Time‐gated interferometric detection can increase Raman scattering to fluorescence signal ratio in biological samples

Abstract. This work describes key improvements made to the in situ laser-induced fluorescence instrument for measuring sulfur dioxide (SO2) that was originally described by Rollins et al. (2016). Here, we report measurements of the SO2 fluorescence emission spectrum. These measurements allow for the determination of the most appropriate bandpass filters to optimize the fluorescence signal, while reducing the instrumental background. Because many aromatic species fluoresce in the same spectral region as SO2, fluorescence spectra were also measured for naphthalene and anisole to determine if ambient SO2 measurements could be biased in the presence of such species. Improvement in the laser system resulted in better tunability, and a significant reduction in the 216.9 nm laser linewidth. This increases the online/offline signal ratio which, in turn, improves the precision and specificity of the measurement. The effects of these improvements on the instrumental sensitivity were determined by analyzing the signal and background of the instrument, using varying optical bandpass filter ranges and cell pressures and calculating the resulting limit of detection. As a result, we report an improvement to the instrumental sensitivity by as much as 50 %.

Download Full-text

Visualizing subcellular structures in neurons with expansion microscopy

10.1101/2020.12.05.408724 ◽

2020 ◽

Author(s):

Logan A. Campbell ◽

Katy E. Pannoni ◽

Niesha A. Savory ◽

Dinesh Lal ◽

Shannon Farris

Keyword(s):

Golgi Apparatus ◽

Brain Tissue ◽

Mouse Brain ◽

Biological Samples ◽

Proteinase K ◽

Fluorescence Signal ◽

Expansion Process ◽

Physical Separation ◽

Subcellular Organelles ◽

The Brain

ABSTRACTProtein expansion microscopy (proExM) is a powerful technique that crosslinks proteins to a swellable hydrogel to physically expand and optically clear biological samples. The resulting increased resolution (~70 nm) and physical separation of labeled proteins make it an attractive tool for studying the localization of subcellular organelles in densely packed tissues, such as the brain. However, the digestion and expansion process greatly reduces fluorescence signals making it necessary to optimize ExM conditions per sample for specific end goals. Here we describe a proExM workflow optimized for resolving subcellular organelles (mitochondria and the Golgi apparatus) and reporter-labeled spines in fixed mouse brain tissue. By directly comparing proExM staining and digestion protocols, we found that immunostaining before proExM and using a Proteinase K based digestion for 8 hours consistently resulted in the best fluorescence signal to resolve subcellular organelles while maintaining sufficient reporter labeling to visualize spines and trace individual neurons. With these methods, we more accurately quantified mitochondria size and number and better visualized Golgi ultrastructure in reconstructed CA2 neurons of the hippocampus.

Download Full-text

Improvements to a laser-induced fluorescence instrument for measuring SO<sub>2</sub>: impact on accuracy and precision

10.5194/amt-2020-435 ◽

2020 ◽

Author(s):

Pamela S. Rickly ◽

Lu Xu ◽

John D. Crounse ◽

Paul O. Wennberg ◽

Andrew W. Rollins

Keyword(s):

Limit Of Detection ◽

Fluorescence Emission ◽

Laser Induced Fluorescence ◽

Fluorescence Signal ◽

Fluorescence Emission Spectrum ◽

Signal Ratio ◽

Accuracy And Precision ◽

Aromatic Species

Abstract. This work describes improvements made to the in-situ laser induced fluorescence SO2 instrument as originally described in Rollins et al. (2016). We report measurements of the SO2 fluorescence emission spectrum. These measurements allow for the determination of the most appropriate bandpass filters to optimize the fluorescence signal while reducing the instrumental background. Because many aromatic species fluoresce in the same spectral region as SO2, fluorescence spectra were also measured for naphthalene and anisole to determine if ambient SO2 measurements may be biased in the presence of such species. In addition, the 216.9 nm laser linewidth was decreased in order to increase the online/offline signal ratio which in-turn increases the precision of the measurement. The effects of these improvements on the instrumental sensitivity were determined by analyzing the signal and background of the instrument using varying optical bandpass filter ranges and cell pressures and calculating the resulting limit of detection. As a result, we report an improvement to the instrumental sensitivity by as much as 50 %.

Download Full-text

Homologous Gold Nanoparticles and Nanoclusters Composites with Enhanced Surface Raman Scattering and Metal Fluorescence for Cancer Imaging

Nanomaterials ◽

10.3390/nano8100819 ◽

2018 ◽

Vol 8 (10) ◽

pp. 819 ◽

Cited By ~ 2

Author(s):

Xiaoxia Wu ◽

Yan Peng ◽

Xiaomei Duan ◽

Lingyan Yang ◽

Jinze Lan ◽

...

Keyword(s):

Gold Nanoparticles ◽

Raman Scattering ◽

Early Stage ◽

Gold Nanoclusters ◽

Diagnostic Techniques ◽

Localized Surface Plasmon ◽

Fluorescence Signal ◽

Gold Nanocluster ◽

Dual Functional ◽

Effective Diagnosis

A large number of deaths from cancer can be attributed to the lack of effective early-stage diagnostic techniques. Thus, accurate and effective early diagnosis is a major research goal worldwide. With the unique phenomenon of localized surface plasmon resonance (LSPR), plasmonic nanomaterials have attracted considerable attention for applications in surface-enhanced Raman scattering (SERS) and metal-enhanced fluorescence (MEF). Both SERS and MEF are ultra-sensitive methods for the detection and identification of early tumor at molecular level. To combine the merits of the fast and accurate imaging of MEF and the stable and clear imaging of SERS, we propose a novel dual functional imaging nanoprobe based on gold nanoparticles and gold nanocluster composites (denoted AuNPC-RGD). The gold nanoparticles are used as LSPR substrates to realized enhancement of Raman or fluorescence signal, while the gold nanoclusters serve as a fluorophore for MEF imaging, and exhibit better biocompatibility and stability. Furthermore, target molecule of cyclic Arg-Gly-Asp (cRGD) is incorporated into the composite to improve delivery efficiency, selectivity and imaging accuracy. These integrated properties endow AuNPC-RGD composites with outstanding biocompatibility and excellent imaging abilities, which could be used to achieve accurate and effective diagnosis for early cancer.

Download Full-text

Ergothioneine, a dietary amino acid with a high relevance for the interpretation of label-free surface enhanced Raman scattering (SERS) spectra of many biological samples

Spectrochimica Acta Part A Molecular and Biomolecular Spectroscopy ◽

10.1016/j.saa.2020.119024 ◽

2021 ◽

Vol 246 ◽

pp. 119024 ◽

Cited By ~ 1

Author(s):

Stefano Fornasaro ◽

Elisa Gurian ◽

Sofia Pagarin ◽

Elena Genova ◽

Gabriele Stocco ◽

...

Keyword(s):

Free Surface ◽

Amino Acid ◽

Raman Scattering ◽

Biological Samples ◽

Surface Enhanced Raman Scattering ◽

Label Free ◽

Surface Enhanced ◽

Dietary Amino Acid ◽

Surface Enhanced Raman ◽

Enhanced Raman Scattering

Download Full-text

An Efficient Statistic for Determining the Diameter of Thin Sectioned Uniform Spheres from Measurements of Biological Samples

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100050585 ◽

1974 ◽

Vol 32 ◽

pp. 114-115

Author(s):

W. R. Schucany ◽

G. H. Kelsoe ◽

V. F. Allison

Keyword(s):

Biological Samples ◽

Traditional Method ◽

Cell Types ◽

Accurate Estimation ◽

Morphological Identification ◽

Sectioned Material ◽

Maximal Diameter ◽

Similar Cell

Accurate estimation of the size of spheroid organelles from thin sectioned material is often necessary, as uniquely homogenous populations of organelles such as vessicles, granules, or nuclei often are critically important in the morphological identification of similar cell types. However, the difficulty in obtaining accurate diameter measurements of thin sectioned organelles is well known. This difficulty is due to the extreme tenuity of the sectioned material as compared to the size of the intact organelle. In populations where low variance is suspected the traditional method of diameter estimation has been to measure literally hundreds of profiles and to describe the “largest” as representative of the “approximate maximal diameter”.

Download Full-text

The Application of Ultra-Thin Sectioning Techniques to Non-Biological Samples

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100101402 ◽

1968 ◽

Vol 26 ◽

pp. 332-333

Author(s):

C. F. Oster

Keyword(s):

Biological Sciences ◽

Epoxy Resin ◽

Cross Sections ◽

Biological Samples ◽

Industrial Applications ◽

Photographic Film ◽

Silver Particles ◽

Thin Sectioning ◽

Embedding Medium

Although ultra-thin sectioning techniques are widely used in the biological sciences, their applications are somewhat less popular but very useful in industrial applications. This presentation will review several specific applications where ultra-thin sectioning techniques have proven invaluable.The preparation of samples for sectioning usually involves embedding in an epoxy resin. Araldite 6005 Resin and Hardener are mixed so that the hardness of the embedding medium matches that of the sample to reduce any distortion of the sample during the sectioning process. No dehydration series are needed to prepare our usual samples for embedding, but some types require hardening and staining steps. The embedded samples are sectioned with either a prototype of a Porter-Blum Microtome or an LKB Ultrotome III. Both instruments are equipped with diamond knives.In the study of photographic film, the distribution of the developed silver particles through the layer is important to the image tone and/or scattering power. Also, the morphology of the developed silver is an important factor, and cross sections will show this structure.

Download Full-text

Artefacts in the x-ray microanalysis of frozen-hydrated biological samples

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100127724 ◽

1987 ◽

Vol 45 ◽

pp. 658-659

Author(s):

Patrick Echlin

Keyword(s):

Electron Scattering ◽

Biological Samples ◽

Fracture Face ◽

Detailed Structure ◽

X Ray ◽

Morphological Appearance ◽

The Poor ◽

Freeze Dried

A number of papers have appeared recently which purport to have carried out x-ray microanalysis on fully frozen hydrated samples. It is important to establish reliable criteria to be certain that a sample is in a fully hydrated state. The morphological appearance of the sample is an obvious parameter because fully hydrated samples lack the detailed structure seen in their freeze dried counterparts. The electron scattering by ice within a frozen-hydrated section and from the surface of a frozen-hydrated fracture face obscures cellular detail. (Fig. 1G and 1H.) However, the morphological appearance alone can be quite deceptive for as Figures 1E and 1F show, parts of frozen-dried samples may also have the poor morphology normally associated with fully hydrated samples. It is only when one examines the x-ray spectra that an assurance can be given that the sample is fully hydrated.

Download Full-text