Two-photon induced red fluorescence emission can avoid overlapping with the autofluorescence emission of biological samples. The uptake and cell viability measurements of the HepG2 cells showed a good biocompatibility and a low toxicity of CdS QDs. Two-photon excited scanning microscopy images revealed that the HepG2 cells incubated with CdS QDs emitted bright red upconversion fluorescence and the fluorescence brightness was 38.2 times of that of the control group. These results support CdS QDs as a good candidate for application in cellular imaging.

Two-photon PSF-engineered image scanning microscopy.

We present two-photon fluorescence image scanning microscopy (ISM) with engineered excitation and detection point-spread-functions enabling 3D imaging in a single 2D scan. This demonstration combines excitation using a holographic multispot array of focused femtosecond pulses with a high-efficiency single-helix PSF phase mask detection. Camera detection along with a multiview reconstruction algorithm allows volumetric imaging of biological samples over a depth of field spanning more than 1500nm with an axial resolution of better than 400nm. The nonlinear two-photon process improves sectioning and the inherent longer wavelengths increase the penetration depth in scattering samples. Our method extends the performance of 3D ISM towards thicker biological samples.

An Off-On Two-Photon Carbazole-Based Fluorescent Probe: Highly Targeting and Super-Resolution Imaging of mtDNA.

Many mitochondria-related diseases are associated with the mutation of mitochondrial DNA (mtDNA). Therefore, visualizing its dynamics in live cells is essential for the understanding of the function of mtDNA transcription and translation. By employing carbazole as the framework and designing a module for DNA minor-groove binding, here we have developed a novel fluorescent probe with a large Stokes shift (λab = 480 nm and λem = 620 nm), CNQ, for mtDNA detection and visualization. It is almost nonfluorescent in PBS buffer and exhibits 182-fold enhancement in fluorescence within 20 s after the application of mtDNA in the solution, with a detection limit of 55.1 μg/L. Using dual-color Hessian-structured illumination microscopy, we have demonstrated that CNQ-labeled mtDNA structures are distinct from those labeled by TFAM-EGFP. Finally, we have used two-photon confocal scanning microscopy (λex = 850 nm) to monitor the nondestructive doxorubicin-induced mtDNA damage in live cells.

Two-photon speckle illumination for super-resolution microscopy.

We present a numerical study of a microscopy setup in which the sample is illuminated with uncontrolled speckle patterns and the two-photon excitation fluorescence is collected on a camera. We show that, using a simple deconvolution algorithm for processing the speckle low-resolution images, this wide-field imaging technique exhibits resolution significantly better than that of two-photon excitation scanning microscopy or one-photon excitation bright-field microscopy.

Two novel two-photon fluorescent probes for low pH values and cell imaging based on spirobifluorene motif

Two novel spirobifluorene derivatives containing arylamine groups, SPF-M and SPF-P were synthesized. As the protonation of the aromatic amine N atoms, SPF-M and SPF-P showed significant pH-dependent spectral properties. The pKa values are below 3.0 and they are suitable for extremely acid conditions. Under the UV light, SPF-M permit detection with the “naked eye” which makes it particularly attractive. To our knowledge, SPF-M and SPF-P represent the first examples that two-photon pH sensors were used to measure the extremely acidic pH value. When the pH is higher than 4.0, SPF-M and SPF-P exhibited strong one- and two-photon excited fluorescence and showed no dramatic spectral changes toward pH variations. Thus, the feasibility of SPF-M and SPF-P as multiphoton luminescence bioprobe under physiological conditions is demonstrated by two-photon scanning microscopy imaging experiments on 293T cells.

Advances in two photon scanning and scanless microscopy technologies for functional neural circuit imaging.

Recent years have seen substantial developments in technology for imaging neural circuits, raising the prospect of large scale imaging studies of neural populations involved in information processing, with the potential to lead to step changes in our understanding of brain function and dysfunction. In this article we will review some key recent advances: improved fluorophores for single cell resolution functional neuroimaging using a two photon microscope; improved approaches to the problem of scanning active circuits; and the prospect of scanless microscopes which overcome some of the bandwidth limitations of current imaging techniques. These advances in technology for experimental neuroscience have in themselves led to technical challenges, such as the need for the development of novel signal processing and data analysis tools in order to make the most of the new experimental tools. We review recent work in some active topics, such as region of interest segmentation algorithms capable of demixing overlapping signals, and new highly accurate algorithms for calcium transient detection. These advances motivate the development of new data analysis tools capable of dealing with spatial or spatiotemporal patterns of neural activity, that scale well with pattern size.

Two novel dyes containing spirobifluorene and triphenylamine: Synthesis, one- and two-photon excited fluorescence and applications as probes for silver ions, water and cell imaging

Two fluorophores, SPF-TNS and SPF-TBB, with electron-rich triphenylamine groups and Ag+ receptor units on the respective biphenyl branches of 9,9′-spirobifluorene were synthesized. They exhibit strong one- and two-photon excited fluorescence, and the two-photon absorption cross-section values are 665 GM for SPF-TNS and 355GM for SPF-TBB. The probing behaviors toward metal ions were investigated via UV/Vis absorption spectra as well as one-photon fluorescence changes. The results indicated that SPF-TNS and SPF-TBB exhibit distinct fluorescent quenching in the presence of silver ions that can be used for highly sensitive and selective naked-eye detection of Ag+. The one-photon excited fluorescence of SPF-TBB can be made to measure water content of organic solvents. The detection limits were calculated to be 0.04% for THF and 0.03% for 1,4-dioxane. The two-photon scanning microscopy experiments suggest that SPF-TNS is a promising label potentially applicable for the tracking of biomolecules.

Cytosolic NADH-NAD(+) Redox Visualized in Brain Slices by Two-Photon Fluorescence Lifetime Biosensor Imaging.

Cytosolic NADH-NAD(+) redox state is central to cellular metabolism and a valuable indicator of glucose and lactate metabolism in living cells. Here we sought to quantitatively determine NADH-NAD(+) redox in live cells and brain tissue using a fluorescence lifetime imaging of the genetically-encoded single-fluorophore biosensor Peredox. We show that Peredox exhibits a substantial change in its fluorescence lifetime over its sensing range of NADH-NAD(+) ratio. This allows changes in cytosolic NADH redox to be visualized in living cells using a two-photon scanning microscope with fluorescence lifetime imaging capabilities (2p-FLIM), using time-correlated single photon counting. Because the lifetime readout is absolutely calibrated (in nanoseconds) and is independent of sensor concentration, we demonstrate that quantitative assessment of NADH redox is possible using a single fluorophore biosensor. Imaging of the sensor in mouse hippocampal brain slices reveals that astrocytes are typically much more reduced (with higher NADH:NAD(+) ratio) than neurons under basal conditions, consistent with the hypothesis that astrocytes are more glycolytic than neurons. Antioxid. Redox Signal. 25, 553-563.