We report on the current version of the optical sectioning programmable array microscope (PAM) implemented with a single digital micro-mirror device (DMD) spatial light modulator utilized as a mask in both the fluorescence excitation and emission paths. The PAM incorporates structured illumination and structured detection operating in synchrony. A sequence of binary patterns of excitation light in high definition format (1920×1080 elements) is projected into the focal plane of the microscope at the 18 kHz binary frame rate of the Texas Instruments 1080p DMD. The resulting fluorescent emission is captured as two distinct signals: conjugate (c, ca. “on-focus”) consisting of light impinging on and deviated from the “on” elements of the DMD, and the non-conjugate (nc, ca. “out-of-focus”) light falling on and deviated from the “off” elements. The two distinct, deflected beams are optically filtered and detected either by two individual cameras or captured as adjacent images on a single camera after traversing an image combiner. The sectioned image is gained from a subtraction of the nc image from the c image, weighted in accordance with the pattern(s) used for illumination and detection and the relative exposure times of the cameras. The widefield image is given by the sum of the c and nc images. This procedure allows a high duty cycle (typically 25-50%) of on-elements in the excitation patterns and thus functions with low light intensities, preventing saturation and minimizing photobleaching of sensitive fluorophores. The corresponding acquisition speed is also very high, limited only by the bandwidth of the camera(s) (100 fps full frame with the sCMOS camera in current use) and the optical power of the light source (lasers, large area LEDs). In contrast to the static patterns typical of SIM systems, the programmable array allows optimization of the patterns (duty cycle, feature size and distribution), thus enabling a wide range of applications, ranging from patterned photobleaching, (e.g., FRAP, FLIP) and photoactivation, spatial superresolution, automated adaptive tracking and minimization of light exposure (MLE), and photolithography.
We report progress on the construction of an optical sectioning programmable array microscope (PAM) implemented
with a digital micro-mirror device (DMD) spatial light modulator (SLM) utilized for both fluorescence illumination and
detection. The introduction of binary intensity modulation at the focal plane of a microscope objective in a computer
controlled pixilated mode allows the recovery of an optically sectioned image. Illumination patterns can be changed very
quickly, in contrast to static Nipkow disk or aperture correlation implementations, thereby creating an optical system
that can be optimized to the optical specimen in a convenient manner, e.g. for patterned photobleaching, photobleaching
reduction, or spatial superresolution.
We present a third generation (Gen-3) dual path PAM module incorporating the 25 kHz binary frame rate TI 1080p
DMD and a newly developed optical system that offers diffraction limited imaging with compensation of tilt angle
distortion.
Quantum dots (QDs) are unique probes due to their special properties (brightness, photostability, narrowband emission
and broadband absorption), and excellent bio(chemical)compatibility for imaging structures and functions of living
cells. When functionalized with ligands, they enable the recognition of specific targets and the tracking of dynamic
processes for extended periods of time, detecting biomolecules with a sensitivity extending to the single molecule level.
Thus, devices and probes based on such nanoparticles are very powerful tools for studying essential processes
underlying the functions and regulation of living cells.
Here we present nanosensors and nanoactuators based on QDs in which the multivalency of these particles plays
an essential role in the functionality and sensing characteristics of the nanodevices. Two examples are discussed, the
first being pH nanosensors based on the interplay of the multivalency and energy transfer between the nanoparticles and
small molecules on their surface, and the second nanoactuators in which a controlled number of molecules of the
amyloid protein α-synuclein (AS) specifically regulate the aggregation of fluorescently labeled bulk AS protein both in
vitro and in live cells.
We report on a new generation, commercial prototype of a programmable array optical sectioning fluorescence
microscope (PAM) for rapid, light efficient 3D imaging of living specimens. The stand-alone module, including light
source(s) and detector(s), features an innovative optical design and a ferroelectric liquid-crystal-on-silicon (LCoS)
spatial light modulator (SLM) instead of the DMD used in the original PAM design. The LCoS PAM (developed in
collaboration with Cairn Research, Ltd.) can be attached to a port of a(ny) unmodified fluorescence microscope. The
prototype system currently operated at the Max Planck Institute incorporates a 6-position high-intensity LED
illuminator, modulated laser and lamp light sources, and an Andor iXon emCCD camera. The module is mounted on an
Olympus IX71 inverted microscope with 60-150X objectives with a Prior Scientific x,y, and z high resolution scanning
stages. Further enhancements recently include: (i) point- and line-wise spectral resolution and (ii) lifetime imaging
(FLIM) in the frequency domain. Multiphoton operation and other nonlinear techniques should be feasible.
The capabilities of the PAM are illustrated by several examples demonstrating single molecule as well as lifetime
imaging in live cells, and the unique capability to perform photoconversion with arbitrary patterns and high spatial
resolution. Using quantum dot coupled ligands we show real-time binding and subsequent trafficking of individual
ligand-growth factor receptor complexes on and in live cells with a temporal resolution and sensitivity exceeding those
of conventional CLSM systems. The combined use of a blue laser and parallel LED or visible laser sources permits
photoactivation and rapid kinetic analysis of cellular processes probed by photoswitchable visible fluorescent proteins
such as DRONPA.
Multifunctional nanoparticles, quantum dots (QDs) are being developed as uniquely sensitive tools for elucidating the (bio)chemical and (bio)physical molecular mechanisms underlying functional states, i.e. the molecular physiology, of biological cells and organisms. Here we present a group of strategies and examples for (i) controlling the spectroscopic properties of QDs via Fluorescence Resonance Energy Transfer (FRET); (ii) determining the emission spectra of individual QDs in a population with an imaging spectrograph (ASI SpectraCube); and (iii) employing such liganded QDs as nano-probes in cellular studies of signal transduction.
Innovations in fluorescence microscopy of live cells involving new reagents and techniques reveal dynamic processes that were not previously observable and therefore unknown. Water soluble, biofunctionalized semiconductor quantum dots (QDs) provide advantages of much greater photostability compared to conventional fluorescent dyes, and, as a consequence, single QDs can be easily detected. QDs coupled to growth factor ligands behave similarly as the natural ligand and serve as highly fluorescent probes of the erbB family of tyrosine kinase receptors in living cells. Continuous confocal laser scanning microscopy and flow cytometry measurements of QDs combined with visible fluorescent fusions of the receptors have elucidated individual steps in the signaling cascades initiated by these receptors. This report highlights advantages and some disadvantages of QDs, such as size and blinking behavior that complicate some live cell imaging applications. The new class of noble metal nanodots constitute an attractive alternative to QDs in that they are not only highly fluorescent and photostable, but also, much smaller and nontoxic. We present a new synthesis method for the production of Au nanodots. We demonstrate that electrochemical synthesis allows the reproducible control of cluster size. The resulting clusters are more monodisperse than those formed by other methods and are stable over many months. We report their characterization using MALDI-TOF mass spectrometry and UV-VIS spectroscopy.
In previous publications we have shown that we can perform enzymatic reactions in nanoarrays by means of a microarray-reader based on a conventional microscope. In this publication we report on a modification of this system in order to monitor the aggregation kinetics of the natively unfolded protein α-synuclein. We describe the motivation for this development, the problems associated with the miniaturization of the aggregation assay, and the validation of our modifications.
Photochromic FRET (pcFRET), a member of the family of acceptor depletion FRET techniques (adFRET), embodies a general conceptual and experimental scheme based on a coupled system of a fluorescent donor and a photchromic acceptor. The procedure involves the reversible and cyclic spectroscopic depletion of the acceptor, and was initially conceived for the determination of FRET efficiency on a continuos, pixel-by-pixel basis in the microscopy of living cells. However, the modulation of donor fluorescence in pcFRET has implications for a wide range of applications. We present the formalism for quantitative interpretations of photostationary and kinetic data, from which the relevant kinetic rate constants and quantum uields for the cyclization and cycloreversion reactions of the photochromic acceptor can be derived. The scheme was applied to a model system consisting of a fluorescent donor (Lucifer Yellow) covalently bound to a diheteroarylethene acceptor. In a Perspectives section, we discuss photochromic probes, instrumentation issues, and the potential of pcFRET for analyizing chemical equilibria and kinetics, in the latter case with a new technique we have denoted Photochromic Relaxation Kinetics (pcRelKin).
Fluorescence anisotropy, a measure of the polarization state of fluorescence emission, is a sensitive measure of molecular rotational motion and of resonance energy transfer (RET). We report here the formalism and application of dynamic and static fluorescence anisotropy measurements primarily intended for implementation in imaging systems. These include confocal lasre scanning microscopes (CLSM) as well as wide-field instruments, in the latter case adapted for anisotropy-based dynamic frequency domain fluorescence lifetime imaging microscopy (FLIM), a method we denote as rFLIM. Anisotropy RET is one of the modalities used for fluorescence RET (FRET) determinations of the association, and proximity of cellular proteins in vivo. A requirement is the existence of intrinsic or extrinsic probes exhibiting homotransfer FRET (in our nomenclature, energy migration or emFRET) between like fluorophores. This phenomenon is particularly useful in studies of the activation and processing of transmembrane receptor tyrosine kinases involved in signal transduction and expressed as fusions with Visible Fluorescence Proteins (VFPs).
We have developed efficient image restoration algorithms for restoration of images that are acquired by conventional and confocal fluorescence microscopy. Assuming additive Gaussian noise or Poisson noise in the image and Gaussian or entropy prior distributions, functionals are formulated that must be minimized to obtain maximum a posteriori (MAP) and maximum likelihood (ML) estimations. We propose computationally efficient algorithms to find the solutions. The quality of the MAP restorations is determined largely by the choice of the regularization parameter, which determines the tradeoff between fitting and smoothing the solution. We propose a normalization method to ease the interactive choice of the regularization parameter if the variance of the noise is known. The performance of the algorithms was tested using simulated fluorescence conventional microscopy and fluorescence confocal laser scanning microscopy. Several error measures and quantitative measurements were used to evaluate the quality of the restoration result. We have tested the super-resolution capabilities and have found that the algorithms are capable of recovering partially the frequencies that were lost. The performance of the algorithms was compared to two existing algorithms that are commonly used for fluorescence imaging: the accelerated EM algorithm of Holmes and the regularized algorithm of Carrington.
Fluorescence lifetime imaging is a relatively new technique for acquiring directly the nanosecond temporal characteristics of the fluorescence emission of a spatially extended object, and for capturing the dynamic features at every pixel of an image simultaneously. In general, the applications of fluorescence lifetime imaging have been mainly in the microscope, but other diverse imaging situations can benefit from the technology. Our instrument employs periodically modulated excitation light, synchronous modulation of the amplification stages of a microchannel plate intensifier, and subsequent digital recording of the image with a charge-coupled device camera. The digitized images can be subsequently analyzed with a variety of different ways. A short description of the lifetime resolved fluorescence imaging instrumentation is given together with typical applications depicting lifetime spatial distributions, multiple lifetime analysis, statistical analysis of the image data, and suppression or enhancement of particular fluorescent species.
The extension of microscope luminescence measurements into the temporal domain provides the possibility of determining time-resolved properties of microscope samples and their surrounding environments, and thereby extends the conventional steady state measurements. `Time resolved imaging microscopy' is a relatively new technique whereby fast kinetic and luminescence decay parameters (decay times and the corresponding time or phase resolved amplitudes) are directly and simultaneously measured throughout an image, pixel by pixel, in an optical microscope. Molecular rotation, solvent and matrix relaxation, quenching mechanisms, reactions, and energy transfer are examples of molecular spectroscopic processes that can be studied best by directly measuring the time dependent properties. Dynamic measurements are generally much more informative than their steady state counterparts. The goal of our work is to develop time-resolved methods that can be applied conveniently and routinely to biological material in the microscope over a wide time domain. In addition to the augmented purely spectroscopic and reaction kinetic information, simultaneous spatial and temporal resolution of an image in a microscope provides significant improvement in image contrast, probe identification and differentiation, background (light scattering and inherent luminescence) reduction, and provides additional parameters for digital image analysis. Time resolution makes it possible to recover structures in an image concealed by background luminescence with a different lifetime. Examples of these procedures are given, and the instrumentation required for the data acquisition and analysis is discussed. The technique employs phase-locked coordination between the modulation of the perturbation and the recording of the luminescence image together with a Photometrics (Tucson, Ariz.) series 200 high resolution slow-scan scientific CCD camera. A normal fluorescence microscope is used.
Biological samples have been imaged using microscopes equipped with slow-scan CCD cameras. Examples are presented of studies based on the detection of light emission signals in the form of fluorescence and phosphorescence. They include applications in the field of cell biology: (a) replication and topology of mammalian cell nuclei; (b) cytogenetic analysis of human metaphase chromosomes; and (c) time-resolved measurements of DNA-binding dyes in cells and on isolated chromosomes, as well as of mammalian cell surface antigens, using the phosphorescence of acridine orange and fluorescence resonance energy transfer of labeled lectins, respectively.
The cell surface receptor for epidermal growth factor (EGFR) is one of the most studied integral membrane proteins. The receptor is widely distributed in cells and tissues of mammalian and avian tissues and plays an important role in growth control. Binding of the epidermal growth factor (EGF) to EGFR initiates a complex biological response, which includes self-phosphorylation of the receptor due to an intrinsic tyrosine kinase activity, phosphorylation of other membrane proteins, increased intake of metabolites, and increased proliferation. Complete amino acid sequence of EGFR revealed a high degree of homology with viral oncogenes and allowed tentative identification of an external hormone binding domain, a transmembrane domain, and a cytoplasmic domain that includes tyrosine kinase activity. EGF binding induces rapid aggregation of EGFR, a process which was also observed on other receptor systems. These and other observations led to a hypothesis that microaggregation of EGFR is a necessary prerequisite for the biological response of EGF. A direct approach to study the processes of oligomerization of cell membrane proteins is to measure their mobility under various conditions. The lateral mobility of the EGFR was studied on mouse 3T3 fibroblasts and on A431 cells. However, an examination of the equations for the lateral and rotational diffusion in membranes shows that only rotational diffusion is strongly dependent on the size of the diffusing entity. A method of measuring protein rotational diffusion by time-resolved phosphorescence has proved to be very useful in the analysis of both in vivo and in vitro systems. The authors apply this method to study the mobility of EGFR on living A431 cells and membrane preparations.
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