Using X-Cite® XLED1 and X-Cite® Power Meter to Calibrate Photo-Uncaging in ATR-FTIR Spectrometer

By AZoNano Staff Writers

Table of Content

Introduction
Application of IR Spectroscopy
Experimental Studies
Results and Discussion
Conclusion
About Lumen Dynamics Group Inc

Introduction

Attenuated Total Reflectance Fourier Transform Infrared (ATR-FTIR) spectroscopy is an advanced and powerful technique used for tracking protein-membrane interactions, including membrane-induced protein refolding.

Information related to the secondary structure of a protein is usually obtained through meticulous analysis of the N-H and C=O stretching and bending modes related to the amide bond. For instance, α-helices will exhibit strong absorbances at ~1650 cm-1 (Amide I) and ~1550 cm-1 (Amide II), while absorbances at ~1620 cm-1 (Amide I) and ~1680 cm-1 (Amide II) are more characteristic of β-pleated sheets.

 

Application of IR Spectroscopy

One specific application of IR spectroscopy is the use of difference spectra to evaluate variations in a protein’s conformation state upon nucleotide hydrolysis, ligand binding, or co-factor addition.

Activating these variations without affecting the sample is important to characterize these events precisely as they occur. For instance, in in-situ photolysis of caged compounds by which Ca2+ or nucleotides are released upon irradiation has proved to be a powerful approach for assessing proteins’ conformational changes.

This is generally carried out using a high power LED, laser, or flash lamp. However, one challenging aspect is to measure the optical power at the sample itself.

 

Experimental Studies

Photolysis experiments are usually carried out in a transmission configuration; however interactions between proteins and membranes, particularly membrane-associated GTPases, are of significant interest.

Hence, for experimental studies, ATR-IR spectroscopy technique was employed. Instruments used were a ThermoNicolet Nexus 670 FT-IR spectrometer equipped with a SmartOrbit ATR-IR accessory, a custom-built flow-through fluid cell consisting of a CaF2 window in a poly(methyl methyacrylate) housing, and a single-bounce diamond internal reflection element. Experiments were performed with the protein bound to lipid bilayers formed on an ATR-FTIR diamond substrate.

Under these experiments, it was critical to obtain IR spectra prior to and subsequent to the introduction of GTP so as to characterize conformational changes associated GTPase activity. Nevertheless, introduction of GTP can modify the baseline of spectra and make it complex to compare data before and after the addition.

Figure 1. The X-Cite® Power Meter is placed within the fluid cell to measure the amount of UV light impinging on the sample.

 

To track these changes sans disturbing the sample, a NPE-caged GTP compound was used; this compound releases free GTP following UV irradiation.

The X-Cite® XLED1 385nm LED module (UVX) was utilized for GTP uncaging. In order to determine the excitation power at the surface of the diamond element and also to account for absorbance by the fluid cell itself, the X-Cite® XP750 Objective Plane Power Sensor joined to the X-Cite® XR2100 Power Meter was positioned within the in-house designed fluid cell to measure the amount of the UV excitation light nominally at the surface of the membrane (Figure 1).

 

Results and Discussion

From the following details, it can be seen that there was significant absorbance of UV light by the fluid cell.

  • Source: X-Cite® XLED1 385nm (UVX) source
  • UV light  reaching the sensor surface: 312mW
  • UV light going through the fluid cell: 100mW

Figure 2. ATR-IR spectra obtained in fluid using a single-bounce diamond ATR element.

The X-Cite® XLED1 system was fitted with a light guide, coupling adaptor, dedicated power supply, and a programmable touch screen controller to automate light output. Figure 2 shows ATR-IR spectra attained in fluid through a single-bounce diamond ATR element.

 

Conclusion

From these experiments, it is clear that the X-Cite® XLED1 system offered a high power UV source for uncaging experiments, while the X-Cite® Power Meter enables quick characterization of photolysis reactions and allows for accurate control of experimental conditions. One can easily tune the power output in a stable and repeatable fashion and this proves advantageous for repeatable experiments.

Moreover, the X-Cite® Power Meter provided a simple and easy approach for calibrating the photolysis reaction and helped in precise recording of the amount of UV light at the preferred uncaging wavelength impinging on the surface of the sample. From the power density and exposure time, the approximate flux related to the uncaging process can then be determined.

 

About Lumen Dynamics Group Inc

Lumen Dynamics, an Excelitas Technologies® Company is a global leader in the design and creation of innovative light delivery solutions inspired by close to 30 years of light expertise in Manufacturing and Life Science applications. We are a technology company propelled forward by our commitment to providing customer solutions through the innovative application of light.

Precision bonding and UV curing is at the heart of the company’s manufacturing solutions for electronics/optoelectronics and medical device manufacturing processes, in addition to digital printing; while Life Sciences focuses on illumination and measurement technologies for bioscience research, microscopy and instrumentation. The comprehensive family of Lumen Dynamics’ products includes its renowned brands: OmniCure® and X-Cite®.

This information has been sourced, reviewed and adapted from materials provided by Lumen Dynamics Group Inc.

For more information on this source, please visit Lumen Dynamics Group Inc.

Date Added: Dec 2, 2013 | Updated: Jan 10, 2014
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