Depth-of-field extension in optical imaging for rapid crystal screening

2021 ◽  
Vol 77 (4) ◽  
pp. 463-470
Author(s):  
Chen Li ◽  
Changqin Ding ◽  
Minghe Li ◽  
Jiayue Rong ◽  
Hilary Florian ◽  
...  
Keyword(s):  
Optical Imaging ◽  
Nonlinear Optical ◽  
Second Harmonic ◽  
Wavelength Dependence ◽  
Optical Microscope ◽  
Field Extension ◽  
Protein Crystal ◽  
Depth Of Field ◽  
Theoretical Predictions ◽  
Second Harmonic Generation Imaging

The depth of field (DoF) was extended 2.8-fold to achieve rapid crystal screening by retrofitting a custom-designed micro-retarder array (µRA) in the optical beam path of a nonlinear optical microscope. The merits of the proposed strategy for DoF enhancement were assessed in applications of second-harmonic generation imaging of protein crystals. It was found that DoF extension increased the number of crystals detected while simultaneously reducing the number of `z-slices' required for screening. Experimental measurements of the wavelength-dependence of the extended DoF were in excellent agreement with theoretical predictions. These results provide a simple and broadly applicable approach to increase the throughput of existing nonlinear optical imaging methods for protein crystal screening.

scholarly journals Intercalating dyes for enhanced contrast in second-harmonic generation imaging of protein crystals

2015 ◽  
Vol 71 (7) ◽  
pp. 1471-1477 ◽  
Author(s):  
Justin A. Newman ◽  
Nicole M. Scarborough ◽  
Nicholas R. Pogranichniy ◽  
Rashmi K. Shrestha ◽  
Richard G. Closser ◽  
...  
Keyword(s):  
Second Harmonic Generation ◽  
Crystal Lattice ◽  
Harmonic Generation ◽  
Nonlinear Optical ◽  
Second Harmonic ◽  
Nonlinear Optical Response ◽  
Protein Crystals ◽  
Kinetics Studies ◽  
Theoretical Predictions ◽  
Second Harmonic Generation Imaging

The second-harmonic generation (SHG) activity of protein crystals was found to be enhanced by up to ∼1000-fold by the intercalation of SHG phores within the crystal lattice. Unlike the intercalation of fluorophores, the SHG phores produced no significant background SHG from solvated dye or from dye intercalated into amorphous aggregates. The polarization-dependent SHG is consistent with the chromophores adopting the symmetry of the crystal lattice. In addition, the degree of enhancement for different symmetries of dyes is consistent with theoretical predictions based on the molecular nonlinear optical response. Kinetics studies indicate that intercalation arises over a timeframe of several minutes in lysozyme, with detectable enhancements within seconds. These results provide a potential means to increase the overall diversity of protein crystals and crystal sizes amenable to characterization by SHG microscopy.

scholarly journals Nonlinear optical imaging of single plasmonic nanoparticles with 30 nm resolution

2015 ◽  
Vol 17 (33) ◽  
pp. 21288-21293 ◽  
Author(s):  
Anke Horneber ◽  
Kai Braun ◽  
Jan Rogalski ◽  
Paul Leiderer ◽  
Alfred J. Meixner ◽  
...  
Keyword(s):  
Second Harmonic Generation ◽  
Optical Imaging ◽  
Optical Microscopy ◽  
Harmonic Generation ◽  
Nonlinear Optical ◽  
Second Harmonic ◽  
Near Field ◽  
Plasmonic Nanoparticles ◽  
Two Photon ◽  
Nonlinear Optical Imaging

Femtosecond-scanning near-field optical microscopy resolves the location-correlated second harmonic generation and two-photon photoluminescence from single nanoparticles with 30 nm resolution.

scholarly journals Integrated nonlinear optical imaging microscope for on-axis crystal detection and centering at a synchrotron beamline

2013 ◽  
Vol 20 (4) ◽  
pp. 531-540 ◽  
Author(s):  
Jeremy T. Madden ◽  
Scott J. Toth ◽  
Christopher M. Dettmar ◽  
Justin A. Newman ◽  
Robert A. Oglesbee ◽  
...  
Keyword(s):  
Nonlinear Optical ◽  
Phenylalanine Hydroxylase ◽  
Trichinella Spiralis ◽  
Second Harmonic ◽  
Protein Crystal ◽  
Cubic Phase ◽  
X Ray ◽  
Raster Scanning ◽  
Fold Reduction ◽  
Diffraction Patterns

Nonlinear optical (NLO) instrumentation has been integrated with synchrotron X-ray diffraction (XRD) for combined single-platform analysis, initially targeting applications for automated crystal centering. Second-harmonic-generation microscopy and two-photon-excited ultraviolet fluorescence microscopy were evaluated for crystal detection and assessed by X-ray raster scanning. Two optical designs were constructed and characterized; one positioned downstream of the sample and one integrated into the upstream optical path of the diffractometer. Both instruments enabled protein crystal identification with integration times between 80 and 150 µs per pixel, representing a ∼103–104-fold reduction in the per-pixel exposure time relative to X-ray raster scanning. Quantitative centering and analysis of phenylalanine hydroxylase fromChromobacterium violaceumcPAH,Trichinella spiralisdeubiquitinating enzyme TsUCH37, human κ-opioid receptor complex kOR-T4L produced in lipidic cubic phase (LCP), intimin prepared in LCP, and α-cellulose samples were performed by collecting multiple NLO images. The crystalline samples were characterized by single-crystal diffraction patterns, while α-cellulose was characterized by fiber diffraction. Good agreement was observed between the sample positions identified by NLO and XRD raster measurements for all samples studied.

scholarly journals Protein Crystal Staining for Second Harmonic Generation Imaging

2014 ◽  
Vol 70 (a1) ◽  
pp. C1154-C1154
Author(s):  
Justin Newman ◽  
Nicole Scarborough ◽  
Nicholas Pogranichnyi ◽  
Richard Closser ◽  
Garth Simpson
Keyword(s):  
Second Harmonic Generation ◽  
Harmonic Generation ◽  
Second Harmonic ◽  
Frequency Doubling ◽  
Theoretical Models ◽  
Time Lapse ◽  
Protein Crystal ◽  
Protein Crystals ◽  
Crystallization Conditions ◽  
Second Harmonic Generation Imaging

Recently, second harmonic generation (SHG) microscopy has become a useful tool in the field of structural biology for the detection of protein crystals. SHG, or the frequency doubling of light, is a process specific to crystalline media lacking inversion centers. Through theoretical models and experimental data, it is estimated that ~84% of the known protein crystal structures give detectable SHG signal using current SHG microscopy instrumentation. Extending this coverage could be extremely useful to structural biologists who routinely screen entire 96 well plates, with hundreds of crystallization conditions, in search of diffraction-quality protein crystals. A series of SHG active dyes, including Malachite Green (MG) and trans-4-[4-(dimethylamino)styryl]-1-methylpyridinium iodide (DMI) were investigated to assess their ability to enhance the nonlinear optical (NLO) response across a broad range of protein crystals with varying degrees of inherent SHG activity. MG and DMI were shown to enhance the SHG activity of tetragonal (P43212) lysozyme crystals, a protein that typically generates little to no SHG signal. SHG enhancements for lysozyme of approximately 16000x and 20x were achieved by intercalation of MG and DMI, respectively. These results are consistent with predictions based on the differences in symmetry and structure for the two dyes. The kinetics of the dye intercalation and uptake were investigated with SHG time-lapse images taken of a lysozyme crystal after the addition of MG dye into the crystallization well. Kinetic results indicate that an increase in SHG activity becomes easily noticeable within minutes of exposure to the dyes. These results show a significant progress towards increasing the coverage of SHG microscopy for protein crystal detection.

scholarly journals Nonlinear Optical Imaging of Cellular Processes in Breast Cancer

2008 ◽  
Vol 14 (6) ◽  
pp. 532-548 ◽  
Author(s):  
Paolo P. Provenzano ◽  
Kevin W. Eliceiri ◽  
Long Yan ◽  
Aude Ada-Nguema ◽  
Matthew W. Conklin ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Optical Imaging ◽  
Cancer Progression ◽  
Nonlinear Optical ◽  
Fluorescent Protein ◽  
Second Harmonic ◽  
Optical Methods ◽  
Additional Advantage ◽  
Biological Studies ◽  
Nonlinear Optical Imaging

AbstractNonlinear optical imaging techniques such as multiphoton and second harmonic generation (SHG) microscopy used in conjunction with novel signal analysis techniques such as spectroscopic and fluorescence excited state lifetime detection have begun to be used widely for biological studies. This is largely due to their promise to noninvasively monitor the intracellular processes of a cell together with the cell's interaction with its microenvironment. Compared to other optical methods these modalities provide superior depth penetration and viability and have the additional advantage in that they are compatible technologies that can be applied simultaneously. Therefore, application of these nonlinear optical approaches to the study of breast cancer holds particular promise as these techniques can be used to image exogeneous fluorophores such as green fluorescent protein as well as intrinsic signals such as SHG from collagen and endogenous fluorescence from nicotinamide adenine dinucleotide or flavin adenine dinucleotide. In this article the application of multiphoton excitation, SHG, and fluorescence lifetime imaging microscopy to relevant issues regarding the tumor-stromal interaction, cellular metabolism, and cell signaling in breast cancer is described. Furthermore, the ability to record and monitor the intrinsic fluorescence and SHG signals provides a unique tool for researchers to understand key events in cancer progression in its natural context.

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