Optical Lock-in Research Articles

Combining traditional and novel techniques to increase our understanding of the lock-in depth of atmospheric gases in polar ice cores – results from the EastGRIP region

Abstract. We investigate the lock-in zone (LIZ) of the East Greenland Ice Core Project (EastGRIP) region, northeastern Greenland, in detail. We present results from the firn air-pumping campaign of the S6 borehole, forward modeling, and a novel technique for finding the lock-in depth (LID, the top of the LIZ) based on the visual stratigraphy of the EastGRIP ice core. The findings in this work help to deepen our knowledge of how atmospheric gases are trapped in ice cores. CO2, δ15N, and CH4 data suggest that the LID lies around 58 to 61 m depth. With the pixel value intensity and bright-spot analysis based on visual stratigraphy, we can pinpoint a change in ice properties to exactly 58.3 m depth, which we define as the optical lock-in depth (OLID). This visual change in ice properties is caused by the formation of rounded and enclosed air bubbles that alter the measured refraction of the light pathways. The results for the LID and OLID agree accurately on the depth. We furthermore use the visual stratigraphy images to obtain information on the sharpness of the open- to closed-porosity transition. Combining traditional methods with the independent optical method presented here, we can now better constrain the bubble closure processes in polar firn.

Untangling light in noisy luminous environments

Electric light sources, including displays and luminaires, often exhibit rapid light fluctuations called temporal light modulation (TLM). TLM can be seen as their electrical footprint. This work demonstrates that TLM can be remotely detected and used to perform in situ photometric and spectroradiometric measurements of very low light levels superimposed to intense backgrounds. Illuminance levels about 500 000 times smaller than the background were reliably measured by lock-in detection, allowing photometric measurements to be performed during daytime, even with sunny conditions. Using a new type of optical lock-in spectrometer, it becomes possible to retrieve spectral quantities through the TLM of the light source. The spectral distribution of an LED lamp was measured against a daylit background more than 1500 times brighter. Lock-in detection is an excellent technique to perform in situ measurements of electric light, even at very low level, in the presence of daylight and other background lights, opening interesting opportunities to characterize anthropogenic light pollution.

A Fiber-Optic Array Spectrometer With Parallel Multichannel Optical Lock-in Detection and its Application to in Situ Lighting Measurements

This paper describes a new optical spectroscopy instrument achieving lock-in detection in all its spectral output channels in a parallel and simultaneous fashion. It is based on two identical compact array spectrometers working in phase quadrature thanks to optical modulators synchronized with an external reference signal. <p xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">In the application example, the optical lock-in spectrometer was used to perform spectral irradiance measurements with commercial LED lamps exhibiting temporal light modulation at different frequencies. A remote sensing optical device was designed to measure from a distance the temporal luminous waveform of a specific LED lamp and send it to the reference input of the optical lock-in spectrometer. When the lamps were operating together on top of a large continuous luminous background, the optical lock-in spectrometer successfully retrieved the respective spectral irradiance distributions and color parameters of each lamp, allowing their characterization to be performed independently of the other light sources illuminating the sensor during the measurements.

Optical Lock-in Spectrometry Reveals Useful Spectral Features of Temporal Light Modulation in Several Light Source Technologies

ABSTRACT This paper presents a study of the spectral characteristics of temporal light modulation in several technologies of lighting products. An optical lock-in spectrometer was designed for this purpose and integrated in a spectral radiant flux measurement facility. It was applied to incandescent and fluorescent lamps, as well as lamps based on white phosphor-converted LEDs and tunable RGB LEDs. The results are well correlated with the light emission processes of each technology. For incandescent lamps, the spectral modulation follows a 1/λ relationship in agreement with the blackbody radiation laws. Measurements performed on halophosphate and tri-phosphor tubes agree well with published data. The modulation and phase spectra of fluorescent lamps reveal a variable modulation rate across the visible range, directly related to the fluorescence lifetimes of the different luminophores, which were estimated from our data using a model of single exponential decay.The spectral modulation of white phosphor-converted LED lamps is nearly constant across the visible spectrum, demonstrating that their color parameters can be assessed from the lock-in modulation amplitude spectrum. In the case of tunable RGB LED lamps using PWM, the spectral modulation widely differs from the steady-state spectral distribution and changes with the user settings, confirming the possible occurrence of temporal color artifacts. Optical lock-in spectrometry can be used to improve spectral and color measurements of solid-state lighting, opening new opportunities for laboratory and remote sensing applications. Other foreseeable applications of optical lock-in spectrometry are also presented.

Polarization modulation with optical lock-in detection reveals universal fluorescence anisotropy of subcellular structures in live cells

The orientation of fluorophores can reveal crucial information about the structure and dynamics of their associated subcellular organelles. Despite significant progress in super-resolution, fluorescence polarization microscopy remains limited to unique samples with relatively strong polarization modulation and not applicable to the weak polarization signals in samples due to the excessive background noise. Here we apply optical lock-in detection to amplify the weak polarization modulation with super-resolution. This novel technique, termed optical lock-in detection super-resolution dipole orientation mapping (OLID-SDOM), could achieve a maximum of 100 frames per second and rapid extraction of 2D orientation, and distinguish distance up to 50 nm, making it suitable for monitoring structural dynamics concerning orientation changes in vivo. OLID-SDOM was employed to explore the universal anisotropy of a large variety of GFP-tagged subcellular organelles, including mitochondria, lysosome, Golgi, endosome, etc. We found that OUF (Orientation Uniformity Factor) of OLID-SDOM can be specific for different subcellular organelles, indicating that the anisotropy was related to the function of the organelles, and OUF can potentially be an indicator to distinguish normal and abnormal cells (even cancer cells). Furthermore, dual-color super-resolution OLID-SDOM imaging of lysosomes and actins demonstrates its potential in studying dynamic molecular interactions. The subtle anisotropy changes of expanding and shrinking dendritic spines in live neurons were observed with real-time OLID-SDOM. Revealing previously unobservable fluorescence anisotropy in various samples and indicating their underlying dynamic molecular structural changes, OLID-SDOM expands the toolkit for live cell research.

Comparison of the Optical Lock-in Thermography using the Reflection Mode and the Transmission Mode

Optical lock-in thermography is a non-destructive testing method. The surface of the sample is excited using the thermal waves and the response is recorded by the thermal camera. This image thermal sequence is processed using the image processing method named lock-in method. Optical lock-in thermography can be used to detect cracks and damages in metal or composite material. Two position modes are used: the reflection and the transmission mode. In this paper, are compared these two modes. An experiment is carried out on a printed composite plane with the square blinded holes placed in the different depths below the surface. The phase images are created using the lock-in method for both position modes. The results are compared and the advantages and the disadvantage of these position modes find out.

Ultra-sensitive lock-in amplifier coupled oscillatory magnetic tweezers for piconewton force manipulation applications

We have developed lock-in amplifier coupled oscillatory magnetic tweezers, aiming to synchronize the oscillatory magnetic force application and single-molecule response detection at the applied oscillatory frequency by incorporating an optical lock-in amplifier detection. The designed home-built lock-in amplifier detection instrument enables an exact reference signal input and hence reveals an accurate and sensitive magnetic response synchronization. We have further demonstrated the approach with the rhodamine 6G stained super-paramagnetic beads by monitoring the below-the-noise-background weak fluorescence signal changes due to the magnetic response of the super-paramagnetic beads under the oscillatory force manipulation. The integration of the lock-in amplifier and the oscillating magnetic tweezers can significantly expand the application of the magnetic tweezers for signal detection below the noise background, such as adapting to the important applications in the detailed exploration of mechanical properties of biomolecules and studies of the protein conformational fluctuation dynamics.

Optical lock-in camera for gravitational wave detectors.

Knowledge of the intensity and phase profiles of spectral components in a coherent optical field is critical for a wide range of high-precision optical applications. One of these is interferometric gravitational wave detectors, which rely on the optical beats between these fields for precise control of the experiment. Here we describe an optical lock-in camera and show that it can be used to record optical beats at MHz or greater frequencies with higher spatial and temporal resolution than previously possible. This improvement is achieved using a Pockels cell as a fast optical switch to transform each pixel on a sCMOS array into an optical lock-in amplifier. We demonstrate that the optical lock-in camera can record fields with 2 Mpx resolution at 10 Hz with a sensitivity of -62 dBc when averaged over 2s.

Role of Gln222 in Photoswitching of Aequorea Fluorescent Proteins: A Twisting and H-Bonding Affair?

Reversibly photoswitchable fluorescent proteins (RSFPs) admirably combine the genetic encoding of fluorescence with the ability to repeatedly toggle between a bright and dark state, adding a new temporal dimension to the fluorescence signal. Accordingly, in recent years RSFPs have paved the way to novel applications in cell imaging that rely on their reversible photoswitching, including many super-resolution techniques such as F-PALM, RESOLFT, and SOFI that provide nanoscale pictures of the living matter. Yet many RSFPs have been engineered by a rational approach only to a limited extent, in the absence of clear structure-property relationships that in most cases make anecdotic the emergence of the photoswitching. We reported [ Bizzarri et al. J. Am Chem Soc. 2010 , 102 , 85 ] how the E222Q replacement is a single photoswitching mutation, since it restores the intrinsic cis-trans photoisomerization properties of the chromophore in otherwise nonswitchable Aequorea proteins of different color and mutation pattern (Q-RSFPs). We here investigate the subtle role of Q222 on the excited-state photophysics of the two simplest Q-RSFPs by a combined experimental and theoretical approach, using their nonswitchable anacestor EGFP as benchmark. Our findings link indissolubly photoswitching and Q222 presence, by a simple yet elegant scenario: largely twisted chromophore structures around the double bond (including hula-twist configurations) are uniquely stabilized by Q222 via H-bonds. Likely, these H-bonds subtly modulate the electronic properties of the chromophore, enabling the conical intersection that connects the excited cis to ground trans chromophore. Thus, Q222 belongs to a restricted family of single mutations that change dramatically the functional phenotype of a protein. The capability to distinguish quantitatively T65S/E222Q EGFP ("WildQ", wQ) from the spectrally identical EGFP by quantitative Optical Lock-In Detection (qOLID) witnesses the relevance of this mutation for cell imaging.

Optical Lock-in Thermography for Structural Health Monitoring – A Study into Infrared Detector Performance

This paper reports on a study comparing the noise performance of two relatively low-cost microbolometers to a high-grade photon detector as applied to optical lock-in thermography, a well-established broad area non-destructive inspection technique. The photon detector is shown to significantly outperform both microbolometers by margins inconsistent with their noise equivalent temperature detectivity specifications. It is demonstrated that this performance gap can be overcome by increasing observation time or optical illumination intensity. For practical situations in which such steps are plausible microbolometers may provide a viable alternative to photon detectors for optical lock-in thermography. This raises the prospect of applying this method to structural health monitoring, where the generally small size and low capital cost of microbolometers would be advantageous.

Quantitative optical lock-in detection for quantitative imaging of switchable and non-switchable components.

Reversible photoswitching has been proposed as a way to identify molecules that are present in small numbers over a large, non-switching, background. This approach, called optical-lock-in-detection (OLID) requires the deterministic control of the fluorescence of a photochromic emitter through optical modulation between a bright (on) and a dark state (off). OLID yields a high-contrast map where the switching molecules are pinpointed, but the fractional intensities of the emitters are not returned. The present work presents a modified OLID approach (quantitative OLID or qOLID) that yields quantitative information of the switching (fSW ) and non-switching (fNS ) components. After the validation of the method with a sample dataset and image sequence, we apply qOLID to measurements in cells that transiently express the photochromic protein EYQ1. We show that qOLID is efficient in separating the modulated from the non-modulated signal, the latter deriving from background/autofluorescence or fluorophores emitting in the same spectral region. Finally, we apply qOLID to Förster (Fluorescence) Resonance Energy Transfer (FRET) imaging. We here demonstrate that qOLID is able to highlight the distribution of FRET intensity in a sample by using a photochromic donor and a non-photochromic acceptor.

High-contrast epi-fluorescence wide-field imaging of biological cells using integrating-bucket method

We present an optical lock-in detection scheme, called the integrating-bucket technique, as a signal-to-background ratio enhancement method for wide-field fluorescence imaging of biological cells. The proposed method uses sinusoidally modulated illumination light and captures four frames of fluorescence images, one per each quarter of the modulation period, by integrating the fluorescence intensity signal. The capability of this technique is demonstrated by imaging fluorescent bead solutions as well as labeled cells. The results show that the method yields a 4–10dB higher signal contrast than conventional fluorescence microscopy, and a background-free fluorescence image can be extracted within a sub-second time scale. Our findings indicate that the proposed method could be advantageous for the long-term study of live cells.

Photothermal optical lock-in optical coherence tomography for in vivo imaging.

Photothermal OCT (PTOCT) provides high sensitivity to molecular targets in tissue, and occupies a spatial imaging regime that is attractive for small animal imaging. However, current implementations of PTOCT require extensive temporal sampling, resulting in slow frame rates and a large data burden that limit its in vivo utility. To address these limitations, we have implemented optical lock-in techniques for photothermal optical lock-in OCT (poli-OCT), and demonstrated the in vivo imaging capabilities of this approach. The poli-OCT signal was assessed in tissue-mimicking phantoms containing indocyanine green (ICG), an FDA approved small molecule that has not been previously imaged in vivo with PTOCT. Then, the effects of in vivo blood flow and motion artifact were assessed and attenuated, and in vivo poli-OCT was demonstrated with both ICG and gold nanorods as contrast agents. Experiments revealed that poli-OCT signals agreed with optical lock-in theory and the bio-heat equation, and the system exhibited shot noise limited performance. In phantoms containing biologically relevant concentrations of ICG (1 µg/ml), the poli-OCT signal was significantly greater than control phantoms (p<0.05), demonstrating sensitivity to small molecules. Finally, in vivo poli-OCT of ICG identified the lymphatic vessels in a mouse ear, and also identified low concentrations (200 pM) of gold nanorods in subcutaneous injections at frame rates ten times faster than previously reported. This work illustrates that future in vivo molecular imaging studies could benefit from the improved acquisition and analysis times enabled by poli-OCT.

Fast wide-field photothermal and quantitative phase cell imaging with optical lock-in detection.

We present a fast, wide-field holography system for detecting photothermally excited gold nanospheres with combined quantitative phase imaging. An interferometric photothermal optical lock-in approach (POLI) is shown to improve SNR for detecting nanoparticles (NPs) on multiple substrates, including a monolayer of NPs on a silanized coverslip, and NPs bound to live cells. Furthermore, the set up allowed for co-registered quantitative phase imaging (QPI) to be acquired in an off-axis holographic set-up. An SNR of 103 was obtained for NP-tagging of epidermal growth factor receptor (EGFR) in live cells with a 3 second acquisition, while an SNR of 47 was seen for 20 ms acquisition. An analysis of improvements in SNR due to averaging multiple frames is presented, which suggest that residual photothermal signal can be a limiting factor. The combination of techniques allows for high resolution imaging of cell structure via QPI with the ability to identify receptor expression via POLI.

Quantitative evaluation of optical lock-in and pulsed thermography for aluminum foam material

In this article, quantitative evaluation of optical thermographic techniques relative to the non-destructive inspection of aluminum foam material is studied. For this purpose, a set of aluminum foam specimens with flat-bottom holes (FBH) was inspected by both optical lock-in thermography (LT) and pulsed thermography (PT). Probability of detection (PoD) analysis, as a quantitative method to estimate the capability and reliability of a particular inspection technique, was studied and compared for both optical LT and PT inspection results.

High-contrast fluorescence imaging in fixed and living cells using optimized optical switches.

We present the design, synthesis and characterization of new functionalized fluorescent optical switches for rapid, all-visible light-mediated manipulation of fluorescence signals from labelled structures within living cells, and as probes for high-contrast optical lock-in detection (OLID) imaging microscopy. A triazole-substituted BIPS (TzBIPS) is identified from a rational synthetic design strategy that undergoes robust, rapid and reversible, visible light-driven transitions between a colorless spiro- (SP) and a far-red absorbing merocyanine (MC) state within living cells. The excited MC-state of TzBIPS may also decay to the MC-ground state emitting near infra-red fluorescence, which is used as a sensitive and quantitative read-out of the state of the optical switch in living cells. The SP to MC transition for a membrane-targeted TzBIPS probe (C12-TzBIPS) is triggered at 405 nm at an energy level compatible with studies in living cells, while the action spectrum of the reverse transition (MC to SP) has a maximum at 650 nm. The SP to MC transition is complete within the 790 ns pixel dwell time of the confocal microscope, while a single cycle of optical switching between the SP and MC states in a region of interest is complete within 8 ms (125 Hz) within living cells, the fastest rate attained for any optical switch probe in a biological sample. This property can be exploited for real-time correction of background signals in living cells. A reactive form of TzBIPS is linked to secondary antibodies and used, in conjunction with an enhanced scope-based analysis of the modulated MC-fluorescence in immuno-stained cells, for high-contrast immunofluorescence microscopic analysis of the actin cytoskeleton.

Reversible optical control of cyanine fluorescence in fixed and living cells: optical lock-in detection immunofluorescence imaging microscopy.

Optical switch probes undergo rapid and reversible transitions between two distinct states, one of which may fluoresce. This class of probe is used in various super-resolution imaging techniques and in the high-contrast imaging technique of optical lock-in detection (OLID) microscopy. Here, we introduce optimized optical switches for studies in living cells under standard conditions of cell culture. In particular, a highly fluorescent cyanine probe (Cy or Cy3) is directly or indirectly linked to naphthoxazine (NISO), a highly efficient optical switch that undergoes robust, 405/532 nm-driven transitions between a colourless spiro (SP) state and a colourful merocyanine (MC) state. The intensity of Cy fluorescence in these Cy/Cy3-NISO probes is reversibly modulated between a low and high value in SP and MC states, respectively, as a result of Förster resonance energy transfer. Cy/Cy3-NISO probes are targeted to specific proteins in living cells where defined waveforms of Cy3 fluorescence are generated by optical switching of the SP and MC states. Finally, we introduce a new imaging technique (called OLID-immunofluorescence microscopy) that combines optical modulation of Cy3 fluorescence from Cy3/NISO co-labelled antibodies within fixed cells and OLID analysis to significantly improve image contrast in samples having high background or rare antigens.

Fast three-dimensional imaging of gold nanoparticles in living cells with photothermal optical lock-in Optical Coherence Microscopy

We introduce photothermal optical lock-in Optical Coherence Microscopy (poli-OCM), a volumetric imaging technique, which combines the depth sectioning of OCM with the high sensitivity of photothermal microscopy while maintaining the fast acquisition speed inherent to OCM. We report on the detection of single 40 nm gold particles with a 0.5 μm lateral and 2 μm axial resolution over a 50 μm depth of field and the three-dimensional localization of gold colloids within living cells. In combination with intrinsic sample contrast measured with dark-field OCM, poli-OCM offers a versatile platform for functional cell imaging.

Reversible Fluorescence Photoswitching in DNA

We describe the engineering of reversible fluorescence photoswitching in DNA with high-density substitution, and its applications in advanced fluorescence microscopy methods. High-density labeling of DNA with cyanine dyes can be achieved by polymerase chain reaction using a modified DNA polymerase that has been evolved to efficiently incorporate Cy3- and Cy5-labeled cytosine base analogues into double-stranded DNA. The resulting biopolymer, “CyDNA”, displays hundreds of fluorophores per DNA strand and is strongly colored and highly fluorescent, although previous observations suggest that fluorescence quenching at such high density might be a concern, especially for Cy5. Herein, we first investigate the mechanisms of fluorescence quenching in CyDNA and we suggest that two different mechanisms, aggregate formation and resonance energy transfer, are responsible for fluorescence quenching at high labeling densities. Moreover, we have been able to re-engineer CyDNA into a reversible fluorescence photoswitchable biopolymer by using the properties of the Cy3–Cy5 pair. This novel biopolymer constitutes a new class of photoactive DNA-based nanomaterial and is of great interest for advanced microscopy applications. We show that reversible fluorescence photoswitching in CyDNA can be exploited in optical lock-in detection imaging. It also lays the foundations for improved and sequence-specific super-resolution fluorescence microscopy of DNA.

A unique series of reversibly switchable fluorescent proteins with beneficial properties for various applications

Reversibly switchable fluorescent proteins (RSFPs) have attracted widespread interest for emerging techniques including repeated tracking of protein behavior and superresolution microscopy. Among the limited number of RSFPs available, only Dronpa is widely employed for most cell biology applications due to its monomeric and other favorable photochemical properties. Here we developed a series of monomeric green RSFPs with beneficial optical characteristics such as high photon output per switch, high photostability, a broad range of switching rate, and pH-dependence, which make them potentially useful for various applications. One member of this series, mGeos-M, exhibits the highest photon budget and localization precision potential among all green RSFPs. We propose mGeos-M as a candidate to replace Dronpa for applications such as dynamic tracking, dual-color superresolution imaging, and optical lock-in detection.