Cooperative 4Pi excitation and detection yields sevenfold sharper optical sections in live-cell microscopy

Although the addition of just the excitation light field at the focus, or of just the fluorescence field at the detector is sufficient for a three- to fivefold resolution increase in 4Pi-fluorescence microscopy, substantial improvements of its optical properties are achieved by exploiting both effects simultaneously. They encompass not only an additional expansion of the optical bandwidth, but also an amplified transfer of the newly gained spatial frequencies to the image. Here we report on the realization and the imaging properties of this 4Pi microscopy mode of type C that also is the far-field microscope with the hitherto largest aperture. We show that in conjunction with two-photon excitation, the resulting optical transfer function displays a sevenfold improvement of axial three-dimensional resolution over confocal microscopy in aqueous samples, and more importantly, a marked transfer of all frequencies within its inner region of support. The latter is present also without the confocal pinhole. Thus, linear image deconvolution is possible both for confocalized and nonconfocalized live-cell 4Pi imaging. Realized in a state-of-the-art scanning microscope, this approach enables robust three-dimensional imaging of fixed and live cells at approximately 80 nm axial resolution.     

Two-photon confocal microscopy: a nondestructive method for studying wound healing

Two-photon confocal microscopy is a new technology useful in nondestructive analysis of tissue. The pattern generated from laser-excited autofluorescence and second harmonic signals can be analyzed to construct a three-dimensional, microanatomical, structural image. The healing of full-thickness guinea pig skin wounds was studied over a period of 28 days using two-photon confocal microscopy. Three-dimensional data were rendered from two-dimensional images and compared with conventional, en face, histologic sections. Two-photon confocal microscopy images show resolution of muscle, fascia fibers, collagen fibers, inflammatory cells, blood vessels, and hair. Although these images do not currently have the resolution of standard histology, the ability to noninvasively acquire three-dimensional images of skin promises to be an important tool in wound-healing studies.     

4Pi microscopy of quantum dot-labeled cellular structures

The most prominent restrictions of fluorescence microscopy are the limited resolution and the finite signal. Established conventional, confocal, and multiphoton microscopes resolve at best approximately 200nm in the focal plane and only 500nm in depth. Additionally, organic fluorophores and fluorescent proteins are bleached after 10(4)-10(5) excitation cycles. To overcome these restrictions, we synergistically combine the 3- to 7-fold improved axial resolution of 4Pi microscopy with the greatly enhanced photostability of semiconductor quantum dots. Co-localization studies of immunolabeled microtubules and mitochondria demonstrate the feasibility of this approach for routine biological measurements. In particular, we visualize the three-dimensional entanglement of the two networks with unprecedented detail.     

4Pi microscopy of the nuclear pore complex

To explore whether super-resolution fluorescence microscopy is able to resolve topographic features of single cellular protein complexes, a two-photon 4Pi microscope was used to study the nuclear pore complex (NPC). The microscope had an axial resolution of 110-130 nm and a two-color localization accuracy of 5-10 nm. In immune-labeled HeLa cells, NPCs could be resolved much better by 4Pi than by confocal microscopy. When two epitopes of the NPC, one localized at the tip of the cytoplasmic filaments and the other at the ring of the nuclear basket, were immune-labeled, they could be clearly resolved in single NPCs, with the distance between them determined to be 152 ± 30 nm. In cells expressing a green fluorescent protein construct localized at the NPC center, the distances between the ring of the nuclear filaments and the NPC center was 76 ± 12 (Potorous tridactylus cells) or 91 ± 21 nm (normal rat kidney cells), whereas the distance between the NPC center and the tips of the cytoplasmic filaments was 84 ± 18 nm, all values in good agreement with previous electron or single-molecule fluorescence estimates. We conclude that super-resolution fluorescence microscopy is a powerful method for analyzing single protein complexes and the cellular nanomachinery in general.     

Continuous fluorescence microphotolysis and correlation spectroscopy using 4Pi microscopy

Continuous fluorescence microphotolysis (CFM) and fluorescence correlation spectroscopy (FCS) permit measurement of molecular mobility and association reactions in single living cells. CFM and FCS complement each other ideally and can be realized using identical equipment. So far, the spatial resolution of CFM and FCS was restricted by the resolution of the light microscope to the micrometer scale. However, cellular functions generally occur on the nanometer scale. Here, we develop the theoretical and computational framework for CFM and FCS experiments using 4Pi microscopy, which features an axial resolution of ∼100 nm. The framework, taking the actual 4Pi point spread function of the instrument into account, was validated by measurements on model systems, employing 4Pi conditions or normal confocal conditions together with either single- or two-photon excitation. In all cases experimental data could be well fitted by computed curves for expected diffusion coefficients, even when the signal/noise ratio was small due to the small number of fluorophores involved.     

Two-photon fluorescence excitation in detection of biomolecules

Two-photon fluorescence excitation has been found to be a very powerful method for enhancing the sensitivity and resolution in far-field light microscopy. Two-photon fluorescence excitation also provides a substantially background-free detection on the single-molecule level. It allows direct monitoring of formation of labelled biomolecule complexes in solution. Two-photon excitation is created when, by focusing an intensive light source, the density of photons per unit volume and per unit time becomes high enough for two photons to be absorbed into the same chromophore. In this case, the absorbed energy is the sum of the energies of the two photons. In two-photon excitation, dye molecules are excited only when both photons are absorbed simultaneously. The probability of absorption of two photons is equal to the product of probability distributions of absorption of the single photons. The emission of two photons is thus a quadratic process with respect to illumination intensity. Thus in two-photon excitation, only the fluorescence that is formed in the clearly restricted three-dimensional vicinity of the focal point is excited. We have developed an assay concept that is able to distinguish optically between the signal emitted from a microparticle in the focal point of the laser beam, and the signal emitted from the surrounding free labelled reagent. Moreover, the free labels outside the focal volume do not contribute any significant signal. This means that the assay is separation-free. The method based on two-photon fluorescence excitation makes possible fast single-step and separation-free immunoassays, for example, for whole blood samples. Since the method allows a separation-free assay in very small volumes, the method is very useful for high-throughput screening assays. Consequently we believe that two-photon fluorescence excitation will make a remarkable impact as a research tool and a routine method in many fields of analysis.     

Two-photon axotomy and time-lapse confocal imaging in live zebrafish embryos

Zebrafish have long been utilized to study the cellular and molecular mechanisms of development by time-lapse imaging of the living transparent embryo. Here we describe a method to mount zebrafish embryos for long-term imaging and demonstrate how to automate the capture of time-lapse images using a confocal microscope. We also describe a method to create controlled, precise damage to individual branches of peripheral sensory axons in zebrafish using the focused power of a femtosecond laser mounted on a two-photon microscope. The parameters for successful two-photon axotomy must be optimized for each microscope. We will demonstrate two-photon axotomy on both a custom built two-photon microscope and a Zeiss 510 confocal/two-photon to provide two examples.Zebrafish trigeminal sensory neurons can be visualized in a transgenic line expressing GFP driven by a sensory neuron specific promoter ¹. We have adapted this zebrafish trigeminal model to directly observe sensory axon regeneration in living zebrafish embryos. Embryos are anesthetized with tricaine and positioned within a drop of agarose as it solidifies. Immobilized embryos are sealed within an imaging chamber filled with phenylthiourea (PTU) Ringers. We have found that embryos can be continuously imaged in these chambers for 12-48 hours. A single confocal image is then captured to determine the desired site of axotomy. The region of interest is located on the two-photon microscope by imaging the sensory axons under low, non-damaging power. After zooming in on the desired site of axotomy, the power is increased and a single scan of that defined region is sufficient to sever the axon. Multiple location time-lapse imaging is then set up on a confocal microscope to directly observe axonal recovery from injury. Open in a separate windowClick here to view.^{(76M, flv)}     

Recording changes in extracellular pH via confocal microscopy during generation of excitation potentials in higher plants

The method of confocal microscopy was applied to pH changes recorded in the apoplast of the stem of a pumpkin seedling during the generation of the action potential and variation potential. To record the change in pH, the fluorescent probe FITC-dextran was used. An analysis of the obtained fluorescent images and the determination of the fluorescence spectra showed that FITC-dextran is located in the stemcell walls. The propagation of the action and variation potentials is accompanied by a transient increase in the probe fluorescence intensity, which indicates the alkalization of cell walls. This transient alkalization is suggested to be due to the temporary inactivation of electrogenic H⁺-pump in the plasma membrane.     

Two-photon excitation in fluorescence polarization receptor-ligand binding assay

Fluorescence polarization is one of the most commonly used homogeneous assay principles in drug discovery for screening of potential lead compounds. In this article, the fluorescence polarization technique is combined with 2-photon excitation of fluorescence. Theoretically, the use of 2-photon excitation of fluorescence increases the volumetric sensitivity and polarization contrast of fluorescence polarization assays. The work in this report demonstrates these predictions for an estrogen receptor ligand binding assay.     

Combined in vivo confocal Raman spectroscopy and confocal microscopy of human skin

In vivo confocal Raman spectroscopy is a noninvasive optical method to obtain detailed information about the molecular composition of the skin with high spatial resolution. In vivo confocal scanning laser microscopy is an imaging modality that provides optical sections of the skin without physically dissecting the tissue. A combination of both techniques in a single instrument is described. This combination allows the skin morphology to be visualized and (subsurface) structures in the skin to be targeted for Raman measurements. Novel results are presented that show detailed in vivo concentration profiles of water and of natural moisturizing factor for the stratum corneum that are directly related to the skin architecture by in vivo cross-sectional images of the skin. Targeting of skin structures is demonstrated by recording in vivo Raman spectra of sweat ducts and sebaceous glands in situ. In vivo measurements on dermal capillaries yielded high-quality Raman spectra of blood in a completely noninvasive manner. From the results of this exploratory study we conclude that the technique presented has great potential for fundamental skin research, pharmacology (percutaneous transport), clinical dermatology, and cosmetic research, as well as for noninvasive analysis of blood analytes, including glucose.     

Two-photon polarization microscopy reveals protein structure and function

Membrane proteins are a large, diverse group of proteins, serving a multitude of cellular functions. They are difficult to study because of their requirement of a lipid membrane for function. Here we show that two-photon polarization microscopy can take advantage of the cell membrane requirement to yield insights into membrane protein structure and function, in living cells and organisms. The technique allows sensitive imaging of G-protein activation, changes in intracellular calcium concentration and other processes, and is not limited to membrane proteins. Conveniently, many suitable probes for two-photon polarization microscopy already exist.     

Two-photon fluorescence excitation of macroscopic areas on planar waveguides

In this paper, we report the first successful demonstration, to our knowledge, of two-photon fluorescence excitation (TPFE) using planar thin-film waveguide structures of macroscopic excitation dimensions (square millimeters to square centimeters in size). The high intensity of excitation light required for TPFE is available not only at a single focus point but along the whole trace of the beam guided in the waveguide structure. Line profiles of the fluorescence excited by TPFE show excellent correlation with the geometry of the launched laser beams. A clear second-order dependence of the fluorescence intensity on the excitation intensity confirms the two-photon character of fluorescence generation. Spectra of the emission generated by one-photon excitation and by two-photon excitation show only minor differences.     

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.     

Modern microscopy in biofilm research: confocal microscopy and other approaches.

Microscopy is the only technique whereby bacterial biofilms can be studied at the single-cell level in situ. Our understanding of biofilm structure, physiology and control hinges on the application of confocal scanning laser microscopy and other advanced microscopic techniques. Gene expression in four dimensions (x,y,z,t), interspecies interactions, and the role of exopolymer are being defined.     

Statistical evaluation of confocal microscopy images

BACKGROUND: The coefficient of variation (CV) is defined as the standard deviation (sigma) of the fluorescent intensity of a population of beads or pixels expressed as a proportion or percentage of the mean (mu) intensity (CV = sigma/mu). The field of flow cytometry has used the CV of a population of bead intensities to determine if the flow cytometer is aligned correctly and performing properly. In a similar manner, the analysis of CV has been applied to the confocal laser scanning microscope (CLSM) to determine machine performance and sensitivity. METHODS: Instead of measuring 10,000 beads using a flow cytometer and determining the CV of this distribution of intensities, thousands of pixels are measured from within one homogeneous Spherotech 10-microm bead. Similar to a typical flow cytometry population that consists of 10,000 beads, a CLSM scanned image consists of a distribution of pixel intensities representing a population of approximately 100,000 pixels. In order to perform this test properly, it is important to have a population of homogeneous particles. A biological particle usually has heterogeneous pixel intensities that correspond to the details in the biological image and thus shows more variability as a test particle. RESULTS: The bead CV consisting of a population of pixel intensities is dependent on a number of machine variables that include frame averaging, photomultiplier tube (PMT) voltage, PMT noise, and laser power. The relationship among these variables suggests that the machine should be operated with lower PMT values in order to generate superior image quality. If this cannot be achieved, frame averaging will be necessary to reduce the CV and improve image quality. There is more image noise at higher PMT settings, making it is necessary to average more frames to reduce the CV values and improve image quality. The sensitivity of a system is related to system noise, laser light efficiency, and proper system alignment. It is possible to compare different systems for system performance and sensitivity if the laser power is maintained at a constant value. Using this bead CV test, 1 mW of 488 nm laser light measured on the scan head yielded a CV value of 4% with a Leica TCS-SP1 (75-mW argon-krypton laser) and a CV value of 1.3% with a Zeiss 510 (25-mW argon laser). A biological particle shows the same relationship between laser power, averaging, PMT voltage, and CV as do the beads. However, because the biological particle has heterogeneous pixel intensities, there is more particle variability, which does not make as useful as a test particle. CONCLUSIONS: This CV analysis of a 10-microm Spherotech fluorescent bead can help determine the sensitivity in a confocal microscope and the system performance. The relationship among the factors that influence image quality is explained from a statistical endpoint. The data obtained from this test provides a systematic method of reducing noise and increasing image clarity. Many components of a CLSM, including laser power, laser stability, PMT functionality, and alignment, influence the CV and determine if the equipment is performing properly. Preliminary results have shown that the bead CV can be used to compare different confocal microscopy systems with regard to performance and sensitivity. The test appears to be analogous to CV tests made on the flow cytometer to assess instrument performance and sensitivity. Published 2001 Wiley-Liss, Inc.     

共聚焦显微技术简介

共聚焦显微镜在生物学研究中得到广泛应用,共聚焦显微技术按照显微镜构造原理的不同分成激光扫描共聚焦和数字共聚焦显微技术两种,共聚焦技术具有成像清晰,获得三维图像,进行多标记观察,活细胞内动态生理反应的实时观察记录,定性定量分析等优势,与共聚焦显微技术相关的技术有荧光染料的选择,荧光指示剂装载以及图像数据处理等。