Increasing fluorescence lifetime for resolution improvement in stimulated emission depletion nanoscopy

Super‐resolution microscopy (SRM) has had a substantial impact on the biological sciences due to its ability to observe tiny objects less than 200 nm in size. Stimulated emission depletion (STED) microscopy represents a major category of these SRM techniques that can achieve diffraction‐unlimited resolution based on a purely optical modulation of fluorescence behaviors. Here, we investigated how the laser beams affect fluorescence lifetime in both confocal and STED imaging modes. The results showed that with increasing illumination time, the fluorescence lifetime in two kinds of fluorescent microspheres had an obvious change in STED imaging mode, compared with that in confocal imaging mode. As a result, the reduction of saturation intensity induced by the increase of fluorescence lifetime can improve the STED imaging resolution at the same depletion power. The phenomenon was also observed in Star635P‐labeled human Nup153 in fixed HeLa cells, which can be treated as a reference for the synthesis of fluorescent labels with the sensitivity to the surrounding environment for resolution improvement in STED nanoscopy.      

STED Nanoscopy with Time-Gated Detection: Theoretical and Experimental Aspects

In a stimulated emission depletion (STED) microscope the region in which fluorescence markers can emit spontaneously shrinks with continued STED beam action after a singular excitation event. This fact has been recently used to substantially improve the effective spatial resolution in STED nanoscopy using time-gated detection, pulsed excitation and continuous wave (CW) STED beams. We present a theoretical framework and experimental data that characterize the time evolution of the effective point-spread-function of a STED microscope and illustrate the physical basis, the benefits, and the limitations of time-gated detection both for CW and pulsed STED lasers. While gating hardly improves the effective resolution in the all-pulsed modality, in the CW-STED modality gating strongly suppresses low spatial frequencies in the image. Gated CW-STED nanoscopy is in essence limited (only) by the reduction of the signal that is associated with gating. Time-gated detection also reduces/suppresses the influence of local variations of the fluorescence lifetime on STED microscopy resolution.     

Dual‐color STED super‐resolution microscope using a single laser source

STED (stimulated emission depletion) microscopy is one of the most promising super‐resolution fluorescence microscopies，due to its fast imaging and ultra‐high resolution. In this paper, we present a dual‐color STED microscope with a single laser source. Polarization beam splitters are used to separate the output from a supercontinuum laser source into four laser beams, including two excitation beams (488, 635 nm) and two depletion beams (592, 775 nm). These four laser beams are then used to build a low cost dual‐color STED system to achieve a spatial resolution of 75 nm in cell samples.     

Live-Cell Superresolution Imaging by Pulsed STED Two-Photon Excitation Microscopy

Two-photon laser scanning microscopy (2PLSM) allows fluorescence imaging in thick biological samples where absorption and scattering typically degrade resolution and signal collection of one-photon imaging approaches. The spatial resolution of conventional 2PLSM is limited by diffraction, and the near-infrared wavelengths used for excitation in 2PLSM preclude the accurate imaging of many small subcellular compartments of neurons. Stimulated emission depletion (STED) microscopy is a superresolution imaging modality that overcomes the resolution limit imposed by diffraction and allows fluorescence imaging of nanoscale features. Here, we describe the design and operation of a superresolution two-photon microscope using pulsed excitation and STED lasers. We examine the depth dependence of STED imaging in acute tissue slices and find enhancement of 2P resolution ranging from approximately fivefold at 20 μm to approximately twofold at 90-μm deep. The depth dependence of resolution is found to be consistent with the depth dependence of depletion efficiency, suggesting resolution is limited by STED laser propagation through turbid tissue. Finally, we achieve live imaging of dendritic spines with 60-nm resolution and demonstrate that our technique allows accurate quantification of neuronal morphology up to 30-μm deep in living brain tissue.     

A new filtering technique for removing anti‐Stokes emission background in gated CW‐STED microscopy

Stimulated emission depletion (STED) microscopy is a prominent approach of super‐resolution optical microscopy, which allows cellular imaging with so far unprecedented unlimited spatial resolution. The introduction of time‐gated detection in STED microscopy significantly reduces the (instantaneous) intensity required to obtain sub‐diffraction spatial resolution. If the time‐gating is combined with a STED beam operating in continuous wave (CW), a cheap and low labour demand implementation is obtained, the so called gated CW‐STED microscope. However, time‐gating also reduces the fluorescence signal which forms the image. Thereby, background sources such as fluorescence emission excited by the STED laser (anti‐Stokes fluorescence) can reduce the effective resolution of the system. We propose a straightforward method for subtraction of anti‐Stokes background. The method hinges on the uncorrelated nature of the anti‐Stokes emission background with respect to the wanted fluorescence signal. The specific importance of the method towards the combination of two‐photon‐excitation with gated CW‐STED microscopy is demonstrated. (© 2014 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Enhanced lateral resolution in continuous wave stimulated emission depletion microscopy using tightly focused annular radially polarized excitation beam

The lateral resolution of continuous wave (CW) stimulated emission depletion (STED) microscopy is enhanced about 12% by applying annular‐shaped amplitude modulation to the radially polarized excitation beam. A focused annularly filtered radially polarized excitation beam provides a more condensed point spread function (PSF), which contributes to enhance effective STED resolution of CW STED microscopy. Theoretical analysis shows that the FWHM of the effective PSF on the detection plane is smaller than for conventional CW STED. Simulation shows the donut‐shaped PSF of the depletion beam and confocal optics suppress undesired PSF sidelobes. Imaging experiments agree with the simulated resolution improvement.      

Visualization of the Immunological Synapse by Dual Color Time-gated Stimulated Emission Depletion (STED) Nanoscopy

Natural killer cells form tightly regulated, finely tuned immunological synapses (IS) in order to lyse virally infected or tumorigenic cells. Dynamic actin reorganization is critical to the function of NK cells and the formation of the IS. Imaging of F-actin at the synapse has traditionally utilized confocal microscopy, however the diffraction limit of light restricts resolution of fluorescence microscopy, including confocal, to approximately 200 nm. Recent advances in imaging technology have enabled the development of subdiffraction limited super-resolution imaging. In order to visualize F-actin architecture at the IS we recapitulate the NK cell cytotoxic synapse by adhering NK cells to activating receptor on glass. We then image proteins of interest using two-color stimulated emission depletion microscopy (STED). This results in <80 nm resolution at the synapse. Herein we describe the steps of sample preparation and the acquisition of images using dual color STED nanoscopy to visualize F-actin at the NK IS. We also illustrate optimization of sample acquisition using Leica SP8 software and time-gated STED. Finally, we utilize Huygens software for post-processing deconvolution of images.     

Two-color STED microscopy of living synapses using a single laser-beam pair

The advent of superresolution microscopy has opened up new research opportunities into dynamic processes at the nanoscale inside living biological specimens. This is particularly true for synapses, which are very small, highly dynamic, and embedded in brain tissue. Stimulated emission depletion (STED) microscopy, a recently developed laser-scanning technique, has been shown to be well suited for imaging living synapses in brain slices using yellow fluorescent protein as a single label. However, it would be highly desirable to be able to image presynaptic boutons and postsynaptic spines, which together form synapses, using two different fluorophores. As STED microscopy uses separate laser beams for fluorescence excitation and quenching, incorporation of multicolor imaging for STED is more difficult than for conventional light microscopy. Although two-color schemes exist for STED microscopy, these approaches have several drawbacks due to their complexity, cost, and incompatibility with common labeling strategies and fluorophores. Therefore, we set out to develop a straightforward method for two-color STED microscopy that permits the use of popular green-yellow fluorescent labels such as green fluorescent protein, yellow fluorescent protein, Alexa Fluor 488, and calcein green. Our new (to our knowledge) method is based on a single-excitation/STED laser-beam pair to simultaneously excite and quench pairs of these fluorophores, whose signals can be separated by spectral detection and linear unmixing. We illustrate the potential of this approach by two-color superresolution time-lapse imaging of axonal boutons and dendritic spines in living organotypic brain slices.     

Nanoscopy in a living multicellular organism expressing GFP

We report superresolution fluorescence microscopy in an intact living organism, namely Caenorhabditis elegans nematodes expressing green fluorescent protein (GFP)-fusion proteins. We also superresolve, by stimulated emission depletion (STED) microscopy, living cultured cells, demonstrating that STED microscopy with GFP can be widely applied. STED with GFP can be performed with both pulsed and continuous-wave lasers spanning a wide wavelength range from at least 556–592 nm. Acquiring subdiffraction resolution images within seconds enables the recording of movies revealing structural dynamics. These results demonstrate that numerous microscopy studies of live samples employing GFP as the marker can be performed at subdiffraction resolution.     

Coaligned Dual-Channel STED Nanoscopy and Molecular Diffusion Analysis at 20 nm Resolution

We report on a fiber laser-based stimulated emission-depletion microscope providing down to ∼20 nm resolution in raw data images as well as 15–19 nm diameter probing areas in fluorescence correlation spectroscopy. Stimulated emission depletion pulses of nanosecond duration and 775 nm wavelength are used to silence two fluorophores simultaneously, ensuring offset-free colocalization analysis. The versatility of this superresolution method is exemplified by revealing the octameric arrangement of Xenopus nuclear pore complexes and by quantifying the diffusion of labeled lipid molecules in artificial and living cell membranes.Since its first demonstration in (live) cell imaging (1), stimulated emission depletion (STED) fluorescence microscopy has been realized in many variants. Particularly, the key phenomenon employed in this method, namely switching fluorophores transiently off by stimulated emission, has been accomplished with laser pulses varying from picoseconds to nanoseconds in duration, and from kHz to MHz in repetition rate. Because continuous-wave beams are suitable as well (2), STED microscopy has been implemented with rather different laser systems, ranging from model-locked femtosecond to continuous-wave laser diodes (3,4). Although it underscores the versatility of STED to modulate the fluorescence capability of a fluorophore, this wide range of options may confuse adopters when balancing simplicity, applicability, and resolution gain. The situation is exacerbated when implementing pairs of excitation and STED beams for dual-color colocalization studies (5,6).Here we report on a simple arrangement providing dual-color STED nanoscopy (Fig. 1) and molecular diffusion quantification down to ∼20 nm in (living) cells. The presented dual-channel STED microscope utilizes a single fiber laser providing a 20-MHz train of 775 nm wavelength pulses of 1.2-ns duration. This compact laser source enables STED on fluorophores emitting in the orange to red range. Specifically, we applied this laser on the orange dyes Atto590 and Atto594 (excitation: 595 nm; detection: 620 ± 20 nm), and the red dyes KK114 and Abberior Star635P (excitation: 640 nm; detection: 670 ± 20 nm). Although the spectra of the dyes are partially overlapping, the individual color channels can be separated without data processing (see Fig. S1 and Fig. S2 in the Supporting Material). Both channels are recorded simultaneously within 50 ns, using temporally interleaved pulsed excitation in combination with time-gated detection (5,7,8).Open in a separate windowFigure 1Fluorescence nanoscopy of protein complexes with a compact near-infrared nanosecond-pulsed STED microscope. (A) STED reveals immunolabeled subunits in amphibian NPC; raw data smoothed with a Gaussian filter extending over 14 nm in FWHM. The diameter of the octameric gp210 ring is established as ∼160 nm. Scale bar, 500 nm. (B) Individual NPC image showing eight antibody-labeled gp210 homodimers as 20–40 nm sized units and a 80 nm-sized localization of the subunits in the central channel.Because in STED microscopy, the STED doughnuts firmly determine the position of the fluorescently active molecules, the use of a single doughnut for both fluorophores guarantees that the two color channels are almost perfectly coaligned. The use of the doughnut even counteracts misalignments of the confocal excitation and detection channels (Fig. 2, and see Fig. S3), making STED microscopy particularly powerful for colocalization measurements.Open in a separate windowFigure 2Determination of the colocalization accuracy. Xenopus A6 cells, labeled with an antiserum against multiple NUP subunits in the central NPC channel and two secondary antibodies decorated with the fluorophores Abberior STAR635P and Atto594 were imaged by STED microscopy. (A) Upon overlaying both channels, a high degree of colocalization is directly visible. Scale bar, 200 nm. (B) Quantification of the colocalization by cross correlation of much larger images (see Fig. S3). The correlation is maximal for zero displacement of the images, proving colocalization. (C) Confocal image of monocolored fluorescent beads taken with improperly coaligned excitation beams (left). Improper coalignment spoils the colocalization accuracy in confocal imaging; the two channels should be perfectly coaligned, but they show a false offset as indicated by the color difference. The offset is quantified by the cross correlation of the two channels (right). (D) The STED image of the same beads (left) not only shows 10-fold improved resolution over the confocal image in panel C, but also improved colocalization, again quantified by cross correlation (right). Thus, by predetermining the position of emission, the STED doughnut counteracts errors induced by imperfect coalignment of the two confocal color channels (for details, see Fig. S3). Scale bars = 100 nm.The cross section for stimulated emission is lower at 775 nm as compared to that found at somewhat shorter wavelengths (5), yet STED pulse energies of ∼7 nJ in the focus are sufficient to yield a resolution of ∼30 nm and ∼20 nm in the orange and red channels, respectively (see Fig. S4). In addition, due to the lower peak intensity, the 1.2 ns pulses are likely to induce less nonlinear absorption and hence less photostress as compared to their more commonly used <0.2 ns counterparts (8,9). On the other hand, the pulses are only 2–4 times shorter than the typical lifetime of the excited state, which lessens their STED efficiency. This slight reduction is neutralized here by detecting photons emitted ∼1 ns after excitation (5,7,8).The potential of this straightforward implementation of STED microscopy is evident when imaging immunolabeled nuclear pore complexes (NPCs) of cultured Xenopus cells. Contrary to the confocal recording, STED microscopy reveals subunits of this protein complex, specifically the typical eightfold symmetry of its peripheral transmembrane protein gp210, along with a set of proteins in the central pore channel (Fig. 1, and see Fig. S5 and Fig. S6). Unlike in stochastic superresolution imaging of gp210 (10), the color channels are inherently coaligned and simultaneously recorded simply by executing a single scan. Apart from a weak smoothing and background subtraction applied to enhance image contrast, the images are raw.Because fluorescence off-switching by STED is an instant process, STED microscopy can be employed to study fast spatial translocations, such as the diffusion of molecules on the nanoscale (3). To benchmark the performance of our setup, we analyzed the diffusion of a fluorescent glycerophospholipid analog (11) by fluorescence correlation spectroscopy (FCS) in membranes of living mammalian PtK2-cells (Fig. 3). STED allowed us to reduce the diameter of the probed area from the 250 nm-sized diffraction limit down to 19 nm (FWHM), representing σ = 8 nm in standard deviation of a Gaussian fit. The attained subdiffraction area is 2.5 times smaller as compared to what has been reported in living cells to date (4). In model membranes, the smallest diameter was 15 nm (σ = 6.4 nm).Open in a separate windowFigure 3Nanoscale molecular diffusion analyzed by STED FCS. (A) For moderate and larger STED beam power P_STED, the resolution scales inversely with its square-root, attaining 15 nm in FWHM of the distribution of fluorescence emission in space, describing the measurement area. Note the relatively small threshold power P_S = 1.4 mW, which implies that a large resolution gain is already attained for P_STED < 100 mW. (Inset) The resolution was determined by measuring the transit time of a fluorescent phospholipid-analog (DSPE-PEG-KK114) in a lipid model membrane through the detection area by FCS. (B) In living mammalian Ptk2-cells, the transit time of the lipid analog scales linearly with the detection area, revealing a diffusion constant D_lat = 0.33 μm²/s, and showing that this lipid analog diffuses largely freely in the plasma membrane down to <20 nm scales.In both measurements, the molecular transit time depends linearly on the probed area, indicating that the labeled lipid molecules diffuse essentially freely down to spatial scales of 20 nm. Accordingly, the anomaly exponent α was close to 1 with values of α > 0.85, showing only minor deviations from free diffusion (see Fig. S7). Because the diameter is inversely proportional to the square-root of the STED beam power, the resolution can be adapted to a particular application need (Fig. 3, A and B).In summary, our arrangement provides up-to-date STED microscopy resolution in offset-free colocalization recordings. The ready-to-use near-infrared laser pulses keep undesired single and multiphoton absorption low and leave the visible spectrum amenable for further studies.     

Achieving λ/10 resolution CW STED nanoscopy with a Ti:Sapphire oscillator

In this report, a Ti:Sapphire oscillator was utilized to realize synchronization-free stimulated emission depletion (STED) microscopy. With pump power of 4.6 W and sample irradiance of 310 mW, we achieved super-resolution as high as 71 nm. With synchronization-free STED, we imaged 200 nm nanospheres as well as all three cytoskeletal elements (microtubules, intermediate filaments, and actin filaments), clearly demonstrating the resolving power of synchronization-free STED over conventional diffraction limited imaging. It also allowed us to discover that, Dylight 650, exhibits improved performance over ATTO647N, a fluorophore frequently used in STED. Furthermore, we applied synchronization-free STED to image fluorescently-labeled intracellular viral RNA granules, which otherwise cannot be differentiated by confocal microscopy. Thanks to the widely available Ti:Sapphire oscillators in multiphoton imaging system, this work suggests easier access to setup super-resolution microscope via the synchronization-free STED.     

Eliminating Unwanted Far-Field Excitation in Objective-Type TIRF. Part I. Identifying Sources of Nonevanescent Excitation Light

Total internal reflection fluorescence microscopy (TIRFM) achieves subdiffraction axial sectioning by confining fluorophore excitation to a thin layer close to the cell/substrate boundary. However, it is often unknown how thin this light sheet actually is. Particularly in objective-type TIRFM, large deviations from the exponential intensity decay expected for pure evanescence have been reported. Nonevanescent excitation light diminishes the optical sectioning effect, reduces contrast, and renders TIRFM-image quantification uncertain. To identify the sources of this unwanted fluorescence excitation in deeper sample layers, we here combine azimuthal and polar beam scanning (spinning TIRF), atomic force microscopy, and wavefront analysis of beams passing through the objective periphery. Using a variety of intracellular fluorescent labels as well as negative staining experiments to measure cell-induced scattering, we find that azimuthal beam spinning produces TIRFM images that more accurately portray the real fluorophore distribution, but these images are still hampered by far-field excitation. Furthermore, although clearly measureable, cell-induced scattering is not the dominant source of far-field excitation light in objective-type TIRF, at least for most types of weakly scattering cells. It is the microscope illumination optical path that produces a large cell- and beam-angle invariant stray excitation that is insensitive to beam scanning. This instrument-induced glare is produced far from the sample plane, inside the microscope illumination optical path. We identify stray reflections and high-numerical aperture aberrations of the TIRF objective as one important source. This work is accompanied by a companion paper (Pt.2/2).     

Eliminating Unwanted Far-Field Excitation in Objective-Type TIRF. Part I. Identifying Sources of Nonevanescent Excitation Light

Total internal reflection fluorescence microscopy (TIRFM) achieves subdiffraction axial sectioning by confining fluorophore excitation to a thin layer close to the cell/substrate boundary. However, it is often unknown how thin this light sheet actually is. Particularly in objective-type TIRFM, large deviations from the exponential intensity decay expected for pure evanescence have been reported. Nonevanescent excitation light diminishes the optical sectioning effect, reduces contrast, and renders TIRFM-image quantification uncertain. To identify the sources of this unwanted fluorescence excitation in deeper sample layers, we here combine azimuthal and polar beam scanning (spinning TIRF), atomic force microscopy, and wavefront analysis of beams passing through the objective periphery. Using a variety of intracellular fluorescent labels as well as negative staining experiments to measure cell-induced scattering, we find that azimuthal beam spinning produces TIRFM images that more accurately portray the real fluorophore distribution, but these images are still hampered by far-field excitation. Furthermore, although clearly measureable, cell-induced scattering is not the dominant source of far-field excitation light in objective-type TIRF, at least for most types of weakly scattering cells. It is the microscope illumination optical path that produces a large cell- and beam-angle invariant stray excitation that is insensitive to beam scanning. This instrument-induced glare is produced far from the sample plane, inside the microscope illumination optical path. We identify stray reflections and high-numerical aperture aberrations of the TIRF objective as one important source. This work is accompanied by a companion paper (Pt.2/2).     

Development of a Multicolor Line-Focus Microscope for Rapid Acquisitions of Excitation Spectra

To conduct rapid microscope observations with the excitation spectral measurement for photosynthetic organisms, a wavelength-dispersive line-focus microscope was developed. In the developed system, fluorescence signals at multiple positions on a sample excited with different wavelengths can be detected as a two-dimensional image on the EMCCD camera at the same time. Using the developed system, one can obtain excitation spectra at every pixel over the excitation wavelength range from 635 to 695 nm, which covers the full range of the Q_y bands of both chlorophyll-a and chlorophyll-b. Recording the reference laser spectra at the same time ensures robust measurement against the moderate spectral fluctuation in the excitation laser. Using an objective lens with a numerical aperture of 0.9, the lateral and axial resolutions of 0.56 and 1.08 μm, respectively, were achieved. The theoretically limited and experimentally estimated spectral resolutions of the excitation spectral measurement were 0.86 and 1.3 nm, respectively. The validity of the system was demonstrated by measuring fluorescent beads and single cells of a model alga, Chlamydomonas reinhardtii. Intrachloroplast inhomogeneity in the relative intensity of the chlorophyll-b band could be visualized in Chlamydomonas cells. The inhomogeneity reflects the intrachloroplast variation in the local peripheral antenna size.     

用激光共聚焦显微镜鉴别可疑笔迹与印章

本文采用激光共聚焦显微镜鉴别可疑笔迹和印章及其它的文件。法医在鉴别可疑文件时通常都是凭经验和用普通光学显微镜,找出其中的一些物理特征。但还是有许多文件的笔迹顺序无法确定,尤其是墨水写的笔迹,为解决法医的这一问题,我们用激光共聚焦显微镜鉴别了72例不同铅笔和圆珠笔写的肉眼难于鉴别的交叉笔顺和其他的一些文字文件。在激光共激光共聚焦显微镜下大多数笔迹和印章都能发出荧光,因此很容易鉴别其笔顺和印章的特征,必要时还可以进行笔顺的三维图象构建,以帮助鉴别。结论：激光共聚焦显微镜可以更准确地鉴别可疑笔迹和印章。印章和笔迹的交叉也很容易分辨出来。     

Frequency division multiplexed multichannel high-speed fluorescence confocal microscope

In this article, we report a new type of fluorescence confocal microscope: frequency division multiplexed multichannel fluorescence confocal microscope, in which we encode the spatial location information into the frequency domain. In this microscope, the exciting laser beam is first split into multiple beams and each beam is modulated at a different frequency. These multiple beams are focused at different locations of the target to form multiple focal points, which further generate multiple fluorescent emission spots. The fluorescent emissions from different focal points are also modulated at different frequencies, because the exciting beams are modulated at different frequencies (or difference carrier frequency). Then, all the fluorescent emissions (modulated at different frequencies) are collected together and detected by a highly sensitive, large-dynamic-range photomultiplier tube. By demodulating the detected signal (i.e., via the Fourier transform), we can distinguish the fluorescent light emitted from the different locations by the corresponding carrier frequencies. The major advantage of this unique fluorescence confocal microscope is that it not only has a high sensitivity because of the use of photomultiplier tube but also can get multiple-point data simultaneously, which is crucial to study the dynamic behavior of many biological process. As an initial step, to verify the feasibility of the proposed multichannel confocal microscope, we have developed a two-channel confocal fluorescence microscope and applied it to study the dynamic behavior of the changes of the calcium ion concentration during the single cardiac myocyte contraction. Our preliminary experimental results demonstrated that we could indeed realize multichannel confocal fluorescence microscopy by utilizing the frequency division multiplexed microscope, which could become an effective tool to study the dynamic behavior of many biological processes.     

Applications of STED fluorescence nanoscopy in unravelling nanoscale structure and dynamics of biological systems

Fluorescence microscopy, especially confocal microscopy, has revolutionized the field of biological imaging. Breaking the optical diffraction barrier of conventional light microscopy, through the advent of super-resolution microscopy, has ushered in the potential for a second revolution through unprecedented insight into nanoscale structure and dynamics in biological systems. Stimulated emission depletion (STED) microscopy is one such super-resolution microscopy technique which provides real-time enhanced-resolution imaging capabilities. In addition, it can be easily integrated with well-established fluorescence-based techniques such as fluorescence correlation spectroscopy (FCS) in order to capture the structure of cellular membranes at the nanoscale with high temporal resolution. In this review, we discuss the theory of STED and different modalities of operation in order to achieve the best resolution. Various applications of this technique in cell imaging, especially that of neuronal cell imaging, are discussed as well as examples of application of STED imaging in unravelling structure formation on biological membranes. Finally, we have discussed examples from some of our recent studies on nanoscale structure and dynamics of lipids in model membranes, due to interaction with proteins, as revealed by combination of STED and FCS techniques.     

Single Plane Illumination Module and Micro-capillary Approach for a Wide-field Microscope

A module for light sheet or single plane illumination microscopy (SPIM) is described which is easily adapted to an inverted wide-field microscope and optimized for 3-dimensional cell cultures, e.g., multi-cellular tumor spheroids (MCTS). The SPIM excitation module shapes and deflects the light such that the sample is illuminated by a light sheet perpendicular to the detection path of the microscope. The system is characterized by use of a rectangular capillary for holding (and in an advanced version also by a micro-capillary approach for rotating) the samples, by synchronous adjustment of the illuminating light sheet and the objective lens used for fluorescence detection as well as by adaptation of a microfluidic system for application of fluorescent dyes, pharmaceutical agents or drugs in small quantities. A protocol for working with this system is given, and some technical details are reported. Representative results include (1) measurements of the uptake of a cytostatic drug (doxorubicin) and its partial conversion to a degradation product, (2) redox measurements by use of a genetically encoded glutathione sensor upon addition of an oxidizing agent, and (3) initiation and labeling of cell necrosis upon inhibition of the mitochondrial respiratory chain. Differences and advantages of the present SPIM module in comparison with existing systems are discussed.     

Far-Red Fluorescent Protein Excitable with Red Lasers for Flow Cytometry and Superresolution STED Nanoscopy

Far-red fluorescent proteins are required for deep-tissue and whole-animal imaging and multicolor labeling in the red wavelength range, as well as probes excitable with standard red lasers in flow cytometry and fluorescence microscopy. Rapidly evolving superresolution microscopy based on the stimulated emission depletion approach also demands genetically encoded monomeric probes to tag intracellular proteins at the molecular level. Based on the monomeric mKate variant, we have developed a far-red TagRFP657 protein with excitation/emission maxima at 611/657 nm. TagRFP657 has several advantages over existing monomeric far-red proteins including higher photostability, better pH stability, lower residual green fluorescence, and greater efficiency of excitation with red lasers. The red-shifted excitation and emission spectra, as compared to other far-red proteins, allows utilizing TagRFP657 in flow cytometry and fluorescence microscopy simultaneously with orange or near-red fluorescence proteins. TagRFP657 is shown to be an efficient protein tag for the superresolution fluorescence imaging using a commercially available stimulated emission depletion microscope.     

Inside Cover: Enhanced lateral resolution in continuous wave stimulated emission depletion microscopy using tightly focused annular radially polarized excitation beam (J. Biophotonics 9/2019)

STED microscopy is a tool that enables superresolution fluorescence imaging by overcoming the diffraction limitation, and has become more useful in various fields such as biology and material science. STED resolution enhancement can be useful in resolving and visualizing sophisticated details of structures of a sample. For this, the excitation focal spot reduction of CW STED microscopy is achieved by PSF engineering using radial polarization and annular aperture, and improved lateral resolution is obtained by STED effect. This leads to a performance improvement that can lower the depletion beam power required to achieve the same superresolution Further details can be found in the article by Geon Lim, Wan‐Chin Kim, Seunghee Oh, Hyungsuk Lee, No‐Cheol Parket ( e201900060 ).