DOI: 10.25338/b8np9p
title: Time and spatially-resolved Fourier-Transform Infrared (FTIR) Spectromicroscopy of cellulose in buffered reactions with Trichoderma reesei Cel7A

description: Synchrotron radiation Fourier transform infrared (SR-FTIR) spectra of
 cellulose undergoing hydrolysis by Trichoderma reesei Cel7A in
 buffer were collected. IR spectra were recorded at 64 locations every ~ 10
 min in an 8x8 grid within a 70 x 70 μm sample area. 
description: Enzyme <em>Trichoderma
 reesei</em> Cel7A purified from Celluclast 1.5L (Sigma
 C2730) Cellulose 
 Algal cellulose fibrils from <em>Cladophora
 aegagropila</em> Reaction
 conditions 
 37<sup>o</sup>C 50 mM sodium
 acetate buffer, pH 5  
 Synchrotron Radiation Fourier Transform Infrared (SR-FTIR)
 spectromicroscopy of cellulose undergoing enzymatic hydrolysis
 Experimental samples for monitoring
 cellulose hydrolysis by SR-FTIR were prepared by depositing and air-drying
 60 mL of
 algal cellulose (0.025mg/mL) fibrils onto the SiNx membrane in an
 open-channel microfluidics (OMM) device, followed by an infiltration of 20
 mL of Cel7A
 (0.5 µmole/g) through the cellulose. The enzyme and substrate was
 equilibrated in air at ambient conditions (~20<sup>o</sup>C)
 for ~ 40 minutes at room temperature, then mounted onto the FTIR
 microscope for optical alignment and location of regions of interest
 (ROI). The experiment was initiated by flowing 1 mM sodium acetate buffer
 at 0.3 mL/min into the
 fluid distribution channel connected to the bottom microchannel, and
 raising the temperature of the FTIR microscope stage to 37°C. The acetate
 solution wicked upwards by capillary forces through the SiNx pores
 maintained a buffered reaction at the membrane surface. At 37
 <sup>o</sup>C the system maintained a relative humidity of
 ~80%.  With the initiation of reaction, infrared absorption measurements
 were collected and monitored to evaluate the quality of the experiment in
 real-time, followed by detailed post-experiment analysis.
 SR-FTIR spectral measurements All SR-FTIR measurements were made at the
 infrared beamline at the Advanced Light Source (ALS) (Lawrence Berkeley
 National Laboratory, CA, USA) using a Nicolet Magna 760 FTIR spectrometer
 equipped with a liquid nitrogen cooled Mercury Cadmium Telluride (MCT)
 detector, and an all-reflective optics infrared microscope (Nicolet
 Nic-Plan, Thermo Scientific, Inc., MA, USA). Mid-infrared photons emitted
 from the synchrotron were focused with a numerical aperture (NA = 0.65)
 objective through the cellulose fibrils onto the gold-coated surface
 inside the OMM device, and reflection SR-FTIR measurements by the
 transflection mode over a mid-infrared wavenumber range of 4000 – 650
 cm<sup>-1</sup> were made over time. Each real-time spectrum
 represents an average of 32 scans at a spectral resolution of 4
 cm<sup>-1</sup> with an absorption peak position accuracy of
 1/100 cm<sup>-1</sup>. To image the spatiotemporal dynamics of
 cellulose during enzymatic hydrolysis, we typically divided the entire
 view-field of the cellulose fibrils into equal-sized 5×5-µm squares before
 raster scanning, collecting full SR-FTIR spectra at each pixel. A 70 x 70
 μm sample area was divided into 8x8 grid such spectra were collected at 64
 points every ~ 10 minutes. Time-elapsed mapping of sample areas were
 conducted using in-house software written in the Python programming
 language (version 3.6). The master program controlled the movement of the
 microscope stage, spectra collection of FTIR spectrometer and data storage
 in the OMNIC software program (version 9.8, Thermo Fisher Scientific,
 Inc.) using the Dynamic Data Exchange (DDE) protocol. The source code of
 our python programs for controlling the microscope and FTIR bench is
 available upon request.
description: # Time and spatially-resolved Fourier-Transform Infrared (FTIR)
 Spectromicroscopy of cellulose in buffered reactions with Trichoderma
 reesei Cel7A This dataset contains FTIR spectral data of cellulose
 undergoing hydrolysis by *Trichoderma reesei* Cel7A in sodium acetate
 buffer at pH 5. Synchrotron radiation infrared imaging was conducted on a
 ~50x50 micrometer area, within which spectral data were collected at 64
 locations in an 8x8 grid, every ~9.5 minutes over ~15 hours. Spectral
 intensities in the wavelength range of 649 - 4000 1/cm were recorded. ##
 Description of the Data and file structure FTIR data were collected at 64
 different locations in an 8x8 grid. Data filenames corresponds to location
 in the 8x8 grid where spectra were obtained ('e.g. '1.csv'
 contain spectra recorded at point 1): | **57.csv** | **58.csv** |
 **59.csv** | **60.csv** | **61.csv** | **62.csv** | **63.csv** |
 **64.csv** | | :--------- | :--------- | :--------- | :--------- |
 :--------- | :--------- | :--------- | :--------- | | 49.csv | 50.csv |
 51.csv | 52.csv | 53.csv | 54.csv | 55.csv | 56.csv | | 41.csv | 42.csv |
 43.csv | 44.csv | 45.csv | 46.csv | 47.csv | 48.csv | | 33.csv | 34.csv |
 35.csv | 36.csv | 37.csv | 38.csv | 39.csv | 40.csv | | 25.csv | 26.csv |
 27.csv | 28.csv | 29.csv | 30.csv | 31.csv | 32.csv | | 17.csv | 18.csv |
 19.csv | 20.csv | 21.csv | 22.csv | 23.csv | 24.csv | | 9.csv | 10.csv |
 11.csv | 12.csv | 13.csv | 14.csv | 15.csv | 16.csv | | 1.csv | 2.csv |
 3.csv | 4.csv | 5.csv | 6.csv | 7.csv | 8.csv | In each csv file, the data
 structure is as follows: * First column enumerates the sequential order in
 which the spectra were collected. ~9.5 minutes separate each step. * First
 row contain wavenumbers in 1/cm at which absorbances were measured * From
 the second row down, spectral data in absorbance units are recorded