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: <i>conjugate</i> (<i>c,</i> ca. “on-focus”) consisting of light impinging on and deviated from the “on” elements of the DMD, and the <i>non-conjugate</i> (<i>nc,</i> 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 <i>c</i> and <i>nc</i> 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
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.
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.
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.