High-order photobleaching of green fluorescent protein inside live cells in two-photon excitation microscopy

Combination of green fluorescent protein (GFP) and two-photon excitation fluorescence microscopy (TPE) has been used increasingly to study dynamic biochemical events within living cells, sometimes even in vivo. However, the high photon flux required in TPE may lead to higher-order photobleaching within the focal volume, which would introduce misinterpretation about the fine biochemical events. Here we first studied the high-order photobleaching rate of GFP inside live cells by measuring the dependence of the photobleaching rate on the excitation power. The photobleaching rate under one- and two-photon excitation increased with 1-power and 4-power of the incident intensity, respectively, implying the excitation photons might interact with excited fluorophore molecules and increase the probability of photobleaching. These results suggest that in applications where two-photon imaging of GFP is used to study dynamic molecular process, photobleaching may ruin the imaging results and attention should be paid in interpreting the imaging results.     

Construction of a two-photon microscope for video-rate Ca imaging

We describe the construction of a video-rate two-photon laser scanning microscope, compare its performance to a similar confocal microscope, and illustrate its use for imaging local Ca(2+) transients from cortical neurons in brain slices. Key features include the use of a Ti-sapphire femtosecond laser allowing continuous tuning over a wide (700-1000 nm) wavelength range, a resonant scanning mirror to permit frame acquisition at 30 Hz, and efficient wide-field fluorescence detection. Two-photon imaging provides compelling advantages over confocal microscopy in terms of improved imaging depth and reduced phototoxicity and photobleaching, but the high cost of commercial instruments has limited their widespread adoption. By constructing one's own system the expense is greatly reduced without sacrifice of performance, and the microscope can be more readily tailored to specific applications.     

Characterization of a range of fura dyes with two-photon excitation

Two-photon excitation (TPE) spectra of Fura-2, -4F, -6F, -FF, and Furaptra were characterized using a tunable (750-850 nM) ultra-short pulse laser. Two-photon fluorescence of these dyes was studied in free solution and in the cytosol of isolated rabbit ventricular cardiomyocytes. The TPE spectra of the Ca(2+)-free and Ca(2+)-bound forms of the dyes were measured in free solution and expressed in terms of the two-photon fluorescence cross section (Goppert-Meyer units). The Fura dyes displayed the same Ca(2+)-free TPE spectrum in the intracellular volume of permeabilized and intact cardiomyocytes. Fluorescence measurements over a range of laser powers confirmed the TPE of both Ca(2+)-free and Ca(2+)-bound forms of the dyes. Single-wavelength excitation at 810 nM was used to determine the effective dissociation constants (K(eff)) and dynamic ranges (R(f)) of Fura-2, -4F, -6F, -FF, and Furaptra dyes (K(eff) = 181 +/- 52 nM, 1.16 +/- 0.016 micro M, 5.18 +/- 0.3 micro M, 19.2 +/- 1 micro M, and 58.5 +/- 2 micro M; and R(f) = 22.4 +/- 3.8, 12.2 +/- 0.34, 6.3 +/- 0.17, 16.1 +/- 2.8, and 25.4 +/- 4, respectively). Single-wavelength excitation of intracellular Fura-4F resolved diastolic and peak [Ca(2+)] in isolated stimulated cardiomyocytes after calibration of the intracellular signal using reversible exposure to low (100 micro M) extracellular [Ca(2+)]. Furthermore, TPE of Fura-4F allowed continuous, long-term (5-10 min) Ca(2+) imaging in ventricular cardiomyocytes using laser-scanning microscopy without significant cellular photodamage or photobleaching of the dye.     

Photobleaching in two-photon excitation microscopy

The intensity-squared dependence of two-photon excitation in laser scanning microscopy restricts excitation to the focal plane and leads to decreased photobleaching in thick samples. However, the high photon flux used in these experiments can potentially lead to higher-order photon interactions within the focal volume. The excitation power dependence of the fluorescence intensity and the photobleaching rate of thin fluorescence samples ( approximately 1 microm) were examined under one- and two-photon excitation. As expected, log-log plots of excitation power versus the fluorescence intensity and photobleaching rate for one-photon excitation of fluorescein increased with a slope of approximately 1. A similar plot of the fluorescence intensity versus two-photon excitation power increased with a slope of approximately 2. However, the two-photon photobleaching rate increased with a slope > or =3, indicating the presence of higher-order photon interactions. Similar experiments on Indo-1, NADH, and aminocoumarin produced similar results and suggest that this higher-order photobleaching is common in two-photon excitation microscopy. As a consequence, the use of multi-photon excitation microscopy to study thin samples may be limited by increased photobleaching.     

Continuous photobleaching in vesicles and living cells: a measure of diffusion and compartmentation

We present a comprehensive and analytical treatment of continuous photobleaching in a compartment, under single photon excitation. In the very short time regime (t<0.1 ms), the diffusion does not play any role. After a transition (or short time regime), one enters in the long time regime (t>0.1-5 s), for which the diffusion and the photobleaching balance each other. In this long time regime, the diffusion is either fast (i.e., the photobleaching probability of a molecule diffusing through the laser beam is low) so that the photobleaching rate is independent of the diffusion constant and dependent only of the laser power, or the diffusion is slow (i.e., the photobleaching probability is high) and the photobleaching rate is mainly dependent on the diffusion constant. We illustrate our theory by using giant unilamellar vesicles ranging from approximately 10 to 100 microm in diameter, loaded with molecules of various diffusion constants (from 20 to 300 microm2/s) and various photobleaching cross sections, illuminated under laser powers between 3 and 100 microW. We also demonstrated that information about compartmentation can be obtained by this method in living cells expressing enhanced green fluorescent proteins or that were loaded with small FITC-dextrans. Our quantitative approach shows that molecules freely diffusing in a cellular compartment do experience a continuous photobleaching. We provide a generic theoretical framework that should be taken into account when studying, under confocal microscopy, molecular interactions, permeability, etc.     

Sample Preparation and Imaging Conditions Affect mEos3.2 Photophysics in Fission Yeast Cells

Photoconvertible fluorescent proteins (PCFPs) are widely used in super-resolution microscopy and studies of cellular dynamics. However, our understanding of their photophysics is still limited, hampering their quantitative application. For example, we do not know the optimal sample preparation methods or imaging conditions to count protein molecules fused to PCFPs by single-molecule localization microscopy in live and fixed cells. We also do not know how the behavior of PCFPs in live cells compares with fixed cells. Therefore, we investigated how formaldehyde fixation influences the photophysical properties of the popular green-to-red PCFP mEos3.2 in fission yeast cells under a wide range of imaging conditions. We estimated photophysical parameters by fitting a three-state model of photoconversion and photobleaching to the time course of fluorescence signal per yeast cell expressing mEos3.2. We discovered that formaldehyde fixation makes the fluorescence signal, photoconversion rate, and photobleaching rate of mEos3.2 sensitive to the buffer conditions likely by permeabilizing the yeast cell membrane. Under some imaging conditions, the time-integrated mEos3.2 signal per yeast cell is similar in live cells and fixed cells imaged in buffer at pH 8.5 with 1 mM DTT, indicating that light chemical fixation does not destroy mEos3.2 molecules. We also discovered that 405-nm irradiation drove some red-state mEos3.2 molecules to enter an intermediate dark state, which can be converted back to the red fluorescent state by 561-nm illumination. Our findings provide a guide to quantitatively compare conditions for imaging mEos3.2-tagged molecules in yeast cells. Our imaging assay and mathematical model are easy to implement and provide a simple quantitative approach to measure the time-integrated signal and the photoconversion and photobleaching rates of fluorescent proteins in cells.     

Attenuation of photobleaching in two-photon excitation fluorescence from green fluorescent protein with shaped excitation pulses

The two-photon excitation fluorescence (TPEF) process of an enhanced green fluorescent protein (EGFP) for fluorescence signals was adaptively controlled by the phase-modulation of femtosecond pulses. After the iteration of pulse shaping, a twofold increase in the ratio of the fluorescence signal to the laser peak power was achieved. Compared with conventional pulses optimized for peak power, phase-optimized laser pulses reduced the bleaching rate of EGFP by a factor of 4 while maintaining the same intensity of the fluorescence signal. Our method will provide a powerful solution to various problems confronting researchers, such as the photobleaching of dyes in two-photon excitation microscopy.     

Theoretical background of light‐emitting diode total internal reflection fluorescence microscopy and photobleaching lifetime analysis of membrane‐associated proteins—Part II

The selective microscopic imaging of the plasma membrane and adjacent structures by total internal reflection fluorescence (TIRF) microscopy is a versatile and frequently used technique in cell biology. A reduction of imaging artifacts in objective‐type TIRF microscopy can be achieved by circular or multi‐spot laser illumination or by using noncoherent light sources that are projected into the back focal plane as a light annulus. Light‐emitting diode (LED)‐based TIRF excitation is a recent advancement of the latter strategy. While some basic principles of LED‐TIRF remain the same as in laser‐based methods, the calculation of penetration depth, the flatness of illumination and the amount of available illumination power differ. This study provides the theoretical framework for the construction and adjustment of LED‐TIRF. Using state‐of‐the art high power LED emitters, LED‐TIRF achieves excitation efficiencies that are comparable to laser‐based systems and homogenously illuminate the entire field of view, thus, allowing variation of the penetration depth or quantitative photobleaching‐assisted imaging protocols. Using autofluorescent transmembrane, soluble and membrane‐attached fusion proteins, we provide examples for a photobleaching‐based assessment of the exchange kinetics of proteins within living human endothelial cells.     

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.     

Imaging of single fluorescent molecules using video-rate confocal microscopy

Single fluorescent molecules in aqueous solution were imaged for the first time at video-rate using Nipkow disk-type confocal microscopy. Performance of this method was evaluated by imaging single kinesin molecules labeled with fluorescent dyes of tetramethylrhodamine (TMR) or IC5. Photodecomposition lifetimes of the fluorophores were approximately 10 s for TMR and approximately 2 s for IC5 under the incident laser power of 0.5 W/mm(2). Both the fluorescence intensity and the photobleaching rate were proportional to the laser power from 0.65 to 3 W/mm(2). 2D sliding movement of single kinesin molecules along microtubules on glass surface and 3D Brownian motion of individual kinesin molecules in viscous solution could be observed using this microscopy. These results indicated that this method could be applicable to the study of single molecular events in living cells at real time.     

Diffusion of green fluorescent protein in the aqueous-phase lumen of endoplasmic reticulum

The endoplasmic reticulum (ER) is the major compartment for the processing and quality control of newly synthesized proteins. Green fluorescent protein (GFP) was used as a noninvasive probe to determine the viscous properties of the aqueous lumen of the ER. GFP was targeted to the ER lumen of CHO cells by transient transfection with cDNA encoding GFP (S65T/F64L mutant) with a C-terminus KDEL retention sequence and upstream prolactin secretory sequence. Repeated laser illumination of a fixed 2-micrometers diameter spot resulted in complete bleaching of ER-associated GFP throughout the cell, indicating a continuous ER lumen. A residual amount (<1%) of GFP-KDEL was perinuclear and noncontiguous with the ER, presumably within a pre- or cis-Golgi compartment involved in KDEL-substrate retention. Quantitative spot photobleaching with a single brief bleach pulse indicated that GFP was fully mobile with a t1/2 for fluorescence recovery of 88 +/- 5 ms (SE; 60x lens) and 143 +/- 8 ms (40x). Fluorescence recovery was abolished by paraformaldehyde except for a small component of reversible photobleaching with t1/2 of 3 ms. For comparison, the t1/2 for photobleaching of GFP in cytoplasm was 14 +/- 2 ms (60x) and 24 +/- 1 ms (40x). Utilizing a mathematical model that accounted for ER reticular geometry, a GFP diffusion coefficient of 0.5-1 x 10(-7) cm2/s was computed, 9-18-fold less than that in water and 3-6-fold less than that in cytoplasm. By frequency-domain microfluorimetry, the GFP rotational correlation time was measured to be 39 +/- 8 ns, approximately 2-fold greater than that in water but comparable to that in the cytoplasm. Fluorescence recovery after photobleaching using a 40x lens was measured (at 23 degrees C unless otherwise indicated) for several potential effectors of ER structure and/or lumen environment: t1/2 values (in ms) were 143 +/- 8 (control), 100 +/- 13 (37 degrees C), 53 +/- 13 (brefeldin A), and 139 +/- 6 (dithiothreitol). These results indicate moderately slowed GFP diffusion in a continuous ER lumen.     

Astrocytes are poised for lactate trafficking and release from activated brain and for supply of glucose to neurons

Brain is a highly-oxidative organ, but during activation, glycolytic flux is preferentially up-regulated even though oxygen supply is adequate. The biochemical and cellular basis of metabolic changes during brain activation and the fate of lactate produced within brain are important, unresolved issues central to understanding brain function, brain images, and spectroscopic data. Because in vivo brain imaging studies reveal rapid efflux of labeled glucose metabolites during activation, lactate trafficking among astrocytes and between astrocytes and neurons was examined after devising specific, real-time, sensitive enzymatic fluorescent assays to measure lactate and glucose levels in single cells in adult rat brain slices. Astrocytes have a 2- to 4-fold faster and higher capacity for lactate uptake from extracellular fluid and for lactate dispersal via the astrocytic syncytium compared to neuronal lactate uptake from extracellular fluid or shuttling of lactate to neurons from neighboring astrocytes. Astrocytes can also supply glucose to neurons as well as glucose can be taken up by neurons from extracellular fluid. Astrocytic networks can provide neuronal fuel and quickly remove lactate from activated glycolytic domains, and the lactate can be dispersed widely throughout the syncytium to endfeet along the vasculature for release to blood or other brain regions via perivascular fluid flow.     

Diffusion of nerve growth factor in rat striatum as determined by multiphoton microscopy

Neurotrophins such as nerve growth factor (NGF) may be useful for treating diseases in the central nervous system; our ability to harness the potential therapeutic benefit of NGF is directly related to our understanding of the fate of exogenously supplied factors in brain tissue. We utilized multiphoton microscopy to quantify the dynamic behavior of NGF in coronal, 400- micro m thick, fresh rat brain tissue slices. We administered a solution containing bioactive rhodamine nerve growth factor conjugate via pressure injection and monitored the dispersion in the striatal region of the coronal slices. Multiphoton microscopy facilitated repeated imaging deep ( approximately 200 micro m) into tissue slices with minimal photodamage of tissue and photobleaching of label. The pressure injection paradigm approximated diffusion from a point source, and we therefore used the corresponding solution to the diffusion equation to estimate an apparent diffusion coefficient in brain tissue (D(b)(34 degrees C)) of 2.75 +/- 0.24 x 10(-7) cm(2)/s (average +/- SE). In contrast, we determined a corresponding free diffusion coefficient in buffered solution (D(f)(34 degrees C)) of 12.6 +/- 0.9 x 10(-7) cm(2)/s using multiphoton fluorescence photobleaching recovery. The tortuosity, defined as the square root of the ratio of D(f) to D(b), was 2.14 and moderate in magnitude.     

基于激光共聚焦同步双扫描技术的LC3脱乙酰化修饰调控自噬的机理研究

激光共聚焦同步双扫描(simultaneous,SIM)技术在常规扫描单元的基础上,引入一个同步扫描单元(SIM scanner),该技术独立控制了两个激光束,一个用于激光光刺激,另一个用于同步成像。本实验中,采用激光共聚焦同步双扫描系统的405 nm和488 nm激光分别对细胞的特定部位进行刺激和同步成像,实时检测了LC3复合物的形成,记录并分析了乙酰化前后LC3的光动力学变化过程,证实了LC3的脱乙酰化修饰是自噬性降解所必须的,本实验体系为激光共聚焦双扫描技术的推广提供了一个很好的平台。SIM技术的应用,解决了刺激过程无法成像的问题,为漂白后荧光恢复(fluorescence recovery after photobleaching,FRAP)、漂白后荧光损失(fluorescence loss in photobleaching,FLIP)和光诱导激活等研究提供了最佳的解决方案,可作为光刺激的一种实验模式在很多实验设计中进行延伸应用。     

Live Imaging of Mitosis in the Developing Mouse Embryonic Cortex

Although of short duration, mitosis is a complex and dynamic multi-step process fundamental for development of organs including the brain. In the developing cerebral cortex, abnormal mitosis of neural progenitors can cause defects in brain size and function. Hence, there is a critical need for tools to understand the mechanisms of neural progenitor mitosis. Cortical development in rodents is an outstanding model for studying this process. Neural progenitor mitosis is commonly examined in fixed brain sections. This protocol will describe in detail an approach for live imaging of mitosis in ex vivo embryonic brain slices. We will describe the critical steps for this procedure, which include: brain extraction, brain embedding, vibratome sectioning of brain slices, staining and culturing of slices, and time-lapse imaging. We will then demonstrate and describe in detail how to perform post-acquisition analysis of mitosis. We include representative results from this assay using the vital dye Syto11, transgenic mice (histone H2B-EGFP and centrin-EGFP), and in utero electroporation (mCherry-α-tubulin). We will discuss how this procedure can be best optimized and how it can be modified for study of genetic regulation of mitosis. Live imaging of mitosis in brain slices is a flexible approach to assess the impact of age, anatomy, and genetic perturbation in a controlled environment, and to generate a large amount of data with high temporal and spatial resolution. Hence this protocol will complement existing tools for analysis of neural progenitor mitosis.     

Evidence for lack of damage during photobleaching measurements of the lateral mobility of cell surface components.

Fluorescence recovery after photobleaching (FRAP) experiments to measure the mobility of cell surface components require a brief, but intense, pulse of light to photobleach the fluorescence in a restricted area of the cell. We studied possible photodamage to the cell surface during the photobleaching step using light and scanning electron microscopy (SEM) and various FRAP measurements themselves. The cell membrane was left impermeable to trypan blue after photobleaching. SEM studies show that the morphology of the cell surface is not altered by photobleaching. Cells can be repeatedly photobleached and/or photobleached using longer bleach times and greater intensities without systematically altering FRAP kinetics. Singlet oxygen quenchers or free radical traps designed to inhibit putative photoreagents produced during photobleaching do not markedly affect the results. Fluorescein and rhodamine labels give similar results. All of these results, obtained with several different monolayer cultures, suggest that photodamage induced during photobleaching is not a serious artefact in the cellular FRAP results obtained to date.     

A Rapid Optical Clearing Protocol Using 2,2′-Thiodiethanol for Microscopic Observation of Fixed Mouse Brain

Elucidation of neural circuit functions requires visualization of the fine structure of neurons in the inner regions of thick brain specimens. However, the tissue penetration depth of laser scanning microscopy is limited by light scattering and/or absorption by the tissue. Recently, several optical clearing reagents have been proposed for visualization in fixed specimens. However, they require complicated protocols or long treatment times. Here we report the effects of 2,2′-thiodiethanol (TDE) solutions as an optical clearing reagent for fixed mouse brains expressing a yellow fluorescent protein. Immersion of fixed brains in TDE solutions rapidly (within 30 min in the case of 400-µm-thick fixed brain slices) increased their transparency and enhanced the penetration depth in both confocal and two-photon microscopy. In addition, we succeeded in visualizing dendritic spines along single dendrites at deep positions in fixed thick brain slices. These results suggest that our proposed protocol using TDE solution is a rapid and useful method for optical clearing of fixed specimens expressing fluorescent proteins.     

Depth penetration and detection of pH gradients in biofilms by two-photon excitation microscopy.

Deep microbial biofilms are a major problem in many industrial, environmental, and medical settings. Novel approaches are needed to understand the structure and metabolism of these biofilms. Two-photon excitation microscopy (TPE) and conventional confocal laser scanning microscopy (CLSM) were compared quantitatively for the ability to visualize bacteria within deep in vitro biofilms. pH gradients within these biofilms were determined by fluorescence lifetime imaging, together with TPE. A constant-depth film fermentor (CDFF) was inoculated for 8 h at 50 ml. h(-1) with a defined mixed culture of 10 species of bacteria grown in continuous culture. Biofilms of fixed depths were developed in the CDFF for 10 or 11 days. The microbial compositions of the biofilms were determined by using viable counts on selective and nonselective agar media; diverse mixed-culture biofilms developed, including aerobic, facultative, and anaerobic species. TPE was able to record images four times deeper than CLSM. Importantly, in contrast to CLSM images, TPE images recorded deep within the biofilm showed no loss of contrast. The pH within the biofilms was measured directly by means of fluorescence lifetime imaging; the fluorescence decay of carboxyfluorescein was correlated with biofilm pH and was used to construct a calibration curve. pH gradients were detectable, in both the lateral and axial directions, in steady-state biofilms. When biofilms were overlaid with 14 mM sucrose for 1 h, distinct pH gradients developed. Microcolonies with pH values of below pH 3.0 were visible, in some cases adjacent to areas with a much higher pH (>5.0). TPE allowed resolution of images at significantly greater depths (as deep as 140 microm) than were possible with CLSM. Fluorescence lifetime imaging allowed the in situ, real-time imaging of pH and the detection of sharp gradients of pH within microbial biofilms.     

Scanning Microphotolysis: Three-Dimensional Diffusion Measurement and Optical Single-Transporter Recording

Scanning microphotolysis (SCAMP) is a combination of fluorescence microphotolysis and confocal laser scanning microscopy. A laser scanning microscope is equipped with an optical switch able to modulate the power or/and wavelength of the laser beam in less than a microsecond while a dedicated computer program is employed to precisely coordinate scanning process and laser beam modulation. By these means it becomes possible to vary the power or/and wavelength of the laser beam during scanning at a precision of one resolution element. Patterns of almost arbitrary design can be written into the object by photolysis, e.g., photobleaching or photoactivation. The dissipation of the photolysis pattern by diffusion or other types of molecular transport can be followed at confocal resolution and used to characterize the transport process. SCAMP can be employed in conjunction with single-photon or multiphoton excitation. Furthermore, it can be easily installed on virtually any confocal laser scanning microscope. We summarize at first the conceptual and practical basis of SCAMP. Then, two novel applications are discussed: (i) measurements of translational diffusion coefficients in truly three-dimensional systems at diffraction-limited resolution, and (ii) optical recording of single transporters in membrane patches.     

Inducible fluorescent speckle microscopy

The understanding of cytoskeleton dynamics has benefited from the capacity to generate fluorescent fiducial marks on cytoskeleton components. Here we show that light-induced imprinting of three-dimensional (3D) fluorescent speckles significantly improves speckle signal and contrast relative to classic (random) fluorescent speckle microscopy. We predict theoretically that speckle imprinting using photobleaching is optimal when the laser energy and fluorophore responsivity are related by the golden ratio. This relation, which we confirm experimentally, translates into a 40% remaining signal after speckle imprinting and provides a rule of thumb in selecting the laser power required to optimally prepare the sample for imaging. This inducible speckle imaging (ISI) technique allows 3D speckle microscopy to be performed in readily available libraries of cell lines or primary tissues expressing fluorescent proteins and does not preclude conventional imaging before speckle imaging. As a proof of concept, we use ISI to measure metaphase spindle microtubule poleward flux in primary cells and explore a scaling relation connecting microtubule flux to metaphase duration.