Microscope Configuration Research Articles

Controlling depth of focus in 3D image reconstructions by flexible and adaptive deformation of digital holograms

We show here that through an adaptive deformation of digital holograms it is possible to manage the depth of focus in 3D imaging reconstruction. Deformation is applied to the original hologram with the aim to put simultaneously in focus, and in one reconstructed image plane, different objects lying at different distances from the hologram plane (i.e., CCD sensor). In the same way, by adapting the deformation it is possible to extend the depth of field having a tilted object entirely in focus. We demonstrate the method in both lensless as well as in microscope configuration.

External linearisation of a MEMS electrothermal scanner with application in a confocal microscope

A compensatory mapping (CM) technique for external linearisation of a MEMS electrothermally actuated optical scanner is reported. Each individual axis of a two-axis MEMS scanner was linearised by the CM technique, and the scanner was then incorporated into a confocal optical microscope configuration. Cross-talk in the scanner when both axes are driven simultaneously, and arising when a drive signal to one axis of the scanner produces a mechanical response in the orthogonal scan axis, was subsequently compensated for by using a look-up-table optimised using a random search algorithm.

Investigation of angular multiplexing and de-multiplexing of digital holograms recorded in microscope configuration

We investigated a method for the angular multiplexing and de-multiplexing of digital holograms recorded in microscope off-axis configuration. The multiplexing has been performed rotating numerically one hologram at different angles and adding all the rotated holograms to obtain a single synthetic digital hologram. Then the digital holograms were de-multiplexed thanks to the unique property of the digital holography to manage numerically the complex wavefields at different image planes. We show that it is possible to retrieve correctly quantitative information about the amplitude and phase maps. The obtained results can be useful to employ the multiplexing technique during the recording process by rotating the CCD array.

Improved single particle localization accuracy with dual objective multifocal plane microscopy

In single particle imaging applications, the number of photons detected from the fluorescent label plays a crucial role in the quantitative analysis of the acquired data. For example, in tracking experiments the localization accuracy of the labeled entity can be improved by collecting more photons from the labeled entity. Here, we report the development of dual objective multifocal plane microscopy (dMUM) for single particle studies. The new microscope configuration uses two opposing objective lenses, where one of the objectives is in an inverted position and the other objective is in an upright position. We show that dMUM has a higher photon collection efficiency when compared to standard microscopes. We demonstrate that fluorescent labels can be localized with better accuracy in 2D and 3D when imaged through dMUM than when imaged through a standard microscope. Analytical tools are introduced to estimate the nanoprobe location from dMUM images and to characterize the accuracy with which they can be determined.

Depth of focus extended microscope configuration for imaging of incorporated groups of molecules, DNA constructs and clusters inside bacterial cells

Imaging of small objects such as single molecules, DNA clusters and single bacterial cells is problematic not only due to the lateral resolution that is obtainable in currently existing microscopy but also, and as much fundamentally limiting, due to the lack of sufficient axial depth of focus to have the full object focused simultaneously. Extension in depth of focus is helpful also for single molecule steady state FRET measurements. In this technique it is crucial to obtain data from many well focused molecules, which are often located in different axial depths. In this paper we present the implementation of an all-optical and a real time technique of extension in the depth of focus that may be incorporated in any high NA microscope system and to be used for the above mentioned applications. We demonstrate experimentally how after the integration of special optical element in high NA 100× objective lens of a single molecule imaging microscope system, the depth of focus is significantly improved while maintaining the same lateral resolution in imaging applications of incorporated groups of molecules, DNA constructs and clusters inside bacterial cells.

Dual Objective Multifocal Plane Microscopy for Single Particle/Molecule Imaging Applications

In single molecule/particle imaging applications, the number of photons detected from the fluorescent label plays a crucial role in the quantitative analysis of the acquired data. For example, in 2D/3D tracking experiments the localization accuracy of the labeled entity is inversely proportional to the square root of the number of detected photons. Hence achieving high photon collection efficiency is important in such studies. Currently, single molecule/particle imaging experiments are typically carried out on either an inverted or an upright microscope, in which the photons are collected from only one side of the sample (i.e., top side or the bottom side).Here, we report the development of dual objective multifocal plane microscopy (dMUM) for single particle/molecule studies. The dMUM configuration uses two opposing objective lenses, where one of the objectives is in an inverted position and the other objective is in an upright position. We show that dMUM has higher photon collection efficiency than a regular (inverted/upright) microscope for a given illumination condition. Because of the presence of two objective lenses, dMUM supports simultaneous imaging of different focal planes. This has been shown to be beneficial for high accuracy 3D localization and tracking of single molecules/particles and sub-cellular objects over a wide spatial range in live cells [1-3]. We demonstrate that fluorescent labels can be localized in 2D/3D with better accuracy when imaged through dMUM than when imaged through a regular (inverted/upright) microscope configuration. Analytical tools are introduced to estimate the 2D/3D location from dMUM images and to characterize the accuracy with which they can be determined.1. Prabhat, P. et al., IEEE Trans. Nanobiosci., 2004, 3, 237-242.2. Prabhat, P. et al., Proc. Natl. Acad. Sci., USA, 2007, 6443, D1-D7.3. Ram S., et al., Biophys. J., 2008, doi:10.1529/biophysj.108.1403

Nanoplasmonic Fluorescence Enhancement Applied to Study of Ion Channels

A metal nanoparticle can act as an antenna capable of increasing both the excitation rate and quantum yield of a fluorophore in close proximity to the nanoparticle. These properties thus enhance the fluorescence yielding an increase in brightness and decrease of the photobleaching rate - a highly useful tool in fluorescence studies of membrane proteins from the macroscopic to single molecule level. We have applied this technique to image purified membrane proteins in supported bilayer. The biological sample is purified KcsA K-channels labeled with TMR-6-M in the bundle crossing and reconstituted as proteoliposomes. We have also studied the membrane fluorophore DiI C18 in supported bilayer as a control. Among the many approaches towards fabrication of effective nanoparticles, we have synthesized spherical silver nanoparticles of ∼100 nm diameter coated with a thin SiO2 outer layer (Ag@SiO2). We have explored different size particles and various SiO2 thicknesses to find experimental conditions for optimal fluorescence enhancement. The SiO2 layer provides protection from chemical attack, acts as a spacer layer to avoid direct metal-fluorophore quenching, and allows surface functionalization. We have conjugated silica-coated silver particles to glass coverslips via polylysine (PL) in order to achieve a high-density silver nanoparticle monolayer. We record fluorescence from the labeled ion channels in an inverted TIRF microscope configuration imaged with a high-speed EMCCD camera. KcsA proteoliposomes are added to an Ag@SiO2-PL coverslip surface to rupture as supported bilayer patches for single molecule imaging. DiI liposomes were used in the same way. The KcsA-TMR and DiI samples show enhancement of at least 4-fold and 10-fold, respectively, compared to the same sample without nanoparticles. These results demonstrate the utility of this technique in fluorescence studies of ion channels or other membrane proteins. Supported by NIH 1R21MH078822 & 1F31NS054532.

Dronpa-1, Dronpa-2 And Dronpa-3 Separation Using Phase Resolved Optical Lock-in Detection (pholid) Microscopy

In living cells, protein complexes are dynamic structures that control many distinct aspects of cell behavior. Addressing the complexity of protein function requires a comprehensive understanding of subcellular localization and protein stoichiometry. Fluorescence microscopy provides a high contrast method to determine specific localizations of individual proteins contained within living cells. Determining localizations of protein pairs is made difficult by inherent photobleaching, cross-talk, and autofluorescence contained within the sample being imaged. Traditionally, to separate fluorescent proteins (FPs) with overlapping spectra, techniques such as linear unmixing and fluorescence lifetime imaging (FLIM) have been used. Such techniques rely on complex microscope configurations and often require a compromise in signal to noise ratios. In these studies we use phase information derived from optical lock in detection (OLID) (Mao et al. Biophys J. 2008 Jun;94(11):4515-2) to separate the photoswitchable FPs Dronpa-1, Dronpa-2, and Dronpa-3 from GFP and autofluorescence with no effect in signal to noise. Additionally, relative stochiometric ratios between each of the Dronpa variants contained within microinjected zebrafish embryos were obtained with a standard confocal microscope.

Benchtop time-resolved magneto-optical Kerr magnetometer

We present here the construction and application of a compact benchtop time-resolved Kerr magnetometer to measure the magnetization precession in magnetic thin films and lithographically patterned elements. As opposed to very expensive femtosecond lasers this system is built upon a picosecond pulsed injection diode laser and electronic pulse and delay generators. The precession is triggered by the electronic pulses of controlled duration and shape, which is launched onto the sample by a microstrip line. We used polarized optical pulses synchronous to the electronic pulses to measure the magneto-optical Kerr rotation. The system is integrated in a conventional upright microscope configuration with separate illumination, imaging, and magneto-optical probe paths. The system offers high stability, relative ease of alignment, sample changing, and a long range of time delay. We demonstrate the measurements of time-resolved dynamics of a Permalloy microwire and microdot using this system, which showed dynamics at two different time scales.

Holographic photolysis of caged neurotransmitters

Stimulation of light-sensitive chemical probes has become a powerful tool for the study of dynamic signaLing processes in living tissue. Classically, this approach has been constrained by limitations of lens-based and point-scanning illumination systems. Here we describe a microscope configuration that incorporates a nematic liquid-crystal spatial light modulator to generate holographic patterns of illumination. This microscope can produce illumination spots of variable size and number, and in patterns shaped to precisely match user-defined elements in a specimen. Using holographic illumination to photolyze caged glutamate in brain slices, we show that shaped excitation on segments of neuronal dendrites and simultaneous, multispot excitation of different dendrites enables precise spatial and rapid temporal control of glutamate receptor activation. By allowing the excitation volume shape to be tailored precisely, the holographic microscope provides an extremely flexible method for activation of various photosensitive proteins and small molecules.

Confocal and Atomic Force Microscopy

A wide range of different forms of microscopy may be applied to the visualisation of biological specimens. Confocal microscopy can provide 3D information about fluorescently labelled cells with the added advantage that it excludes out of focus light. Atomic force microscopy (AFM) provides direct high resolution images of surface features of a sample. Here we look at the microscope configuration necessary for simultaneous confocal and AFM, and examine how the combination of the two techniques together with the ‘Direct Overlay’ function can be used to great advantage in cell research for correlating surface and internal features.

Progress in the application of ATR-FTIR microscopy to the study of multi-layered cross-sections from works of art

As a non-invasive or micro-invasive technique attenuated total reflectance Fourier transform infrared spectroscopic (ATR-FTIR) microscopy is a valuable tool for the analysis of materials in works of art. An application for which it has received growing interest is in the analysis of paint cross-sections. However, FTIR microscope configurations, objectives' geometries and low spatial resolutions, and issues of sample preparation have often hampered the characterization of individual layers or features in cross-sections. With the use of case studies, it is demonstrated here that an ATR-FTIR microscope featuring a crystal of optimized geometry and a viewing capability feature allows characterization of individual layers, or areas within layers, of 10 microm thickness or less in single measurements. Of particular value is a remote aperturing feature which allows the analysis of selected areas within the contact footprint of the ATR crystal. Since the technique is non-destructive, the same area can be analyzed by complementary microscopic techniques such as Raman spectroscopy and scanning electron microscopy with energy-dispersive spectroscopy. Pyrolysis gas chromatography-mass spectrometry was also used in some cases to corroborate the spectroscopic data. The analyses presented provided data which were important in informing art historical interpretation and conservation of the artworks examined.

Polarization shifting interferometric profilometer

A phase sensitive Michelson interferometer based on interference microscope configuration with a polarization adjustment approach is proposed to determine the two-dimensional (2-D) surface profile of optical grating with real time capability. In the proposed method, nonlinear behavior of a PZT phase shifter is avoided by use of polarization stepping and a phase map is developed with the four-bucket algorithm. The phase map is unwrapped to give the true surface profile of the sample. A close agreement of measurements is found between the measured result determined by the proposed method and that determined by an atomic force microscope (AFM). We also analyzed the estimated uncertainty of measurement in the nanometer range for the random fluctuations of the experimental parameters.

Submicrometer photoresponse mapping of nanowire superconducting single-photon detectors

We report on the photoresponse mapping of nanowire superconducting single-photon detectors using a focal spot significantly smaller than the device area (10×10μm2). Using a confocal microscope configuration and solid immersion lens, we achieve a spot size of 320nm full width at half maximum onto the device at 470nm wavelength. We compare the response maps of two devices: The higher detection efficiency device gives a uniform response, whereas the lower detection efficiency device is limited by a single defect or constriction.

Activity-dependent Regulation of h Channel Distribution in Hippocampal CA1 Pyramidal Neurons

The hyperpolarization-activated cation current, I(h), plays an important role in regulating intrinsic neuronal excitability in the brain. In hippocampal pyramidal neurons, I(h) is mediated by h channels comprised primarily of the hyperpolarization-activated cyclic nucleotide-gated (HCN) channel subunits, HCN1 and HCN2. Pyramidal neuron h channels within hippocampal area CA1 are remarkably enriched in distal apical dendrites, and this unique distribution pattern is critical for regulating dendritic excitability. We utilized biochemical and immunohistochemical approaches in organotypic slice cultures to explore factors that control h channel localization in dendrites. We found that distal dendritic enrichment of HCN1 is first detectable at postnatal day 13, reaching maximal enrichment by the 3rd postnatal week. Interestingly we found that an intact entorhinal cortex, which projects to distal dendrites of CA1 but not area CA3, is critical for the establishment and maintenance of distal dendritic enrichment of HCN1. Moreover blockade of excitatory neurotransmission using tetrodotoxin, 6-cyano-7-nitroquinoxaline-2,3-dione, or 2-aminophosphonovalerate redistributed HCN1 evenly throughout the dendrite without significant changes in protein expression levels. Inhibition of calcium/calmodulin-dependent protein kinase II activity, but not p38 MAPK, also redistributed HCN1 in CA1 pyramidal neurons. We conclude that activation of ionotropic glutamate receptors by excitatory temporoammonic pathway projections from the entorhinal cortex establishes and maintains the distribution pattern of HCN1 in CA1 pyramidal neuron dendrites by activating calcium/calmodulin-dependent protein kinase II-mediated downstream signals.

Quantitative Phase Microscopy of microstructures with extended measurement range and correction of chromatic aberrations by multiwavelength digital holography

Quantitative Phase Microscopy (QPM) by interferometric techniques can require a multiwavelength configuration to remove 2pi ambiguity and improve accuracy. However, severe chromatic aberration can affect the resulting phase-contrast map. By means of classical interference microscope configuration it is quite unpractical to correct such aberration. We propose and demonstrate that by Digital Holography (DH) in a microscope configuration it is possible to clear out the QPM map from the chromatic aberration in a simpler and more effective way with respect to other approaches. The proposed method takes benefit of the unique feature of DH to record in a plane out-of-focus and subsequently reconstruct numerically at the right focal image plane. In fact, the main effect of the chromatic aberration is to shift differently the correct focal image plane at each wavelength and this can be readily compensated by adjusting the corresponding reconstruction distance for each wavelength. A procedure is described in order to determine easily the relative focal shift among different imaging wavelengths by performing a scanning of the numerical reconstruction along the optical axis, to find out the focus and to remove at the same time the chromatic aberration.

Diamond and boron carbide field emission in medium vacuum

AbstractField electron emission current–time and current–voltage measurements were made on single field emitters of synthetic diamond and boron carbide in medium vacuum (10−5–10−8 Torr), and two diamond specimens were characterized by scanning electron microscopy (SEM) and field ion microscopy (FIM). Generally, we found that the boron carbide emitters were less noisy than the diamond emitters, and that emission current stability improved with increasing current, at least up to 6 × 10−6 A. Use of the conventional field electron microscope (FEM) configuration enabled direct visualization of the surface condition during emission current and voltage measurements, and the combination of FIM and FEM permitted an estimation of current density for one diamond specimen, 1 × 106 A/cm2. Copyright © 2007 John Wiley & Sons, Ltd.

Simulation of a theta line-scanning confocal microscope

We describe a 2-D computational model of the optical propagation of coherent light from a laser diode within human skin to better understand the performance of a confocal reflectance theta microscope. The simulation uses finite-difference time domain (FDTD) computations to solve Maxwell's equations in a synthetic skin model that includes melanin, mitochondria, and nuclei. The theta line-scanning confocal microscope configuration experiences more localized decreases in the signal than the confocal common-path point-scanning microscope. We hypothesize that these decreases result from the bistatic imaging configuration, the imaging geometry, and the inhomogeneity of the index of refraction of the skin. All these factors result in the source path having aberrations different than those of the receiver path. The model predicts signal decreases that are somewhat greater than those seen in experiments. New details on the reflection from a spherical object show that imaging with the theta line scanner leads to somewhat different results than would be seen with a common-path point scanner. The model is used to optimize the design of the theta line-scanning confocal microscope.

Imaging of semiconducting polymer blend systems using environmental scanning electron microscopy and environmental scanning transmission electron microscopy

This study has investigated the potential of environmental electron microscopy techniques for studying the structure of polymer-based electronic devices. Polymer blend systems composed of F8BT and PFB were examined. Excellent contrast, both topographical and compositional, can be achieved using both conventional environmental scanning electron microscopy (ESEM) and a transmission detector giving an environmental scanning transmission electron microscope (ESTEM) configuration. Controllable charging effects present in the ESEM were observed, giving rise to a novel voltage contrast. This shows the potential of such contrast to provide excellent images of phase structure and charge distributions.

Identifying Chemical Changes in Subchondral Bone Taken from Murine Knee Joints Using Raman Spectroscopy

Application of Raman spectroscopy to analysis of subchondral bone is described. The effect of cartilage health on subchondral bone has been widely studied using radiological and histological methods; however, there is no method to directly assay mineral components. We present Raman spectra of femur condyles and observe mineral bands that arise from the subchondral bone. In two separate experiments, transgenic mouse models of early-onset osteoarthritis (OA) and lipoatrophy were compared to tissue from wild-type mice. Raman spectroscopy was used to identify chemical changes in the mineral of subchondral bone that may accompany or precede morphological changes that can be observed by histology. The transgenic mice were compared to age-matched wild-type mice. Subtle alterations in the mineral or collagen matrix were observed by Raman spectroscopy using established Raman markers such as the carbonate-to-phosphate ratio, mineral-to-matrix ratio (MTMR), and amide I ratio. The Raman microscope configuration enabled rapid collection of Raman spectra from the mineralized layer that lies under an intact layer of non-mineralized articular cartilage. The effect of the cartilage layer on collection of spectra is discussed. The technique proposed is capable of providing insight into the chemical changes that occur in subchondral bone on a molecular level.