Optimizing the spatial resolution of Channelrhodopsin-2 activation

Over the past few years, the light-gated cation channel Channelrhodopsin-2 (ChR2) has seen a remarkable diversity of applications in neuroscience. However, commonly used wide-field illumination provides poor spatial selectivity for cell stimulation. We explored the potential of focal laser illumination to map photocurrents of individual neurons in sparsely transfected hippocampal slice cultures. Interestingly, the best spatial resolution of photocurrent induction was obtained at the lowest laser power. By adjusting the light intensity to a neuron's spike threshold, we were able to trigger action potentials with a spatial selectivity of less than 30 microm. Experiments with dissociated hippocampal cells suggested that the main factor limiting the spatial resolution was ChR2 current density rather than scattering of the excitation light. We conclude that subcellular resolution can be achieved only in cells with a high ChR2 expression level and that future improved variants of ChR2 are likely to extend the spatial resolution of photocurrent induction to the level of single dendrites.     

Three-photon induced fluorescence of the calcium probe Indo-1.

We report the calcium-dependent emission spectral properties of the calcium probe Indo-1 for three-photon excitation. We found that Indo-1 could be readily excited with the femtosecond pulses from a mode-locked Ti:sapphire laser at 885 nm. This wavelength is too long for two-photon excitation, which is expected to occur for wavelengths no longer than twice the longest single-photon absorption wavelength of 400 nm. For excitation at 885 nm the emission intensity was found to depend on the cube of the laser power, as expected for simultaneous interaction with three photons. At wavelengths below 840 nm the emission intensity depends on the square of the laser power, indicating two-photon excitation at shorter wavelengths. The intensity decays of Indo-1 were found to be dependent on Ca2+ and essentially identical for one- and three-photon excitation. The emission anisotropy of Indo-1 was found to be considerably higher for three-photon excitation than for one-photon excitation, consistent with cos6 theta photoselection, as compared with cos2 theta photoselection for one-photon excitation. The high values of the anisotropy are in agreement with those expected for a three-photon process. Calcium-dependent emission spectra were observed for Indo-1 with three-photon excitation, demonstrating that three-photon excitation of Indo-1 can be used for calcium imaging by emission intensity ratio measurements. The calcium-dependent emission spectra indicate a higher three-photon cross-section for the calcium-free form of Indo-1 than for the calcium-bound form. The possible advantages of three-photon excitation include the availability of the appropriate wavelengths with solid-state lasers, enhanced spatial resolution due to a reduced size of the excited volume, absence of light quenching, and possibly high selectivity of the three-photon excitation process.     

Laser tweezers and multiphoton microscopes in life sciences

Near infrared (NIR) laser microscopy enables optical micromanipulation, piconewton force determination, and sensitive fluorescence studies by laser tweezers. Otherwise, fluorescence images with high spatial and temporal resolution of living cells and tissues can be obtained via non-resonant fluorophore excitation with multiphoton NIR laser scanning microscopes. Furthermore, NIR femtosecond laser pulses at TW/cm2 intensities can be used to realize non-invasive contact-free surgery of nanometer-sized structures within living cells and tissues. Applications of these novel versatile NIR laser-based tools for the determination of motility forces, coenzyme and chlorophyll imaging, three-dimensional multigene detection, non-invasive optical sectioning of tissues ("optical biopsy"), functional protein imaging, and nanosurgery of chromosomes are described.     

Assessment of fluorochromes for two-photon laser scanning microscopy of biofilms

A major limitation for the use of two-proton laser scanning microscopy (2P-LSM) in biofilm and other studies is the lack of a thorough understanding of the excitation-emission responses of potential fluorochromes. In order to use 2P-LSM, the utility of various fluorochromes and probes specific for a range of biofilm constituents must be evaluated. The fluorochromes tested in this study included classical nucleic acid-specific stains, such as acridine orange (AO) and 4",6"-diamidino-2-phenylindole (DAPI), as well as recently developed stains. In addition, stains specific for biofilm extracellular polymeric substances (EPS matrix components) were tested. Two-photon excitation with a Ti/Sapphire laser was carried out at wavelengths from 760 to 900 nm in 10-nm steps. It was found that autofluorescence of phototrophic organisms (cyanobacteria and green algae) resulted in strong signals for the entire excitation range. In addition, the coenzyme F(420)-related autofluorescence of methanogenic bacteria could be used to obtain images of dense aggregates (excitation wavelength, 780 nm). The intensities of the emission signals for the nucleic acid-specific fluorochromes varied. For example, the intensities were similar for excitation wavelengths ranging from 780 to 900 nm for AO but were higher for a narrower range, 780 to 810 nm, for DAPI. In selective excitation, fading, multiple staining, and combined single-photon-two-photon studies, the recently developed nucleic acid-specific fluorochromes proved to be more suitable regardless of whether they are intended for living or fixed samples. Probes specific for proteins and glycoconjugates allowed two-photon imaging of polymeric biofilm constituents. Selective excitation-emission was observed for Calcofluor White M2R (780 to 800 nm) and SyproOrange (880 to 900 nm). In addition, fluor-conjugated concanavalin A lectins were examined and provided acceptable two-photon emission signals at wavelengths ranging from 780 to 800 nm. Finally, CellTracker, a fluorochrome suitable for long-term labeling of microbial eucaryote cells, was found to give strong emission at wavelengths ranging from 770 to 810 nm. If fluorochromes have the same two-photon excitation cross section, they are suitable for multiple staining and multichannel recording. Generally, if an appropriate excitation wavelength and fluorochrome were used, it was possible to obtain more highly resolved images for thick biofilm samples with two-photon laser microscopy than with conventional single-photon laser microscopy. Due to its potential for higher resolution in light-scattering tissue-like material, such as biofilms, and extremely localized excitation, 2P-LSM is a valuable addition to conventional confocal laser scanning microscopy with single-photon excitation. However, further development of the method and basic research are necessary to take full advantage of nonlinear excitation in studies of interfacial microbial ecology.     

Ca2+ fluorescence imaging with pico- and femtosecond two-photon excitation: signal and photodamage.

The signal and limitations of calcium florescence imaging using nonresonant multiphoton absorption of near-infrared femto- and picosecond laser pulses were examined. The fluorescence changes of various Ca(2+)-indicators induced by transient increases of the intradendritic calcium concentration were evaluated by evoking physiological activity in neocortical neurons in rat brain slices. Photodamage was noticeable as irreversible changes in the parameters describing the calcium fluorescence transients. At higher two-photon excitation rates, a great variety of irregular functional and structural alterations occurred. Thus, signal and observation time were limited by phototoxic effects. At lower excitation rates, photodamage accumulated linearly with exposure time. Femtosecond and picosecond laser pulses were directly compared with respect to this cumulative photodamage. The variation of the pulse length at a constant two-photon excitation rate indicated that a two-photon excitation mechanism is mainly responsible for the cumulative photodamage within the investigated window of 75 fs to 3.2 ps. As a direct consequence, at low excitation rates, the same image quality is achieved irrespective of whether two-photon Ca(2+)-imaging is carried out with femto- or picosecond laser pulses.     

Highly nonlinear photodamage in two-photon fluorescence microscopy

Two-photon fluorescence excitation is being increasingly used in laser scan microscopy due to very low photodamage induced by this technique under normal operation. However, excitation intensity has to be kept low, because nonlinear photodamage sets in when laser power is increased above a certain threshold. We studied this kind of damage in bovine adrenal chromaffin cells, using two different indicators of damage: changes in resting [Ca(2+)] level and the degranulation reaction. In agreement with previous studies, we found that, for both criteria, damage is proportional to the integral (over space and time) of light intensity raised to a power approximately 2.5. Thus, widening the laser pulse shape at constant average intensity both in time and in focal volume is beneficial for avoiding this kind of damage. Both measures, of course, reduce the two-photon fluorescence excitation. However, loss of signal can be compensated by increasing excitation power, such that, at constant damaging potential, signals may be even larger with long pulses and large focal volumes, because the exponent of the power law of damage is higher (mu approximately 2.5) than that of the two-photon signal (mu approximately 2).     

Two-photon activation and excitation properties of PA-GFP in the 720-920-nm region

This report covers the two-photon activation and excitation properties of the PA-GFP, a photoactivatable variant of the Aequorea victoria green fluorescent protein in the spectral region from 720 to 920 nm. It is known from this special form of the molecule that it has an increased level of fluorescence emission when excited at 488 nm after irradiation at lambda approximately 413 nm, under single-photon excitation conditions. Here, we show that upon two-photon irradiation, PA-GFP yields activation in the spectral region from 720 to 840 nm. After photoactivation, the excitation spectrum shifts maintaining the very same emission spectrum of the single-photon case for the native and photoactivated protein. Additionally, when comparing the conventional photoactivation at lambda = 405 nm with a two-photon one, a sharper and better controllable three-dimensional volume of activation is obtained.     

In vivo two-photon laser-scanning microscopy of Ca2+ dynamics in visual motion-sensitive neurons

We applied two-photon laser-scanning microscopy (TPLSM) to motion-sensitive visual interneurons of the fly to study Ca(2+) dynamics in vivo at a higher spatial and temporal resolution than possible with conventional fluorescence microscopy. Based on a custom-built two-photon microscope, we performed line scans to measure changes in presynaptic Ca(2+) concentrations elicited by visual stimulation. We used a fast avalanche photodiode (APD) with a high quantum efficiency to detect even low levels of emitted fluorescence. Our experiments show that our in vivo preparation is amenable to TPLSM: with excitation intensities low enough not to cause photodamage, activity-dependent fluorescence changes of Ca(2+)-sensitive dyes can be detected in small neuronal branches. The performance of two-photon and conventional Ca(2+) imaging carried out consecutively at the same neuron is compared and it is demonstrated that two-photon imaging allows us to detect differences in Ca(2+) dynamics between individual neurites.     

光敏感通道(Channelrhodopsin-2)——神经回路功能和神经系统疾病研究的新工具

光敏感通道(channelrhodopsin-2,ChR2)是一种受光脉冲控制的具有7次跨膜结构的非选择性阳离子通道蛋白,自1991年从莱茵衣藻中发现后被许多实验室所关注.依据ChR2可以快速形成光电流,使细胞发生去极化反应的电生理特性,ChR2已被广泛应用于神经系统的研究.与传统的神经系统研究方法如电生理技术、神经药理学方法相比,用光脉冲控制带有ChR2的神经元的活动,具有更高的空间选择性和特异性.ChR2作为光基因技术的核心组成部分,对神经科学是一个崭新的应用前景广泛的研究工具.近年来ChR2不仅应用于视觉、躯体感觉、听觉和嗅觉等多条感觉神经回路的形态和功能研究,还被应用于各种临床神经系统疾病的研究.本文总结了目前ChR2在神经系统中的研究进展,并对ChR2未来的应用前景作了进一步展望.     

Optogenetic versus Electrical Stimulation of Human Cardiomyocytes: Modeling Insights

Optogenetics provides an alternative to electrical stimulation to manipulate membrane voltage, and trigger or modify action potentials (APs) in excitable cells. We compare biophysically and energetically the cellular responses to direct electrical current injection versus optical stimulation mediated by genetically expressed light-sensitive ion channels, e.g., Channelrhodopsin-2 (ChR2). Using a computational model of ChR2(H134R mutant), we show that both stimulation modalities produce similar-in-morphology APs in human cardiomyocytes, and that electrical and optical excitability vary with cell type in a similar fashion. However, whereas the strength-duration curves for electrical excitation in ventricular and atrial cardiomyocytes closely follow the theoretical exponential relationship for an equivalent RC circuit, the respective optical strength-duration curves significantly deviate, exhibiting higher nonlinearity. We trace the origin of this deviation to the waveform of the excitatory current—a nonrectangular self-terminating inward current produced in optical stimulation due to ChR2 kinetics and voltage-dependent rectification. Using a unifying charge measure to compare energy needed for electrical and optical stimulation, we reveal that direct electrical current injection (rectangular pulse) is more efficient at short pulses, whereas voltage-mediated negative feedback leads to self-termination of ChR2 current and renders optical stimulation more efficient for long low-intensity pulses. This applies to cardiomyocytes but not to neuronal cells (with much shorter APs). Furthermore, we demonstrate the cell-specific use of ChR2 current as a unique modulator of intrinsic activity, allowing for optical control of AP duration in atrial and, to a lesser degree, in ventricular myocytes. For self-oscillatory cells, such as Purkinje, constant light at extremely low irradiance can be used for fine control of oscillatory frequency, whereas constant electrical stimulation is not feasible due to electrochemical limitations. Our analysis offers insights for designing future new energy-efficient stimulation strategies in heart or brain.     

Inhibition of Murine Bladder Cancer Cell Growth In Vitro by Photocontrollable siRNA Based on Upconversion Fluorescent Nanoparticles

This study provides a unique approach to activate caged small interfering RNAs (siRNAs) using indirect UV light emitted by the near-infrared (NIR)-to-UV upconversion process to achieve high spatial and temporal gene interference patterns. siRNA molecules against the anti-apoptotic gene survivin was caged by light-sensitive molecules (4,5-dimethoxy-2-nitroacetophenone, DMNPE), which rendered them temporarily non-functional. NIR-to-UV NaYF₄:Yb,Tm upconversion nanoparticles (UCPs) served as delivery vehicles and activators of the caged siRNA molecules in murine bladder cancer cells (MB49 cell line). Upconverted UV light at 355 nm was emitted from the NIR-irradiated UCPs, which well coincided with the wavelength needed to uncage DMNPE. Consequently, UV light acted as a switch to uncage the delivered siRNA molecule, thereby rendering fully functional for exerting its therapeutic effect in the bladder cancer cells. To achieve the highest RNA interference efficiency, conditions such as time after cellular uptake, excitation time, UCPs concentration and laser power were optimized. Results showed that 200 µg/mL nanoparticle concentration combined with 12 h incubation with MB49 cells and excitation with NIR laser at 100 mW power for 15 min provided the ideal interference efficiency and strongest induction of MB49 cell death. Our findings demonstrate the potential biological application of UCPs in treating bladder cancer by a novel therapeutic approach.     

Channelrhodopsin-2 and optical control of excitable cells

Electrically excitable cells are important in the normal functioning and in the pathophysiology of many biological processes. These cells are typically embedded in dense, heterogeneous tissues, rendering them difficult to target selectively with conventional electrical stimulation methods. The algal protein Channelrhodopsin-2 offers a new and promising solution by permitting minimally invasive, genetically targeted and temporally precise photostimulation. Here we explore technological issues relevant to the temporal precision, spatial targeting and physiological implementation of ChR2, in the context of other photostimulation approaches to optical control of excitable cells.     

Femtosecond near-infrared laser pulses elicit generation of reactive oxygen species in mammalian cells leading to apoptosis-like death

Two-photon excitation-based near-infrared (NIR) laser scanning microscopy is currently emerging as a new and versatile alternative to conventional confocal laser scanning microscopy, particularly for vital cell imaging in life sciences. Although this innovative microscopy has several advantages such as highly localized excitation, higher penetration depth, reduced photobleaching and photodamage, and improved signal to noise ratio, it has, however, recently been evidenced that high-power NIR laser irradiation can drastically inhibit cell division and induce cell death. In the present study we have investigated the cellular responses of unlabeled rat kangaroo kidney epithelium (PtK2) cells to NIR femtosecond laser irradiation. We demonstrate that NIR 170-fs laser pulses operating at 80-MHz pulse repetition frequency and at mean power of > or = 7 mW evoke generation of reactive oxygen species (ROS) such as H2O2 that can be visualized in situ by standard in vivo cytochemical analysis using Ni-3,3'-diaminobenzidine (Ni-DAB) as well as with a recently developed fluorescent probe Jenchrom px blue. The formation of the Ni-DAB reaction product as well as that of Jenchrom was relatively more pronounced when irradiated cells were incubated in alkaline solution (pH 8) than in those incubated in acidic solution (pH 6), suggesting peroxisomal localization of these reaction products. Two-photon time-lapse imaging of the internalization of the cell impermeate fluorescent dye propidium iodide revealed that the integrity of the plasma membrane of NIR irradiated cells is drastically compromised. Visualization of the nuclei with DNA-specific fluorescent probes such as 4',6-diamidino-2-phenylindole 24 h postirradiation further provided tangible evidence that the nuclei of these cells undergo several deformations and eventual fragmentation. That these NIR irradiated cells die by apoptosis has been established by in situ detection of DNA strand breaks using the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling method. Because the reactive oxygen species such as H2O2 and OH* can cause noxious effects such as cell membrane injury by peroxidation of polyunsaturated lipids and proteins and oxidative phosphorylation, and alterations of ATP-dependent Ca2+ pumps, these ROS are likely to contribute to drastic cytological alterations observed in this study following NIR irradiation. Taken together, we have established that NIR laser irradiations at mean power > or = 7 mW delivered at pulse duration time of 170 fs generally used in two- and multiphoton microscopes cause oxidative stress (1) evoking production of ROS, (2) resulting in membrane barrier dysfunction, (3) inducing structural deformations and fragmentation of the nuclei as well as DNA strand breaks, (4) leading to cell death by apoptosis.     

Expanding Two-Photon Intravital Microscopy to the Infrared by Means of Optical Parametric Oscillator

Chronic inflammation in various organs, such as the brain, implies that different subpopulations of immune cells interact with the cells of the target organ. To monitor this cellular communication both morphologically and functionally, the ability to visualize more than two colors in deep tissue is indispensable. Here, we demonstrate the pronounced power of optical parametric oscillator (OPO)-based two-photon laser scanning microscopy for dynamic intravital imaging in hardly accessible organs of the central nervous and of the immune system, with particular relevance for long-term investigations of pathological mechanisms (e.g., chronic neuroinflammation) necessitating the use of fluorescent proteins. Expanding the wavelength excitation farther to the infrared overcomes the current limitations of standard Titanium:Sapphire laser excitation, leading to 1), simultaneous imaging of fluorophores with largely different excitation and emission spectra (e.g., GFP-derivatives and RFP-derivatives); and 2), higher penetration depths in tissue (up to 80%) at higher resolution and with reduced photobleaching and phototoxicity. This tool opens up new opportunities for deep-tissue imaging and will have a tremendous impact on the choice of protein fluorophores for intravital applications in bioscience and biomedicine, as we demonstrate in this work.     

Non-Scanning Fiber-Optic Near-Infrared Beam Led to Two-Photon Optogenetic Stimulation In-Vivo

Stimulation of specific neurons expressing opsins in a targeted region to manipulate brain function has proved to be a powerful tool in neuroscience. However, the use of visible light for optogenetic stimulation is invasive due to low penetration depth and tissue damage owing to larger absorption and scattering. Here, we report, for the first time, in-depth non-scanning fiber-optic two-photon optogenetic stimulation (FO-TPOS) of neurons in-vivo in transgenic mouse models. In order to optimize the deep-brain stimulation strategy, we characterized two-photon activation efficacy at different near-infrared laser parameters. The significantly-enhanced in-depth stimulation efficiency of FO-TPOS as compared to conventional single-photon beam was demonstrated both by experiments and Monte Carlo simulation. The non-scanning FO-TPOS technology will lead to better understanding of the in-vivo neural circuitry because this technology permits more precise and less invasive anatomical delivery of stimulation.     

Living cell functions and morphology revealed by two-photon microscopy in intact neural and secretory organs

Laser light microscopy enables observation of various simultaneously occurring events in living cells. This capability is important for monitoring the spatiotemporal patterns of the molecular interactions underlying such events. Two-photon excited fluorescence microscopy (two-photon microscopy), a technology based on multiphoton excitation, is one of the most promising candidates for such imaging. The advantages of two-photon microscopy have spurred wider adoption of the method, especially in neurological studies. Multicolor excitation capability, one advantage of two-photon micro-scopy, has enabled the quantification of spatiotemporal patterns of [Ca(2+)](i) and single episodes of fusion pore openings during exocytosis. In pancreatic acinar cells, we have successfully demonstrated the existence of "sequential compound exocytosis" for the first time, a process which has subsequently been identified in a wide variety of secretory cells including exocrine, endocrine and blood cells. Our newly developed method, the two-photon extracellular polar-tracer imaging-based quantification (TEPIQ) method, can be used for determining fusion pores and the diameters of vesicles smaller than the diffraction-limited resolution. Furthermore, two-photon microscopy has the demonstrated capability of obtaining cross-sectional images from deep layers within nearly intact tissue samples over long observation times with excellent spatial resolution. Recently, we have successfully observed a neuron located deeper than 0.9 mm from the brain cortex surface in an anesthetized mouse. This microscopy also enables the monitoring of long-term changes in neural or glial cells in a living mouse. This minireview describes both the current and anticipated capabilities of two-photon microscopy, based on a discussion of previous publications and recently obtained data.     

Light activation of channelrhodopsin-2 in excitable cells of Caenorhabditis elegans triggers rapid behavioral responses

For studying the function of specific neurons in their native circuitry, it is desired to precisely control their activity. This often requires dissection to allow accurate electrical stimulation or neurotransmitter application , and it is thus inherently difficult in live animals, especially in small model organisms. Here, we employed channelrhodopsin-2 (ChR2), a directly light-gated cation channel from the green alga Chlamydomonas reinhardtii, in excitable cells of the nematode Caenorhabditis elegans, to trigger specific behaviors, simply by illumination. Channelrhodopsins are 7-transmembrane-helix proteins that resemble the light-driven proton pump bacteriorhodopsin , and they also utilize the chromophore all-trans retinal, but to open an intrinsic cation pore. In muscle cells, light-activated ChR2 evoked strong, simultaneous contractions, which were reduced in the background of mutated L-type, voltage-gated Ca2+-channels (VGCCs) and ryanodine receptors (RyRs). Electrophysiological analysis demonstrated rapid inward currents that persisted as long as the illumination. When ChR2 was expressed in mechanosensory neurons, light evoked withdrawal behaviors that are normally elicited by mechanical stimulation. Furthermore, ChR2 enabled activity of these neurons in mutants lacking the MEC-4/MEC-10 mechanosensory ion channel . Thus, specific neurons or muscles expressing ChR2 can be quickly and reversibly activated by light in live and behaving, as well as dissected, animals.     

Quantitative determination of localized tissue oxygen concentration in vivo by two-photon excitation phosphorescence lifetime measurements.

This study describes the use of two-photon excitation phosphorescence lifetime measurements for quantitative oxygen determination in vivo. Doubling the excitation wavelength of Pd-porphyrin from visible light to the infrared allows for deeper tissue penetration and a more precise and confined selection of the excitation volume due to the nonlinear two-photon effect. By using a focused laser beam from a 1,064-nm Q-switched laser, providing 10-ns pulses of 10 mJ, albumin-bound Pd-porphyrin was effectively excited and oxygen-dependent decay of phosphorescence was observed. In vitro calibration of phosphorescence lifetime vs. oxygen tension was performed. The obtained calibration constants were kq = 356 Torr(-1) x s(-1) (quenching constant) and tau0 = 550 micros (lifetime at zero-oxygen conditions) at 37 degrees C. The phosphorescence intensity showed a squared dependency to the excitation intensity, typical for two-photon excitation. In vivo demonstration of two-photon excitation phosphorescence lifetime measurements is shown by step-wise PO2 measurements through the cortex of rat kidney. It is concluded that quantitative oxygen measurements can be made, both in vitro and in vivo, using two-photon excitation oxygen-dependent quenching of phosphorescence. The use of two-photon excitation has the potential to lead to new applications of the phosphorescence lifetime technique, e.g., noninvasive oxygen scanning in tissue at high spatial resolution. To our knowledge, this is the first report in which two-photon excitation is used in the setting of oxygen-dependent quenching of phosphorescence lifetime measurements.     

A custom confocal and two-photon digital laser scanning microscope

We describe a custom one-photon (confocal) and two-photon all-digital (photon counting) laser scanning microscope. The confocal component uses two avalanche photodiodes (APDs) as the fluorescence detector to achieve high sensitivity and to overcome the limited photon counting rate of a single APD ( approximately 5 MHz). The confocal component is approximately nine times more efficient than our commercial confocal microscope (fluorophore fluo 4). Switching from one-photon to two-photon excitation mode (Ti:sapphire laser) is accomplished by moving a single mirror beneath the objective lens. The pulse from the Ti:sapphire laser is 109 fs in duration at the specimen plane, and average power is approximately 5 mW. Two-photon excited fluorescence is detected by a fast photomultiplier tube. With a x63 1.4 NA oil-immersion objective, the resolution of the confocal system is 0.25 microm laterally and 0.52 microm axially. For the two-photon system, the corresponding values are 0.28 and 0.82 microm. The system is advantageous when excitation intensity must be limited, when fluorescence is low, or when thick, scattering specimens are being studied (with two-photon excitation).     

Two-Photon Excitation STED Microscopy in Two Colors in Acute Brain Slices

Many cellular structures and organelles are too small to be properly resolved by conventional light microscopy. This is particularly true for dendritic spines and glial processes, which are very small, dynamic, and embedded in dense tissue, making it difficult to image them under realistic experimental conditions. Two-photon microscopy is currently the method of choice for imaging in thick living tissue preparations, both in acute brain slices and in vivo. However, the spatial resolution of a two-photon microscope, which is limited to ∼350 nm by the diffraction of light, is not sufficient for resolving many important details of neural morphology, such as the width of spine necks or thin glial processes. Recently developed superresolution approaches, such as stimulated emission depletion microscopy, have set new standards of optical resolution in imaging living tissue. However, the important goal of superresolution imaging with significant subdiffraction resolution has not yet been accomplished in acute brain slices. To overcome this limitation, we have developed a new microscope based on two-photon excitation and pulsed stimulated emission depletion microscopy, which provides unprecedented spatial resolution and excellent experimental access in acute brain slices using a long-working distance objective. The new microscope improves on the spatial resolution of a regular two-photon microscope by a factor of four to six, and it is compatible with time-lapse and simultaneous two-color superresolution imaging in living cells. We demonstrate the potential of this nanoscopy approach for brain slice physiology by imaging the morphology of dendritic spines and microglial cells well below the surface of acute brain slices.