Advanced laboratory equipment

Raman measurements

Biological applications

Generic applications

• Precancer/cancer detection
• Raman investigation on viruses
• Microorganisms: Direct identification of clinically relevant microorganisms on solid culture media
• Agrofood: Rapid detection of foodborne microorganisms on food surface, Protein analysis in food systems, rapid analysis of sugars
• Pharmaceutical applications: pharmacokinetics
• Confocal microscopy

Tissue applications

• Raman measurements of plant tissue (in example cell walls)
• Mammalian tissue (flies brain tissue)
• Human tissue (skin, thyroid: cancer imaging)
• Ιdentification of unknown particles on a biological tissue
• Analysis of proteins, lipids, nucleic acids and sugars in tissues

Cells (fixed or live)

• In vivo discrimination between normal and pathological cells: carcinoma, melanoma, human thyroid, leukemia, prostate, blood, brain, skin, epithelial, lung, breast, bladder
• In vivo characterization of different types of human skin cells, lymphocytes, lung cancer cells, lymphoma cells etc.
• microorganisms such as bacterial cells and fungi
• Protein/lipid and sugar distribution in cells

Biofluids

• Lipid discrimination: Investigation the Atherosclerosis Process by Monitoring Lipid Deposits Including Cholesterol and Free Fatty Acids
• DNA/RNA:
  • Raman analysis of Sperm Nuclear DNA Integrity
  • methylation
  • acetylation
  • RNA detection in DNA samples
• Protein characterization in biological systems
• Nucleic acid assemblies analysis

Characterization techniques

Raman spectroscopy

• Description: Raman spectroscopy enables the characterization of chemical bonds vibration. It is a well-defined spectroscopic technique sensitive to the molecular vibrations of materials, since it provides fingerprint-like information regarding the molecular structure of the analyzed samples. It has been extensively used for the non-destructive and label-free cell characterization, particularly on the qualitative and quantitative estimation of amino acids, lipids, nucleic acids, and carbohydrates. This technique has promising applications for biological materials characterization, the early diagnosis, categorization and assessment of pathologies, and the putative drug efficacy on targeted diseases. It is important to note that Raman signal remains uninfluenced by water and contains information regarding concentration, chemical phase and crystallinity of the sample.

• Substrates

Raman grade Calcium Fluoride (CaF₂) substrates (Crystran)

Raman Grade CaF₂ offers many advantages:

► Wide transmission range from deep UV to mid IR

► Complete flexibility of laser between 130nm to 10µm

► One single Raman peak only at 321cm^-1 specific to CaF₂

► No other Raman peaks to confuse the results (does not interfere with the Raman signal)

► Minimal fluorescence

► Durable and insoluble

• microscopic slide – (75 x 25 x 1.0mm)
• Round and square Coverslips-200 or 500 μm thickness, 12 mm diameter covers the biological specimen in a microscope specimen. Used basically for liquid samples formation into flat layer of uniform thickness.

Stainless steel microscopic slide (Renishaw)

Provides improved signal for Raman spectroscopy of tissue and cells with minimum background

Raman hyperspectral imaging (mapping)

• Description: Raman spectroscopy coupled with optical microscopy achieves the acquisition of spatial and spectral information from a sample at the same time. Raman hyperspectral imaging allows the biomolecular characterization of the sample (different chemical content) within the field of view of the objective lens by adding distribution information on the spatial domain. Data are collected in the form of a three-dimensional hyperspectral datacube (x,y and λ) with x and y as spatial and λ as spectral dimensions.
• Characteristics: Hyperspectral data are acquired with the use of a XYZ scanner for mapping. Analysis is based on chemometrics and may be divided in three main parts: (i) preprocessing of the raw spectral data, aiming at extracting only the relevant information by limiting the effect of undesired phenomena, (ii) processing of preprocessed data based on univariate (peak height, area or ratio) or multivariate data analysis (for instance PCA, ICA, MCR-ALS or PLS) depending on the data complexity and (iii) post-processing of the image. Each chemometric tool has its own specificities and will be used depending on the available data and the desired results.
• Applications
  • Tissue imaging and medical diagnosis
  • Food safety inspection and control (Microbial growth localization)
  • Inedible bodies in food (such as fish bones in fillets)
  • Microbiology (single cell and microorganism imaging)
  • Cell biology (Stem cell research, identification of cell cycle phase, monitoring of cell death, cellular responses to drugs)

SERS

• Surface-enhanced Raman spectroscopy or surface-enhanced Raman scattering (SERS) is a surface-sensitive technique that enhances Raman scattering by molecules adsorbed on rough metal surfaces or by nanostructures such as plasmonic-magnetic silica nanotubes. In this way we have scattering enhancement and therefore Raman signaling enhancement.
• Types of SERS
  • Deposition of a liquid sample onto a nanostructured noble metal surface (Ag, Au)
  • Dispersion of metal nanoparticles (Ag, Au, Cu) in the sample
• Nanoparticles for SERS
• AgNPs (Sigma-Aldrich Purchased Product Number: 730785)
• AuNPs; Silver, dispersion nanoparticles, 10 nm particle size (TEM), 0.02 mg/mL in aqueous buffer, contains sodium citrate as stabilizer (manufactured in laboratory)
• AgNPs by our lab with spermine (manufactured in laboratory)
• Potential Applications
  • Intracellular imaging
  • Biological mechanisms (such as “Insights into thrombosis mechanisms)
  • Biosensing used for various cancers, Alzheimer’s disease, Parkinson’s disease, Diabetes Type 2 (SERS-based in vivo glucose sensor for diabetic patients)
  • Live Single cells (such as lymphocytes)
  • Food science (edible leaves examination)
  • Nanotoxicology
  • miRNA detection (from human blood, cancer cells etc.)
  • DNA signaling enhancement from plants, mammalian, microorganisms, viruses, cell free and synthetic
  • Single molecule (SMSERS)

Horiba LabRAM HR Evolution Raman microspectrometer

“HUH7” live cell

Instruments

• A “Horiba LabRAM HR Evolution” Raman microspectrometer (30 – 4000 cm^-1) is with an open space Confocal microscope. It includes high throughput achromatic coupling optics optimized to work from UV to NIR with maximum efficiency, icluding also Open Electrode CCD air-cooled to -60°C. The Horiba LabRAM HR Evolution Raman microspectrometer is attached to two air-cooled solid state laser kits, at 532 nm (532 nm/100 mW), and at 785 nm (785 nm/ 100 mW).
  • Olympus Objective lenses with the following characteristics; 10x (numerical aperture of 0.25), long distance 50x (NA=0.5), water immersion 60x (NA=1.2), 100x (NA=0.9), and 2 gratings (600 and 1800 grooves/mm, respectively) and another two lens (2.5x, 4x) custom made
  • “Linkam PE120” thermoelectrically cooled stage
  • x,y,z micro positioning stage
  • Köhler Illumination (μlight transmission system based on LEDs)

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