The OLIS Integrating Sphere
||NEW For 100% immunity to scatter, opening up successful studies (including rapid-scanning ones) of living, intact cells and other 'impossibly' turbid suspensions, choose the Olis CLARiTY!
Two common applications supported by use of an Integrating Sphere are: (1) scattered transmission spectroscopy and (2) diffuse reflectance spectroscopy.
OLIS Integrating Sphere Gallery
Olis Diffuse Reflectance Accessory* for Liquid Samples
Olis 14 Diffuse Reflectance Model
This Olis 14 Diffuse Reflectance model is being used in a long term human study of renal dialysis patients by a group of vision and kidney researchers at the University of Ottawa who are developing a novel technique to rapidly recognize patterns in whole blood by measuring how it absorbs light at many different wavelengths. The instrument is capable of simultaneous transmittance and diffuse reflectance measurements in the 185 - 2600 nm range; it also has the capability of measuring forward and backward scatter from whole blood samples by strategic positioning of the sample holders.
Reference position, North Pole of sphere
Sample position, South Pole of sphere
Dr. Robert Glaum at the University of Bonn uses this Olis 14I Diffuse Reflectance model to characterize transition metal ions in powder form. The Olis 14 Infrasil optical system with the Olis Module for Diffuse Reflectance* studies of powder samples includes a sphere with separate sample and reference vesicles (pictured above) to support dual beam diffuse reflectance measurements in the 225 - 3000 nm range.
Sample & Reference Integrating Spheres for Dual Beam Diffuse Reflectance Measurements
Olis 14 with Absorbance, Fluorescence, & Diffuse Reflectance Capabilities
Faculty in the Physics department at UFMG in Brazil worked with us to create this multiple purpose Olis 14F model which supports dual beam UV/Vis/NIR absorbance, diffuse reflectance, and scanning emission fluorescence of single-walled carbon nanotube samples in solution and deposited on thin films. An integrating sphere is mounted in both the sample and reference chambers to support dual beam diffuse reflectance measurements (pictured above).
Scattered Transmission Spectroscopy
Collecting absorbance spectra of clear solutions is a routine operation in many laboratories and many types of spectrophotometers are available which can make the measurement. The situation is dramatically different when one is dealing with turbid or cloudy samples, such as whole cells and cell organelles (such as mitochondria). Turbid samples scatter light, disrupting the measuring beam in most spectrophotometers to the extent that meaningful measurements are impossible. Features of a spectrum are often 'washed out' by apparently high absorbances, which are due in fact to scattering of light rather than to absorbance of light by the sample.
Over the past decades, several special-purpose instruments (e.g., the Aminco DW-2 and DW-2000 spectrophotometers) have been available for studying scattering samples using one of two strategies to improve the fidelity of measurements. The first strategy is the use of a large-diameter photodetector positioned as close to the scattering sample as possible so as to gather light which has been scattered out of the usual light path. The second strategy is the use of dual-wavelength measurements. The idea here is that one measures the apparent absorbance at two wavelengths; one wavelength-the measurement wavelength-is chosen to coincide with the absorbance maximum of the sample; e.g., 450 nm if one is measuring a flavoprotein. The other wavelength-termed the 'reference'-is chosen so as to avoid absorbance due to sample and instead to measure apparent absorbance due to scatter of the measuring beam. Thus, one wavelength provides "sample + scatter" data and the other provides "scatter" data; obviously, subtracting the latter from the former produces data due to 'sample' only. Both strategies produce more meaningful results than those attainable using standard spectroscopic methods but still do not produce spectra the quality of those collectible using clear samples.
The OLIS RSM rapid scanning spectrophotometer is capable of reliable absorbance measurements on turbid samples. It features a photodetector positioned very close to the sample and it scans multiple wavelengths, making possible appropriate corrections for scatter much like the use of dual wavelengths. Numerous measurements prove that RSM spectrophotometer systems provide better spectra of turbid samples than do special-purpose instruments of the dual-wavelength type.
Recently, test of an integrating sphere incorporated into the RSM sample chamber proved that additional meaningful improvements in the quality of data collected on a turbid sample are possible. The test arrangement is shown schematically in Figures 1-3.
Figure 1 shows the normal dual-beam optical arrangement of the RSM. An incoming light beam is split into a reference beam and a sample beam. Each beam is detected by a photodetector. If one places a very turbid sample, e.g., a suspension of yeast cells, in the sample cuvette, one notes a dramatic loss of signal at the sample PMT. Our sample produced a loss of greater than 1,200 fold! That is, less than 0.1% of the original light beam reached the sample PMT; the rest was either absorbed by the sample or scattered away from the detector by the highly turbid cell preparation. In any case, the loss of signal can be overcome by increasing the HV supplied to it. However, this increase in HV is unavoidably accompanied by an increase in noise (or uncertainty). The only way to avoid this increase in noise is to increase the amount of light reaching the photodetector. The integrating sphere is intended to do just that, i.e., to ensure that as much light as possible reaches the detector.
Figure 2 shows the optical arrangement tested. The sample cuvette was placed at the center of a sphere with a 150-mm diameter. The interior surface of the sphere is coated with a highly efficient reflective coating. The photodetector is placed so as to be contiguous with the inner surface of the sphere. In principle, any photon hitting the inner surface will reflect and "bounce around" until it encounters the PMT, at which point it becomes part of the measurement. That is, with a perfect sphere having a perfect reflective surface, all photons will eventually reach the photodetector, perhaps after hundreds or thousands of "bounces" off of the inside surface of the sphere; thus, there will be no apparent losses due to scatter by the sample. All photons not absorbed will be detected, making turbidity irrelevant. Of course, a perfect sphere is not possible, so one cannot expect to eliminate all of the effects due to turbidity. However, significant improvements do occur. In the case of the system shown in Figure 2, greater than a 10-fold increase in light reaching the detector is realized. This is a meaningful improvement and results in the use of much lower HV to the PMT and, therefore, much-improved S/N.
In summary, the RSM spectrophotometer is superior to most instruments in dealing with turbid samples. Using an integrating sphere around the sample to gather scattered photons results in a better detection system than those previously available for the study of highly turbid samples.
Features that make the RSM so effective with turbid samples:
1. large-area end-on PMT detectors
2. PMTs close to sample
3. PMTs have enormous gain range and can be adjusted to measure light of very widely varying intensity.
As a side note, none of the above three features is possible when dealing with diode array detector systems.
NEW For 100% immunity to scatter, opening up successful studies (including rapid-scanning ones) of living, intact cells and other 'impossibly' turbid suspensions, choose the Olis CLARiTY!
Diffuse Reflectance Spectroscopy
The integrating sphere can also be used in the configuration shown in Figure 3 for diffuse reflectance spectroscopy. The PMT has been moved to another port on the sphere and the sample has been placed in the path of the measuring beam. No light reaches the PMT directly; only light scattered from the sample and gathered by the sphere is measured, producing a diffuse reflectance spectrum. This technique is used to study solid samples, powders, painted surfaces, and other opaque objects.
*Integrating spheres manufactured by SphereOptics and customized by Olis, Inc.