Single molecule microscopy methods for the study of DNA origami structures

Single molecule microscopy techniques play an important role in the investigation of advanced DNA structures such as those created by the DNA origami method. Three single molecule microscopy techniques are particularly interesting for the investigation of complex self-assembled three-dimensional (3D) DNA nanostructures, namely single molecule fluorescence microscopy, atomic force microscopy (AFM), and cryogenic transmission electron microscopy (cryo-EM). Here we discuss the strengths of these three techniques and demonstrate how their interplay can yield very important and unique new insights into the structure and conformation of advanced biological nanostructures. The applications of the three single molecule microscopy techniques are illustrated by focusing on a self-assembled DNA origami 3D box nanostructure. Its size and structure were studied by AFM and cryo-EM, while the lid opening, which can be controlled by the addition of oligonucleotide keys, was recorded by F?rster/fluorescence resonance energy transfer (FRET) spectroscopy.     

Fluorescence resonance energy transfer from cyan to yellow fluorescent protein detected by acceptor photobleaching using confocal microscopy and a single laser

One manifestation of fluorescence resonance energy transfer (FRET) is an increase in donor fluorescence after photobleaching the acceptor. Published acceptor‐photobleaching methods for FRET have mainly used wide‐field microscopy. A laser scanning confocal microscope enables faster and targeted bleaching within the field of view, thereby improving speed and accuracy. Here we demonstrate the approach with CFP and YFP, the most versatile fluorescent markers now available for FRET. CFP/YFP FRET imaging has been accomplished with a single laser (argon) available on virtually all laser‐scanning confocal microscopes. Accordingly, we also describe the conditions that we developed for dual imaging of CFP and YFP with the 458 and 514 argon lines. We detect FRET in a CFP/YFP fusion and also between signalling molecules (TNF‐Receptor‐Associated‐Factors or TRAFs) that are known to homo‐ and heterotrimerize. Importantly, we demonstrate that appropriate controls are essential to avoid false positives in FRET by acceptor photobleaching. We use two types of negative control: (a) an internal negative control (non‐bleached areas of the cell) and (b) cells with donor in the absence of the acceptor (CFP only). We find that both types of negative control can yield false FRET. Given this false FRET background, we describe a method for distinguishing true positive signals. In summary, we extensively characterize a simple approach to FRET that should be adaptable to most laser‐scanning confocal microscopes, and demonstrate its feasibility for detecting FRET between several CFP/YFP partners.     

Characterization of two-photon excitation fluorescence lifetime imaging microscopy for protein localization

Two-photon excitation fluorescence resonance energy transfer (2P-FRET) imaging microscopy can provide details of specific protein molecule interactions inside living cells. Fluorophore molecules used for 2P-FRET imaging have characteristic absorption and emission spectra that introduce spectral cross-talk (bleed-through) in the FRET signal that should be removed in the 2P-FRET images, to establish that FRET has actually occurred and to have a basis for distance estimations. These contaminations in the FRET signal can be corrected using a mathematical algorithm to extract the true FRET signal. Another approach is 2P-FRET fluorescence lifetime imaging (FLIM). This methodology allows studying the dynamic behavior of protein-protein interactions in living cells and tissues. 2P-FRET-FLIM was used to study the dimerization of the CAATT/enhancer binding protein alpha (C/EBPalpha). Results show that the reduction in donor lifetime in the presence of acceptor reveals the dimerization of the protein molecules and also determines more precisely the distance between the donor and acceptor. We describe the development and characterization of the 2P-FRET-FLIM imaging system with the Bio-Rad Radiance2100 confocal/multiphoton microscopy system.     

Scanning near-field optical microscopy using semiconductor nanocrystals as a local fluorescence and fluorescence resonance energy transfer source

Local fluorescence probes based on CdSe semiconductor nanocrystals were prepared and tested by recording scanning near‐field optical microscopy (SNOM) images of calibration samples and fluorescence resonance energy transfer SNOM (FRET SNOM) images of acceptor dye molecules inhomogeneously deposited onto a glass substrate. Thousands of nanocrystals contribute to the signal when this probe is used as a local fluorescence source while only tens of those (the most apical) are involved in imaging for the FRET SNOM operation mode. The dip‐coating method used to make the probe enables diminishing the number of active fluorescent nanocrystals easily. Prospects to realize FRET SNOM based on a single fluorescence centre using such an approach are briefly described.     

Single-molecule near-field optical energy transfer microscopy with dielectric tips

The fluorescence lifetime and the fluorescence rate of single molecules are recorded as a function of the position of a Si₃N₄ atomic force microscopy tip with respect to the molecule. We observe a decrease of the excited state lifetime and the fluorescence rate when the tip apex is in close proximity to the molecule. These effects are attributed to the fact that the dielectric tip converts non‐propagating near‐fields to propagating fields within the dielectric tip effectively quenching the fluorescence. The spatial extension of the quenching area is of subwavelength dimensions. The results are discussed in terms of molecular fluorescence in a system of stratified media. The experiment provides surprising new insights into the interactions between a fluorescent molecule and a dielectric tip. The methodology holds promise for applications in ultra high‐resolution near‐field optical imaging at the level of single fluorophores.     

Fluorescence microscopy: established and emerging methods, experimental strategies, and applications in immunology

Cutting-edge biophysical technologies including total internal reflection fluorescence microscopy, single molecule fluorescence, single channel opening events, fluorescence resonance energy transfer, high-speed exposures, two-photon imaging, fluorescence lifetime imaging, and other tools are becoming increasingly important in immunology as they link molecular events to cellular physiology, a key goal of modern immunology. The primary concern in all forms of microscopy is the generation of contrast; for fluorescence microscopy contrast can be thought of as the difference in intensity between the cell and background, the signal-to-noise ratio. High information-content images can be formed by enhancing the signal, suppressing the noise, or both. As improved tools, such as ICCD and EMCCD cameras, become available for fluorescence imaging in molecular and cellular immunology, it is important to optimize other aspects of the imaging system. Numerous practical strategies to enhance fluorescence microscopy experiments are reviewed. The use of instrumentation such as light traps, cameras, objectives, improved fluorescent labels, and image filtration routines applicable to low light level experiments are discussed. New methodologies providing resolution well beyond that given by the Rayleigh criterion are outlined. Ongoing and future developments in fluorescence microscopy instrumentation and technique are reviewed. This review is intended to address situations where the signal is weak, which is important for emerging techniques stressing super-resolution or live cell dynamics, but is less important for conventional applications such as indirect immunofluorescence. This review provides a broad integrative discussion of fluorescence microscopy with selected applications in immunology.     

Harmonic optical microscopy and fluorescence lifetime imaging platform for multimodal imaging

In this work, we proposed and built a multimodal optical setup that extends a commercially available confocal microscope (Olympus VF300) to include nonlinear second harmonic generation (SHG) and third harmonic generation (THG) optical (NLO) microscopy and fluorescence lifetime imaging microscopy (FLIM). We explored all the flexibility offered by this commercial confocal microscope to include the nonlinear microscopy capabilities. The setup allows image acquisition with confocal, brightfield, NLO/multiphoton and FLIM imaging. Simultaneously, two‐photon excited fluorescence (TPEF) and SHG are well established in the biomedical imaging area, because one can use the same ultrafast laser and detectors set to acquire both signals simultaneously. Because the integration with FLIM requires a separated modulus, there are fewer reports of TPEF+SHG+FLIM in the literature. The lack of reports of a TPEF+SHG+THG+FLIM system is mainly due to difficulties with THG because the present NLO laser sources generate THG in an UV wavelength range incompatible with microscope optics. In this article, we report the development of an easy‐to‐operate platform capable to perform two‐photon fluorescence (TPFE), SHG, THG, and FLIM using a single 80 MHz femtosecond Ti:sapphire laser source. We described the modifications over the confocal system necessary to implement this integration and verified the presence of SHG and THG signals by several physical evidences. Finally, we demonstrated the use of this integrated system by acquiring images of vegetables and epithelial cancer biological samples. Microsc. Res. Tech. 2012. © 2012 Wiley Periodicals, Inc.     

Versatile single-molecule multi-color excitation and detection fluorescence setup for studying biomolecular dynamics

Single-molecule fluorescence imaging is at the forefront of tools applied to study biomolecular dynamics both in vitro and in vivo. The ability of the single-molecule fluorescence microscope to conduct simultaneous multi-color excitation and detection is a key experimental feature that is under continuous development. In this paper, we describe in detail the design and the construction of a sophisticated and versatile multi-color excitation and emission fluorescence instrument for studying biomolecular dynamics at the single-molecule level. The setup is novel, economical and compact, where two inverted microscopes share a laser combiner module with six individual laser sources that extend from 400 to 640 nm. Nonetheless, each microscope can independently and in a flexible manner select the combinations, sequences, and intensities of the excitation wavelengths. This high flexibility is achieved by the replacement of conventional mechanical shutters with acousto-optic tunable filter (AOTF). The use of AOTF provides major advancement by controlling the intensities, duration, and selection of up to eight different wavelengths with microsecond alternation time in a transparent and easy manner for the end user. To our knowledge this is the first time AOTF is applied to wide-field total internal reflection fluorescence (TIRF) microscopy even though it has been commonly used in multi-wavelength confocal microscopy. The laser outputs from the combiner module are coupled to the microscopes by two sets of four single-mode optic fibers in order to allow for the optimization of the TIRF angle for each wavelength independently. The emission is split into two or four spectral channels to allow for the simultaneous detection of up to four different fluorophores of wide selection and using many possible excitation and photoactivation schemes. We demonstrate the performance of this new setup by conducting two-color alternating excitation single-molecule fluorescence resonance energy transfer (FRET) and a technically challenging four-color FRET experiments on doubly labeled duplex DNA and quadruple-labeled Holliday junction, respectively.     

Fluorescence resonance energy transfer of GFP and YFP by spectral imaging and quantitative acceptor photobleaching

To study protein–protein interactions by fluorescence energy transfer (FRET), the proteins of interest are tagged with either a donor or an acceptor fluorophore. For efficient FRET, fluorophores need to have a reasonable overlap of donor emission and acceptor excitation spectra. However, given the relatively small Stokes shift of conventional fluorescent proteins, donor and acceptor pairs with high FRET efficiencies have emission spectra that are difficult to separate. GFP and YFP are widely used in fluorescence microscopy studies. The spectral qualities of GFP and YFP make them one of the most efficient FRET donor–acceptor couples available. However, the emission peaks of GFP (510 nm) and YFP (527 nm) are spectrally too close for separation by conventional fluorescence microscopy. Difficulties in simultaneous detection of GFP and YFP with a fluorescence microscope are eliminated when spectral imaging and subsequent linear unmixing are applied. This allows FRET microscopy using these tags to study protein–protein interactions. We adapted the linear unmixing procedure from commercially available software (Zeiss) for use with acceptor photobleaching FRET using GFP and YFP as FRET pair. FRET efficiencies up to 52% for a GFP-YFP fusion protein were measured. To investigate the applicability of the procedure, we used two constituents of the nucleotide excision repair system, which removes UV-induced single-strand DNA damage. ERCC1 and XPF form a heterodimeric 5' endonuclease in nucleotide excision repair. FRET between ERCC1-GFP and XPF-YFP occurs with an efficiency of 30%.     

Combining AFM and FRET for high resolution fluorescence microscopy

Here we demonstrate a new microscopic method that combines atomic force microscopy (AFM) with fluorescence resonance energy transfer (FRET). This method takes advantage of the strong distance dependence in Förster energy transfer between dyes with the appropriate donor/acceptor properties to couple an optical dimension with conventional AFM. This is achieved by attaching an acceptor dye to the end of an AFM tip and exciting a sample bound donor dye through far-field illumination. Energy transfer from the excited donor to the tip immobilized acceptor dye leads to emission in the red whenever there is sufficient overlap between the two dyes. Because of the highly exponential distance dependence in this process, only those dyes located at the apex of the AFM tip, nearest the sample, interact strongly. This limited and highly specific interaction provides a mechanism for obtaining fluorescence contrast with high spatial resolution. Initial results in which 400 nm resolution is obtained through this AFM/FRET imaging technique are reported. Future modifications in the probe design are discussed to further improve both the fluorescence resolution and imaging capabilities of this new technique.     

Biosensor Förster resonance energy transfer detection by the phasor approach to fluorescence lifetime imaging microscopy

We present here the phasor approach to biosensor Förster resonance energy transfer (FRET) detection by fluorescence lifetime imaging microscopy (FLIM) and show that this method of data representation is robust towards biosensor design as well as the fluorescence artifacts inherent to the cellular environment. We demonstrate this property on a series of dual and single chain biosensors, which report the localization of Rac1 and RhoA activity, whilst performing concomitant ratiometric FRET analysis on the acquired FLIM data by the generalized polarization (GP) approach. We then evaluate and compare the ability of these two methods to quantitatively image biosensor FRET signal as a function of time and space. We find that with lifetime analysis in the phasor plot each molecular species is transformed into a two‐dimensional coordinate system where independent mixtures of fluorophores can be distinguished from changes in lifetime due to FRET. This enables the fractional contribution of the free and bound state of a dual chain biosensor or the low and high FRET species of a single chain biosensor to be quantified in each pixel of an image. The physical properties intrinsic to each biosensor design are also accurately characterized by the phasor analysis; thus, this method could be used to inform biosensor optimization at the developmental stage. We believe that as biosensors become more sophisticated and are multiplexed with other fluorescent molecular tools, biosensor FRET detection by the phasor approach to FLIM will not only become imperative to their use but also their advancement. Microsc. Res. Tech., 2011. © 2011 Wiley‐Liss, Inc.     

Fluorescence lifetime imaging--techniques and applications

Fluorescence lifetime imaging (FLIM) uses the fact that the fluorescence lifetime of a fluorophore depends on its molecular environment but not on its concentration. Molecular effects in a sample can therefore be investigated independently of the variable, and usually unknown concentration of the fluorophore. There is a variety of technical solutions of lifetime imaging in microscopy. The technical part of this paper focuses on time‐domain FLIM by multidimensional time‐correlated single photon counting, time‐domain FLIM by gated image intensifiers, frequency‐domain FLIM by gain‐modulated image intensifiers, and frequency‐domain FLIM by gain‐modulated photomultipliers. The application part describes the most frequent FLIM applications: Measurement of molecular environment parameters, protein‐interaction measurements by Förster resonance energy transfer (FRET), and measurements of the metabolic state of cells and tissue via their autofluorescence. Measurements of local environment parameters are based on lifetime changes induced by fluorescence quenching or conformation changes of the fluorophores. The advantage over intensity‐based measurements is that no special ratiometric fluorophores are needed. Therefore, a much wider selection of fluorescence markers can be used, and a wider range of cell parameters is accessible. FLIM‐FRET measures the change in the decay function of the FRET donor on interaction with an acceptor. FLIM‐based FRET measurement does not have to cope with problems like donor bleedthrough or directly excited acceptor fluorescence. This relaxes the requirements to the absorption and emission spectra of the donors and acceptors used. Moreover, FLIM‐FRET measurements are able to distinguish interacting and noninteracting fractions of the donor, and thus obtain independent information about distances and interacting and noninteracting protein fractions. This is information not accessible by steady‐state FRET techniques. Autofluorescence FLIM exploits changes in the decay parameters of endogenous fluorophores with the metabolic state of the cells or the tissue. By resolving changes in the binding, conformation, and composition of biologically relevant compounds FLIM delivers information not accessible by steady‐state fluorescence techniques.     

Field enhancement and apertureless near-field optical spectroscopy of single molecules

We report on fluorescence enhancement in near field optical spectroscopy by apertureless microscopy. Our apertureless microscope is designed around a confocal fluorescence microscope associated with an AFM head. First, we show that the confocal microscope alone allows single molecule imaging and single molecule fluorescence analysis. When associated with the AFM head, we demonstrate, for the first time to our knowledge, that single molecule fluorescence is enhanced under the silicon tip. We tentatively attribute this effect to field enhancement under the tip.     

Field enhancement and apertureless near-field optical spectroscopy of single molecules

We report on fluorescence enhancement in near field optical spectroscopy by apertureless microscopy. Our apertureless microscope is designed around a confocal fluorescence microscope associated with an AFM head. First, we show that the confocal microscope alone allows single molecule imaging and single molecule fluorescence analysis. When associated with the AFM head, we demonstrate, for the first time to our knowledge, that single molecule fluorescence is enhanced under the silicon tip. We tentatively attribute this effect to field enhancement under the tip.     

Ultrafast optical pulse delivery with fibers for nonlinear microscopy

Nonlinear microscopies including multiphoton excitation fluorescence microscopy and multiple-harmonic generation microscopy have recently gained popularity for cellular and tissue imaging. The optimization of these imaging methods for minimally invasive use requires optical fibers to conduct light into tight space, where free-space delivery is difficult. The delivery of high-peak power laser pulses with optical fibers is limited by dispersion resulting from nonlinear refractive index responses. In this article, we characterize a variety of commonly used optical fibers in terms of how they affect pulse profile and imaging performance of nonlinear microscopy; the following parameters are quantified: spectral bandwidth and temporal pulse width, two-photon excitation efficiency, and optical resolution. A theoretical explanation for the measured performance of these fibers is also provided.     

Novel lambda FRET spectral confocal microscopy imaging method

We report a highly specific, sensitive, and robust method for analyzing fluorescence resonance energy transfer (FRET) based on spectral laser scanning confocal microscopy imaging. The lambda FRET (lambdaFRET) algorithm comprises imaging of a FRET sample at multiple emission wavelengths rendering a FRET spectrum, which is separated into its donor and acceptor components to obtain a pixel-based calculation of FRET efficiency. The method uses a novel off-line precalibration procedure for spectral bleed-through correction based on the acquisition of reference reflection images, which simplifies the method and reduces variability. LambdaFRET method was validated using structurally characterized FRET standards with variable linker lengths and stoichiometries designed for this purpose. LambdaFRET performed better than other well-established methods, such as acceptor photobleaching and sensitized emission-based methods, in terms of specificity, reproducibility, and sensitivity to distance variations. Moreover, lambdaFRET analysis was unaffected by high fluorochrome spectral overlap and cellular autofluorescence. The lambdaFRET method demonstrated outstanding performance in intra- and intermolecular FRET analysis in both fixed and live cell imaging studies.     

Single molecule mapping of the optical field distribution of probes for near-field microscopy

The most difficult task in near-field scanning optical microscopy (NSOM) is to make a high quality subwavelength aperture probe. Recently, we have developed high definition NSOM probes by focused ion beam (FIB) milling. These probes have a higher brightness, better polarization characteristics, better aperture definition and a flatter end face than conventional NSOM probes. We have determined the quality of these probes in four independent ways: by FIB imaging and by shear-force microscopy (both providing geometrical information), by far-field optical measurements (yielding throughput and polarization characteristics), and ultimately by single molecule imaging in the near-field. In this paper, we report on a new method using shear-force microscopy to study the size of the aperture and the end face of the probe (with a roughness smaller than 1.5 nm). More importantly, we demonstrate the use of single molecules to measure the full three-dimensional optical near-field distribution of the probe with molecular spatial resolution. The single molecule images exhibit various intensity patterns, varying from circular and elliptical to double arc and ring structures, which depend on the orientation of the molecules with respect to the probe. The optical resolution in the measurements is not determined by the size of the aperture, but by the high optical field gradients at the rims of the aperture. With a 70 nm aperture probe, we obtain fluorescence field patterns with 45 nm FWHM. Clearly, this unprecedented near-field optical resolution constitutes an order of magnitude improvement over far-field methods like confocal microscopy.     

Imaging of endodontic biofilms by combined microscopy (FISH/cLSM – SEM)

Scanning electron microscopy is a useful imaging approach for the visualization of bacterial biofilms in their natural environments including their medical and dental habitats, because it allows for the exploration of large surfaces with excellent resolution of topographic features. Most biofilms in nature, however, are embedded in a thick layer of extracellular matrix that prevents a clear identification of individual bacteria by scanning electron microscopy. The use of confocal laser scanning microscopy on the other hand in combination with fluorescence in situ hybridization enables the visualization of matrix embedded bacteria in multi-layered biofilms. In our study, fluorescence in situ hybridization/confocal laser scanning microscopy and scanning electron microscopy were applied to visualize bacterial biofilm in endodontic root canals. The resulting fluorescence in situ hybridization /confocal laser scanning microscopy and scanning electron microscopy and pictures were subsequently combined into one single image to provide high-resolution information on the location of hidden bacteria. The combined use of scanning electron microscopy and fluorescence in situ hybridization / confocal laser scanning microscopy has the potential to overcome the limits of each single technique.