Conventional Two-photon Microscopy Research Articles

Multi-color two-photon scanning structured illumination microscopy imaging of live cells.

Multi-color two-photon microscopy imaging of live cells is essential in biology. However, the limited diffraction resolution of conventional two-photon microscopy restricts its application to subcellular organelle imaging. Recently, we developed a laser scanning two-photon non-linear structured illumination microscope (2P-NLSIM), whose resolution improved three-fold. However, its ability to image polychromatic live cells under low excitation power has not been verified. Here, to improve the reconstruction super-resolution image quality under low excitation power, we increased the image modulation depth by multiplying the raw images with the reference fringe patterns in the reconstruction process. Simultaneously, we optimized the 2P-NLSIM system to image live cells, including the excitation power, imaging speed, and field of view. The proposed system could provide a new imaging tool for live cells.

Single-scan volumetric imaging throughout thick tissue specimens by one-touch installable light-needle creating device

Biological tissues and their networks frequently change dynamically across large volumes. Understanding network operations requires monitoring their activities in three dimensions (3D) with single-cell resolution. Several researchers have proposed various volumetric imaging technologies. However, most technologies require large-scale and complicated optical setups, as well as deep expertise for microscopic technologies, resulting in a high threshold for biologists. In this study, we propose an easy-to-use light-needle creating device for conventional two-photon microscopy systems. By only installing the device in one position for a filter cube that conventional fluorescent microscopes have, single scanning of the excitation laser light beam excited fluorophores throughout over 200 μm thickness specimens simultaneously. Furthermore, the developed microscopy system successfully demonstrated single-scan visualization of the 3D structure of transparent YFP-expressing brain slices. Finally, in acute mouse cortical slices with a thickness of approximately 250 μm, we detected calcium activities with 7.5 Hz temporal resolution in the neuronal population.

Two-photon microscopy with enhanced resolution and signal-to-background ratio using hollow Gaussian beam excitation.

Two-photon microscopy (TPM) offers deeper imaging depth inside the scattering medium, however, it suffers from limited resolution owing to the longer excitation wavelength. We demonstrate the use of a hollow Gaussian beam (HGB) at the therapeutic window to improve the resolution and signal-to-background ratio (SBR). The HGB was produced by omitting the azimuthal phase term from the vortex mode, and the excitation point spread function (PSF) can be readily tuned by the mode order. The performance of the TPM with HGB was evaluated by experimentally imaging 100 nm fluorescent beads to estimate the PSF. The HGB improved the lateral resolution of the TPM by 36% in contrast to the conventional TPM. The HGB also furnishes an improvement of SBR by eliminating the out-of-focus light owing to its ring shape. Furthermore, we have used a translating lens-based module for additional lateral resolution tuning and reduced the resolution further down to 44% with respect to conventional TPM. Finally, we have performed imaging with merely two-dimensional scanning of a 50 µm thick mouse brain slice (Thy-YFP H-line) using the developed TPM with HGB. Our compact, robust, and low-cost design of the HGB generation scheme can easily be integrated into the commercial TPM to accommodate the improvements.

Two-photon scanned light sheet fluorescence microscopy with axicon imaging for fast volumetric imaging.

.Significance: Two-photon microscopy has become the standard platform for deep-tissue fluorescence imaging. However, the use of point scanning in conventional two-photon microscopy limits the speed of volumetric image acquisition.Aim: To obtain fast and deep volumetric images, we combine two-photon light sheet fluorescence microscopy (2p-LSFM) and axicon imaging that yields an extended depth of field (DOF) in 2p-LSFM.Approach: Axicon imaging is achieved by imposing an axicon lens in the detection part of LSFM.Results: The DOF with axicon imaging is extended more than 20-fold over that of a conventional imaging lens, liberating the synchronized scanning in LSFM. We captured images of dynamic beating hearts and red blood cells in zebrafish larvae at volume acquisition rates up to 30 Hz.Conclusions: We demonstrate the fast three-dimensional imaging capability of 2p-LSFM with axicon imaging by recording the rapid dynamics of physiological processes.

Long-term in vivo two-photon imaging of the neuroinflammatory response to intracortical implants and micro-vessel disruptions in awake mice

Our understanding of biomaterials in the brain have been greatly enhanced by advancements in in vivo imaging technologies such as two-photon microscopy. However, when applied to chronic studies, two-photon microscopy enables high-resolution imaging only in superficial regions due to inflammatory responses introduced by the craniotomy and insertion of foreign biomaterials. Microprisms provide a unique vertical view from brain surface to ~1 mm deep or more (depending on the size of the microprisms) which may break through this limitation on imaging depth. Although microprism has been used in the field of neuroscience, the in vivo foreign body responses to the microprism implant have yet to be fully elucidated. This is of important concern in broader applications of this approach, especially for neuroinflammation-sensitive studies. In this work, we first assessed the activation of microglia/macrophages for 16 weeks after microprism implantation using two-photon microscopy in awake CX3CR1-GFP mice. The imaging window became clear from bleedings after ~2 weeks and the maximum imaging distance (in the horizontal direction) stabilized at around 500 μm after ~5 weeks. We also quantified the microglial morphology from week 3 to week 16 post-implantation. Compared to non-implant controls, microglia near the microprism showed higher cell density, smaller soma, and shorter and less branched processes in the early-chronic phase. After week 5, microglial morphology further than 100 μm from the microprism was generally similar to microglia in the control group. In addition, time-lapse imaging confirmed that microglial processes were surveying normally from week 3, even for microglia as close as 50 μm away. These morphological analyses and dynamic imaging results suggest that microglia around chronically implanted microprism eventually exhibit inactive phenotypes. Next, we examined microglial/macrophage responses following laser induced micro-vessel disruptions as an example application of microprism implantation for neuroinflammation related studies. Through the microprism, we captured microglial/macrophage polarization and migration, as well as blood flow changes after the insult for additional 16 weeks. To our surprise, microglia/macrophage aggregation around the insult site was sustained over the 16-week observation period. This work demonstrates the feasibility of using microprisms for long-term characterizations of inflammatory responses to other injuries including implantable devices at deeper depths than that achievable by conventional two-photon microscopy.

Intravital three-photon microscopy of T-cell migration in the T-cell zones over the entire depth of mouse popliteal lymph node.

Abstract Most T cells in a popliteal lymph node are located in T-cell zones at ~150 to ~600 μm below the cortical surface of the lymph node. For the last 20 years, intravital two-photon microscopy has been a powerful tool for observing the three dimensional T-cell migration in the T-cell zones. However, two-photon imaging can only visualize superficial T-cell zones to ~300 μm depth. In this work, we successfully visualized T-cell migration in T-cell zones over the entire depth of a mouse popliteal lymph node with intravital three-photon microscopy. We measured the change in lymphocyte velocity with increasing laser powers at 600 μm depth to determine the safe laser power range for visualizing T-cell migration. We found that, with less than 80 mW at 1300 nm excitation, the migration of EGFP-expressing lymphocytes were not perturbed by the laser illumination. Using the safe power of 1300 nm excitation, we observed dynamic migration of EGFP-expressing naïve CD4 T cells and DsRed-expressing naïve CD8 T cells simultaneously in T-cell zones at various depths. The naïve T cells were sparse at 0 ~150 μm depth in cortical side (follicular and interfollicular regions), dense in the T-cell zones, and less dense in the medulla, which was identified by labeling with eFluor615-conjugated LYVE-1 antibody in vivo. We found that the density distributions of CD4 and CD8 T cells were different in the T-cell zones. We also measured the migration velocity and motility coefficient for naïve CD4 and CD8 T cells in the T-cell zones over the entire depth of the lymph node. This work shows that three-photon microscopy has the potential to shed light on immune cell dynamics in the deep regions of lymph nodes that are typically inaccessible to conventional two-photon microscopy.

Axial resolution improvement of two-photon microscopy by multi-frame reconstruction and adaptive optics.

Two-photon microscopy (TPM) has been widely used in biological imaging owing to its intrinsic optical sectioning and deep penetration abilities. However, the conventional TPM suffers from poor axial resolution, which makes it difficult to recognize some three-dimensional fine features. We present multi-frame reconstruction two-photon microscopy (MR-TPM) using a liquid lens as a fast axial scanning engine. A sensorless adaptive optics (AO) approach is adopted to correct the aberrations caused by both the liquid lens and the optical system. By overcoming the effect of optical aberrations, inadequate sampling, and poor focusing capability of a conventional TPM, the axial resolution can be improved by a factor of 3 with a high signal-to-noise ratio. The proposed technology is compatible with the conventional TPM and requires no optical post-processing. We demonstrate the proposed method by imaging fluorescent beads, in vitro imaging of the neural circuit of mouse brain slice, and in vivo time-lapse imaging of the morphological changes of microglial cells in septic mice model. The results suggest that the axon of the neural circuit and the process of microglia along the axial direction, which cannot be resolved using conventional TPM, become distinguishable using the proposed AO MR-TPM.

Three-photon imaging of synthetic dyes in deep layers of the neocortex

Multiphoton microscopy has emerged as the primary imaging tool for studying the structural and functional dynamics of neural circuits in brain tissue, which is highly scattering to light. Recently, three-photon microscopy has enabled high-resolution fluorescence imaging of neurons in deeper brain areas that lie beyond the reach of conventional two-photon microscopy, which is typically limited to ~ 450 µm. Three-photon imaging of neuronal calcium signals, through the genetically-encoded calcium indicator GCaMP6, has been used to successfully record neuronal activity in deeper neocortical layers and parts of the hippocampus in rodents. Bulk-loading cells in deeper cortical layers with synthetic calcium indicators could provide an alternative strategy for labelling that obviates dependence on viral tropism and promoter penetration, particularly in non-rodent species. Here we report a strategy for visualized injection of a calcium dye, Oregon Green BAPTA-1 AM (OGB-1 AM), at 500–600 µm below the surface of the mouse visual cortex in vivo. We demonstrate successful OGB-1 AM loading of cells in cortical layers 5–6 and subsequent three-photon imaging of orientation- and direction- selective visual responses from these cells.

In vivo two-photon microscopic observation and ablation in deeper brain regions realized by modifications of excitation beam diameter and immersion liquid.

In vivo two-photon microscopy utilizing a nonlinear optical process enables, in living mouse brains, not only the visualization of morphologies and functions of neural networks in deep regions but also their optical manipulation at targeted sites with high spatial precision. Because the two-photon excitation efficiency is proportional to the square of the photon density of the excitation laser light at the focal position, optical aberrations induced by specimens mainly limit the maximum depth of observations or that of manipulations in the microscopy. To increase the two-photon excitation efficiency, we developed a method for evaluating the focal volume in living mouse brains. With this method, we modified the beam diameter of the excitation laser light and the value of the refractive index in the immersion liquid to maximize the excitation photon density at the focal position. These two modifications allowed the successful visualization of the finer structures of hippocampal CA1 neurons, as well as the intracellular calcium dynamics in cortical layer V astrocytes, even with our conventional two-photon microscopy system. Furthermore, it enabled focal laser ablation dissection of both single apical and single basal dendrites of cortical layer V pyramidal neurons. These simple modifications would enable us to investigate the contributions of single cells or single dendrites to the functions of local cortical networks.

Intravital three-photon microscopy of entire mouse lymph node

Abstract For the last 20 years, intravital confocal and two-photon microscopy have been powerful tools to explore dynamic immune cell behavior in mouse lymph node. However, they can only image ~100 and ~300 μm depth respectively while a peripheral lymph node of adult mouse has 600 – 1000 μm thickness. Here, we visualized whole thickness of adult mouse popliteal lymph node with intravital three-photon microscopy. Three-photon excitation significantly improved a signal-to-background ratio compared to two-photon excitation even at the same excitation wavelength. The capability to image the full depth enabled to achieve the 3D volume of entire lymph node vasculature in vivo by tiling multiple z-stack images. We observed DsRed-expressing lymphocyte migration in LYVE1+ lymphatic sinus at 600 μm depth of lymph node where conventional two-photon microscopy was normally inaccessible. In addition, we demonstrated the capability multi-color imaging by using Cγ1-Confetti mice of which each germinal center B cells stochastically expresses one of 4 different fluorescent proteins (CFP, GFP, YFP and RFP). Intravital three-photon microscopy has the potential to shed light on unknown immune cell behavior in deeper regions of the lymph node in vivo just as two-photon microscopy did during the last 20 years at the shallower depth.

Scanless two-photon excitation with temporal focusing.

Temporal focusing, with its ability to focus light in time, enables scanless illumination of large surface areas at the sample with micrometer axial confinement and robust propagation through scattering tissue. In conventional two-photon microscopy, widely used for the investigation of intact tissue in live animals, images are formed by point scanning of a spatially focused pulsed laser beam, resulting in limited temporal resolution of the excitation. Replacing point scanning with temporally focused widefield illumination removes this limitation and represents an important milestone in two-photon microscopy. Temporal focusing uses a diffusive or dispersive optical element placed in a plane conjugate to the objective focal plane to generate position-dependent temporal pulse broadening that enables axially confined multiphoton absorption, without the need for tight spatial focusing. Many techniques have benefitted from temporal focusing, including scanless imaging, super-resolution imaging, photolithography, uncaging of caged neurotransmitters and control of neuronal activity via optogenetics.

Simultaneous label-free autofluorescence and multi-harmonic imaging reveals in vivo structural and metabolic changes in murine skin.

Simultaneous quantification of multifarious cellular metabolites and the extracellular matrix in vivo has been long sought. Simultaneous label-free autofluorescence and multi-harmonic (SLAM) microscopy has achieved simultaneous four-channel nonlinear imaging to study tissue structure and metabolism. In this study, we implemented two laser systems and directly compared SLAM microscopy with conventional two-photon microscopy for in vivo imaging. We found that three-photon imaging of adenine dinucleotide (phosphate) (NAD(P)H) in SLAM microscopy using our tailored laser source provided better resolution, contrast, and background suppression than conventional two-photon imaging of NAD(P)H. We also integrated fluorescence lifetime imaging with SLAM microscopy, and enabled differentiation of free and bound NAD(P)H. We imaged murine skin in vivo and showed that changes in tissue structure, cell dynamics, and metabolism can be monitored simultaneously in real-time. We also discovered an increase in metabolism and protein-bound NAD(P)H in skin cells during the early stages of wound healing.

Simultaneous two-plane, two-photon imaging based on spatial multiplexing.

The two-photon microscope is a powerful tool in life science. Conventional two-photon microscopy can image only a plane of a particular axial position at a time. Axial scanning can get the volumetric information, but it gets signals from different axial positions serially, which means that the exposure time at every plane is limited. Here we demonstrate a novel method, to the best of our knowledge, that can simultaneously record images from two planes at different xyz positions. The demultiplexing of the signal is realized using a confocal strategy. The experimental results show that it can be used for simultaneous two-photon imaging at two focal planes with little cross talk.

Designing a large field-of-view two-photon microscope using optical invariant analysis.

.Conventional two-photon microscopy (TPM) is capable of imaging neural dynamics with subcellular resolution, but it is limited to a field-of-view (FOV) diameter . Although there has been recent progress in extending the FOV in TPM, a principled design approach for developing large FOV TPM (LF-TPM) with off-the-shelf components has yet to be established. Therefore, we present a design strategy that depends on analyzing the optical invariant of commercially available objectives, relay lenses, mirror scanners, and emission collection systems in isolation. Components are then selected to maximize the space-bandwidth product of the integrated microscope. In comparison with other LF-TPM systems, our strategy simplifies the sequence of design decisions and is applicable to extending the FOV in any microscope with an optical relay. The microscope we constructed with this design approach can image lateral and axial resolution over a 7-mm diameter FOV, which is a 100-fold increase in FOV compared with conventional TPM. As a demonstration of the potential that LF-TPM has on understanding the microarchitecture of the mouse brain across interhemispheric regions, we performed in vivo imaging of both the cerebral vasculature and microglia cell bodies over the mouse cortex.

Contrast and resolution enhanced optical sectioning in scattering tissue using line-scanning two-photon structured illumination microscopy.

Optical sectioning imaging with high spatial resolution deep inside scattering samples such as mammalian brain is of great interest in biological study. Conventional two-photon microscopy deteriorates in focus when light scattering increases. Here we develop an optical sectioning enhanced two-photon technique which incorporates structured illumination into line-scanning spatial-temporal focusing microscopy (LTSIM), and generate patterned illumination via laser intensity modulation synchronized with scanning. LTSIM brings scattering background elimination and in-focus contrast enhancement, and realizes nearly 2-fold increase in spatial resolution to ∼208 nm laterally and ∼0.94 µm axially. In addition, the intensity modulated line-scanning implementation of LTSIM enables fast and flexible generation of structured illumination, permitting adjustable spatial frequency profiles to optimize image contrast. The highly qualified optical sectioning ability of our system is demonstrated on samples including tissue phantom, C. elegans and mouse brain at depths over hundreds of microns.

Image formation in image scanning microscopy, including the case of two-photon excitation.

The effect of combining the image scanning microscopy (ISM) technique with two-photon fluorescence microscopy is analyzed. The effective spatial frequency cutoff can be doubled, as compared with conventional two-photon fluorescence microscopy, and the magnitude of the optical transfer function near the cutoff of conventional two-photon microscopy is increased by orders of magnitude. For the two-photon case, it is found that the optimum pixel reassignment factor in ISM is not equal to one half, as is often assumed in single-photon fluoresence image scanning microscopy, because the excitation and detection point spread functions are different. The optimum reassignment factor depends on the noise level, and in general the useful cutoff spatial frequency is about 1.8 times that for conventional two-photon microscopy. The effect of altering the reassignment factor in single-photon fluorescence ISM with a Stokes shift is also investigated. Illumination using pupil filters, such as by a Bessel beam, is considered. Using a ring detector array is found to result in good imaging behavior, exhibiting a sharpening of the point spread function by a factor of 1.7 compared with conventional fluorescence. Image formation in ISM can be considered in a four-dimensional spatial frequency space, giving new insight into the imaging properties. This approach is related to phase space representations such as the Wigner distribution function and the ambiguity function. A noniterative algorithm for image restoration is proposed.

Saturated two-photon excitation fluorescence microscopy with core-ring illumination.

We demonstrated resolution improvement in two-photon excitation microscopy by combining saturated excitation (SAX) of fluorescence and pupil manipulation. We theoretically estimated the resolution improvement and the sidelobe effect in the point spread function with various pupil designs and found that the combination of SAX and core-ring illumination can effectively enhance the spatial resolution in 3D and suppress sidelobe artifacts. The experimental demonstration shows that the proposed technique is effective for observation with a depth of 100μm in a tissue phantom and can be applied to 3D observations of tissue samples with higher spatial resolution than conventional two-photon excitation microscopy.

Painting with Rainbows: Patterning Light in Space, Time, and Wavelength for Multiphoton Optogenetic Sensing and Control.

Photons are a fascinating reagent, flowing and reacting quite differently compared to more massive and less ephemeral particles of matter. The optogenetic palette comprises an ever growing set of light-responsive proteins, which open the possibility of using light to perturb and to measure biological processes with great precision in space and time. Yet there are limits on what light can achieve. Diffraction limits the smallest features, and scattering in tissue limits the largest. Photobleaching, diffusion of photogenerated products, and optical crosstalk between overlapping absorption spectra further muddy the optogenetic picture, particularly when one wants to use multiple optogenetic tools simultaneously. But these obstacles are surmountable. Most light-responsive proteins and small molecules undergo more than one light-driven transition, often with different action spectra and kinetics. By overlapping multiple laser beams, carefully patterned in space, time, and wavelength, one can steer molecules into fluorescent or nonfluorescent, active or inactive conformations. By doing so, one can often circumvent the limitations of simple one-photon excitation and achieve new imaging and stimulation capabilities. These include subdiffraction spatial resolution, optical sectioning, robustness to light scattering, and multiplexing of more channels than can be achieved with simple one-photon excitation. The microbial rhodopsins are a particularly rich substrate for this type of multiphoton optical control. The natural diversity of these proteins presents a huge range of starting materials. The spectroscopy and photocycles of microbial rhodopsins are relatively well understood, providing states with absorption maxima across the visible spectrum, which can be accessed on experimentally convenient time scales. A long history of mutational studies in microbial rhodopsins allows semirational protein engineering. Mutants of Archaerhodopsin 3 (Arch) come in all the colors of the rainbow. In a solution of purified Arch-eGFP, a focused green laser excites eGFP fluorescence throughout the laser path, while a focused red laser excites fluorescence of Arch only near the focus, indicative of multiphoton fluorescence. This nonlinearity occurs at a laser intensity ∼1010-fold lower than in conventional two-photon microscopy! The mutant Arch(D95H) shows photoswitchable optical bistability. In a lawn of E. coli expressing this mutant, illumination with patterned blue light converts the molecule into a state that is fluorescent. Illumination with red light excites this fluorescence, and gradually resets the molecules back to the non-fluorescent state. This review describes the new types of molecular logic that can be implemented with multi-photon control of microbial rhodopsins, from whole-brain activity mapping to measurements of absolute membrane voltage. Part of our goal in this Account is to describe recent work in nonlinear optogenetics, but we also present a variety of interesting things one could do if only the right optogenetic molecules were available. This latter component is intended to inspire future spectroscopic, protein discovery, and protein engineering work.

High-Speed Two-Photon Imaging

The small size of neuronal dendrites and spines combined with the high speed of neurophysiological signals, such as transients in membrane potential or ion concentration, necessitates that any functional study of these structures uses recording methods with both high spatial and high temporal resolutions. In this regard, conventional two-photon microscopy, in combination with fluorescent indicators sensitive to physiological parameters, has proved to be only a partial solution by providing near-diffraction-limited spatial resolution even when imaging structures deep inside light-scattering tissue. This is because the relatively slow beam-scanning methods used in most conventional two-photon microscopes severely limit the extent to which functional data can be recorded. Here, we detail developments to create high-speed two-photon imaging systems that overcome this limitation and discuss important considerations that must be taken into account when attempting to construct such systems.

SHG nanoprobes: Advancing harmonic imaging in biology

Second harmonic generating (SHG) nanoprobes have recently emerged as versatile and durable labels suitable for in vivo imaging, circumventing many of the inherent drawbacks encountered with classical fluorescent probes. Since their nanocrystalline structure lacks a central point of symmetry, they are capable of generating second harmonic signal under intense illumination - converting two photons into one photon of half the incident wavelength - and can be detected by conventional two-photon microscopy. Because the optical signal of SHG nanoprobes is based on scattering, rather than absorption as in the case of fluorescent probes, they neither bleach nor blink, and the signal does not saturate with increasing illumination intensity. When SHG nanoprobes are used to image live tissue, the SHG signal can be detected with little background signal, and they are physiologically inert, showing excellent long-term photostability. Because of their photophysical properties, SHG nanoprobes provide unique advantages for molecular imaging of living cells and tissues with unmatched sensitivity and temporal resolution.