Commercial Two-photon Microscope Research Articles

A simple Bessel module with tunable focal depth and constant resolution for commercial two-photon microscope

The volumetric imaging of two-photon microscopy expands the focal depth and improves the throughput, which has unparalleled superiority for three-dimension samples, especially in neuroscience. However, emerging in volumetric imaging is still largely customized, which limits the integration with commercial two-photon systems. Here, we analyzed the key parameters that modulate the focal depth and lateral resolution of polarized annular imaging and proposed a volumetric imaging module that can be directly integrated into commercial two-photon systems using conventional optical elements. This design incorporates the beam diameter adjustment settings of commercial two-photon systems, allowing flexibility to adjust the depth of focus while maintaining the same lateral resolution. Further, the depth range and lateral resolution of the design were verified, and the imaging throughput was demonstrated by an increase in the number of imaging neurons in the awake mouse cerebral cortex.

The DIVER Microscope for Imaging in Scattering Media.

We describe an advanced DIVER (Deep Imaging Via Emission Recovery) detection system for two-photon fluorescence microscopy that allows imaging in multiple scattering media, including biological tissues, up to a depth of a few mm with micron resolution. This detection system is more sensitive to low level light signals than conventional epi-detection used in two-photon fluorescence microscopes. The DIVER detector efficiently collects scattered emission photons from a wide area of turbid samples at almost any entrance angle in a 2π spherical angle. Using an epi-detection scheme only photons coming from a relatively small area of a sample and at narrow acceptance angle can be detected. The transmission geometry of the DIVER imaging system makes it exceptionally suitable for Second and Third Harmonic Generation (SHG, THG) signal detection. It also has in-depth fluorescence lifetime imaging (FLIM) capability. Using special optical filters with sin-cos spectral response, hyperspectral analysis of images acquired in-depth in scattering media can be performed. The system was successfully employed in imaging of various biological tissues. The DIVER detector can be plugged into a standard microscope stage and used as an external detector with upright commercial two-photon microscopes.

Schlieren two-photon microscopy for phase-contrast imaging.

While simultaneous phase-contrast and two-photon fluorescence imaging in microscopy can bring abundant biomedical information, it is difficult to retrieve phase information from conventional two-photon microscopes. To realize low-cost, in situ phase-contrast and two-photon fluorescence imaging, we propose Schlieren two-photon microscopy, a method that implements phase-contrast imaging on two-photon microscopes. This method involves spatially modulated fluorescence plates, which are made of two-photon fluorescence dyes or upconversion nanoparticles. We demonstrate that the fluorescence intensity fluctuation reflects the phase gradients of the specimen via theoretical analysis, simulations, and experiments. The proposed method is fully compatible with commercial two-photon microscopes, thus enabling widespread applications in live tissue imaging.

Light-Triggered Genome Editing: Cre Recombinase Mediated Gene Editing with Near-Infrared Light.

A light-activated genome editing platform based on the release of enzymes from a plasmonic nanoparticle carrier when exposed to biocompatible near-infrared light pulses is described. The platform relies on the robust affinity of polyhistidine tags to nitrilotriacetic acid in the presence of copper which is attached to double-stranded nucleic acids self-assembled on the gold nanoparticle surface. A protein fusion of the Cre recombinase containing a TAT internalization peptide sequence to achieve endosomal localization is also employed. High-resolution gene knock-in of a red fluorescent reporter is observed using a commercial two-photon microscope. High-throughput irradiation is described to generate useful quantities of edited cells.

Nonlinear optical imaging of extracellular matrix proteins.

Over the last 2 decades, nonlinear imaging methods such as multiharmonic imaging microscopy (MHIM) have become powerful approaches for the label-free visualization of biological structures. Multiharmonic signals are generated when an intense electromagnetic field propagates through a sample that either has a specific molecular orientation or exhibits certain physical properties. It can provide complementary morphological information when integrated with other nonlinear optical imaging techniques such as two-photon excitation (TPE). Here, we present the necessary methodology to implement an integrated approach for multiharmonic and TPE imaging of the mouse aorta using a commercial two-photon microscope. This approach illustrates how to differentiate the microstructure of the mouse aorta that are due to collagen fibrils and elastic laminae under 820 and 1230nm excitation. Our method also demonstrates how to perform multiharmonic generation by reflectance of the forwardly propagating emission signal. The ability to visualize biological samples without additional genetically targeted or chemical stains makes MHIM well suited for studying the morphology of the mouse aorta and has the potential to be applied to other collagen and elastin-rich tissues.

Interfacing with Neural Activity via Femtosecond Laser Stimulation of Drug-Encapsulating Liposomal Nanostructures.

External control over rapid and precise release of chemicals in the brain potentially provides a powerful interface with neural activity. Optical manipulation techniques, such as optogenetics and caged compounds, enable remote control of neural activity and behavior with fine spatiotemporal resolution. However, these methods are limited to chemicals that are naturally present in the brain or chemically suitable for caging. Here, we demonstrate the ability to interface with neural functioning via a wide range of neurochemicals released by stimulating loaded liposomal nanostructures with femtosecond lasers. Using a commercial two-photon microscope, we released inhibitory or excitatory neurochemicals to evoke subthreshold and suprathreshold changes in membrane potential in a live mouse brain slice. The responses were repeatable and could be controlled by adjusting laser stimulation characteristics. We also demonstrate the release of a wider range of chemicals-which previously were impossible to release by optogenetics or uncaging-including synthetic analogs of naturally occurring neurochemicals. In particular, we demonstrate the release of a synthetic receptor-specific agonist that exerts physiological effects on long-term synaptic plasticity. Further, we show that the loaded liposomal nanostructures remain functional for weeks in a live mouse. In conclusion, we demonstrate new techniques capable of interfacing with live neurons, and extendable to in vivo applications.

Practical intravital two‐photon microscopy for immunological research: faster, brighter, deeper

With its emphasis on minimally invasive high-speed imaging of intact tissues at depth, video-rate two-photon microscopy has revolutionized cell biology. This is particularly true in immunology, where the orchestration of cell migration, cell-cell interactions and intracellular signalling events in multiple distinct anatomical compartments within secondary lymphoid organs is fundamental for achieving an effective immune response. Until recently, access to this powerful tool has been limited to a handful of laboratories with the necessary skills and resources to either custom-build or purchase a commercial two-photon microscope. However, with the entry of more commercial vendors into the market and availability of turnkey solutions, two-photon microscopy is now becoming more accessible. Here, we discuss the practical aspects of establishing a basic intravital two-photon microscopy facility specifically for immunological research and how recent advances in ultrafast lasers, non-linear optics and localized photochemistry can be used to build more sophisticated instruments to support applications such as photoactivation and photobleaching, spectral fingerprinting and automated single-cell tracking. In addition, we discuss the next generation of fluorescent dyes and reporter mice and some of the microsurgical principles required to expose the relevant biology to interrogation by two-photon excitation.

Enhanced resolution in two-photon imaging using a TM_01 laser beam at a dielectric interface

We have experimentally demonstrated that the resolution of a commercial two-photon microscope is improved using a TM(01) laser beam. With a water immersion objective having a 1.2 NA, the measured point- spread function has an area of 0.15lambda(2). We used a plane interface between dielectrics instead of an annular aperture to increase the relative contribution of the longitudinal field of the TM(01) laser beam. The results are in agreement with the vectorial diffraction theory established by Richards and Wolf [Proc. R. Soc. Lond. A253, 358 (1959)]. The TM(01) laser beam was produced with a quadrant of half-wave plates. We have also used the same mode converter to generate a TE(01) laser beam with a zero at the beam center.

A two-photon and second-harmonic microscope

Two-photon microscopy has revolutionized life sciences by enabling long-term imaging of living preparations in highly scattering tissue while minimizing photodamage. At the same time, commercial two-photon microscopes are expensive and this has prevented the widespread application of this technique to the biological community. As an alternative to commercial systems, we provide an update of our efforts designing custom-built two-photon instruments by modifying the Olympus FluoView laser scanning confocal microscope. With the newer version of our instrument we modulate the intensity of the laser beam in arbitrary spatiotemporal patterns using a Pockels cell and software control over the scanning. We can also perform simultaneous optical imaging and optical stimulation experiments and combine them with second harmonic generation measurements.