Materials and methods: Thin sections, focal stacking, and analytical techniques in paleomycology
5. Analytical techniques and protocols
Paleomycological studies are in the early stages of integrating analytical techniques and new systems of methodology. One such analytical technique includes biomarker analysis.
Biomarkers can be any type of molecule indicating the existence, past or present, of living organisms (Olcott, 2005). Biomarker analysis can be useful when there is no detectable signs or symptoms of microorganisms on or in a specimen. In order to determine if a specimen is suitable for biomarker analysis, Raman spectroscopy can be conducted on a sample to detect if a specific highly modified type of carbon is present, which is required for samples to use in biomarker analysis. Raman spectroscopy is a non-destructive vibrational spectroscopy used to study the structure and composition of compounds; it relies on inelastic or Raman scattering of a
monochromatic light, typically a laser in either the visible, near infrared or near ultraviolet range (Nasdala et al., 2012). Raman spectroscopy can be used a screening method to see if geologic material is suitable for biomarker analysis. Other spectroscopic techniques include Fourier transform infrared spectroscopy (FTIR) analysis, a technique widely used in the study of extant wood decay by fungi (e.g., Pandey and Pitman, 2003; 2004). FTIR is a technique typically used to obtain infrared spectra of absorption, emission, photoconductivity or Raman scatting of a solid, liquid, or gas (Marshall et al., 2005). One of the major advantages of FTIR is that the spectra is produced are organic compounds which have well defined frequency valves, which can easily discerned and referenced in a organic chemistry FTIR table (e.g., Lin-Vien et al., 1991).
5.1 Biomarker protocol
Numerous permineralized samples were prepared from Permian sites. All samples were processed serially, rather than in parallel, to avoid cross-contamination. About 50 g of rock was washed then sonicated in distilled water for ~10 s. Samples were air-dried at room temperature and crushed into <5 cm pieces with a jaw-type rock crusher that had been cleaned 4x each with acetone then dichloromethane (DCM). These large pieces were then sonicated with ~250 ml 9:1 DCM:MeOH for 2 minutes. Solvent was collected and rock pieces were crushed to <1 cm on a
smaller rock crusher cleaned as before. Ultrasonic extraction was repeated, and the sample was powdered in a shatter box that was cleaned by grinding quartz sand followed by 4x acetone and DCM cleaning.
The powdered rock was extracted in a microwave-accelerated reaction system (MARSXpress): 20 g of rock was split equally between 5 clean Teflon vessels, 25 ml of 9:1 DCM/MeOH was added to each vessel, and the samples were extracted at 100°C for 15 minutes with stirring. Extracts were filtered through combusted glass-fiber filters to remove particulates, and solvent was evaporated to ~30ml under nitrogen at 35°C, taking care not to allow samples to completely dry. Elemental sulfur was removed by filtration through activated copper (~3.5g, -40+100 mesh), and the S°-free extract was evaporated to near-dryness under N
2. Extract was transferred to a vial with hexane, solvent volume was reduced under N
2 to 100µl, and then analyzed by gas chromatography/mass spectrometry (GC/MS). Extracts from the two
preliminary extractions were prepared and analyzed following the same procedures. All samples were analyzed on a ThermoFinnigan Trace GC-DSQ quadrupole MS equipped with a DB-5MS capillary column (30 m x 0.25 mm x 0.25 µm film thickness). 1 µl aliquots were injected into a PTV injector (35°C hold for 3 min, 14.5°C/s to 200°C then 12°C/s to 350°C with a 3 minute hold). The column oven was programmed at 20°C/min to 130°C, then 5°C/min to 320°C with a 20 minute hold.
After initial GC/MS analyses, each of the final extracts was separated into fractions by column chromatography. Polar compounds (mainly phthalates and other plasticizers) were first separated from hydrocarbons on 1.0g silica gel (100-200 mesh, 5% deactivated) dry-packed into Pasteur pipettes. Hydrocarbons were eluted with 3.75 ml 8:2 Hexane:DCM (F1) and polar compounds were eluted with 6ml 7:3 DCM:MeOH (F2). Saturates and aromatics were then separated on silica gel with 10% AgNO
3 dry-packed into Pasteur pipettes. Aliphatic compounds
were eluted with 5ml hexane (F1a) and aromatic compounds were eluted with 4ml DCM (F1b).
The three fractions (polar, aromatic, aliphatic) were concentrated under N
2, and analyzed by GC-MS using the same conditions as above.
Laboratory blanks of the solvents, copper, silica gel, silver-impregnated silica gel and MARS vessels were analyzed, and a block of pre-baked basalt was spiked with a standard lipid solution and then subjected to the entire analytical procedure. No hydrocarbons, oil residues or UCM were observed in any of the blanks. Biomarker yields were confirmed with replicate extractions of several samples.
5.2 Raman spectroscopy
Raman spectra were acquired from each Permian wood sample using a Renishaw inVia Reflex Raman microprobe. A 325 nm wavelength line of an air-cooled Kimmon HeCd 20 mW laser was used to excite the samples, with a beam measuring 1 µm in diameter. UV excitation was used rather than green excitation at 514.5 or 532 nm, which is typically used for
carbonaceous materials, in order to mitigate autofluorescence emission from such thermally immature organic matter. The Raman scattered light was dispersed with a 3600 mm/line diffraction grating, and the signal was analyzed with a Peltier cooled charge-coupled device (CCD) camera at room temperature (1,024×256 pixels). A Leica DMLM microscope coupled to the system, and two UV objectives (LMU x15/NUV 0.32 and LMU x40/NUV 0.50) were used to view and analyze the samples. A diamond sample was used to calibrate Raman shift at one accumulation for 10 seconds using the F2g mode at 1332 cm-1. Spectra were acquired using 100%
laser power for one accumulation at an exposure time of 30 seconds. Spectra were collected from three areas of the spores and pollen grains (intracellular inclusion, cell lumen, and surrounding matrix) of three cells from each taxon. Raman spectra were normalized using Renishaw software
(e.g., Olcott Marshall and Marshall, 2015), converted to SPC files using Batch File Converter, and analyzed using GRAMS/32 software to obtain ID and IG values, as well as carbonate band positions. Additionally, GRAMS/32 software was used for the removal of cosmic rays, and several spectra were baseline corrected using Renishaw WiRE 3.3TM 158 software.
5.3 FTIR spectroscopy
Specimens of permineralized wood with signs of pocket rot, based on reports of
previously described signs of fungal infected woods, were selected for analysis (Stubblefield and Taylor, 1986a; Stubblefield and Taylor, 1986b) were approximately 1 to 2 cm in width, by 2 to 4 cm in length, and 1 to 3 cm in depth and had clear signs of pocket rot 2 to 5 mm in diameter throughout the entire specimen. The samples were initially cut on a Buehler Isomet low speed saw, 11-1180-160 saw into wafers approximately ~2 mm in thickness. Wafers were then subsequently ground to desired thickness on a glass plate with 600 carborundum grit by hand.
Measurements of wafer thickness were taken by a Mitutoyo Digital Micrometer H-2780. Due to the delicate nature of the wafers measurements were taken by: (1) recording the thickness of a standard transmittance light microscope slide, (2) placing the wafers on standard transmittance light microscopy slides, (3) then recording the thickness of the wafer and the glass slide together, (4) and finally subtracting the initial glass slide measurement. Using this method results in an accurate measurement of the wafer without damage to it by the micrometer accidentally crushing or chipping the sample. The wafers were ground unevenly on one side to produce a “wedge”
shape on the specimen. The “wedge” or gradient of thickness from 2 µm at the thinnest point to 15 µm at the thickest portion, made it possible to examine and analyze different thickness of sample using FTIR analysis of the same specimen in order to compare and contrast which is the optimal thickness for a wafer for FTIR analysis.
Measured wafers were carefully placed via forceps into a beaker of DI water in order to clean and remove all dust and carborundum grit particles. Samples were removed from the beaker and subsequently placed in a desiccation chamber for three days to eliminate and
evaporate all residual water. The wafers were then placed on an IR microscope slide and washed with ethanol as an additional cleaning step, but to also help adhere the specimen to the IR slide.
Once the ethanol evaporated, the wafer was analyzed with FTIR spectroscopy.
5.3.1 FTIR spectroscopy protocol
IR spectra were measured using a Smiths Detection IlluminatIR IITM Infrared Microprobe coupled to a Leica DM 2500 microscope. The spectrometer uses a
mercurycadmium-telluride (MCT) photoconductive liquid nitrogen–cooled detector and contains a KBr beamsplitter with a spectral range of 4000 to 650 cm21. FTIR microspectroscopic
measurements were made using the reflection-absorption ARO objective (315, 0.88 NA). The aperture controls the size and shape of the region to be analyzed by the IR beam. An aperture area of 20 x 20 mm was used to collect the IR spectra, which gave adequate signal to noise ratios. The inferogram for the background and the microfossils were collected for 256 to 2000 scans over a spectral region from 4000 to 650 cm21 at 4 cm-1 spectral resolution. The
spectrometer is controlled by SynchronizIRTM software. The spectra were exported into
GRAMS/AITM software for further processing. Processing included converting the spectra into absorbance and baseline correcting them using a fifth-order polynomial.
5.4 Scanning Electron Microscopy (SEM)
A Versa 3D dual beam Scanning Electron Microscope/ Focused Ion Beam (FEI, Hillsboro, OR, USA) with a silicon drift EDX detector (Oxford Instruments, X-Max, UK) was
used to measure the surface morphology, elemental composition, and distribution of elements.
All the SEM data reported were obtained at low vacuum of 0.53 Torr, acceleration voltage of 15kV, spot size 4.0 and the images were collected with a Low Vacuum Secondary Electron detector (LVSED). The elemental mapping and energy spectrums were acquired with Aztec tools (Oxford Instruments, UK).