The chemistry associated with kidney stone formation and growth is not well understood and has been the focus of many studies for the past seventy years. Fully formed urinary stones can be studied much like a geologist studies sedimentary rock to elucidate the chemistry during the growth of a stone. However, to study the cause for initial kidney stone formation, small mineral inclusions in tissue biopsies must be analyzed.1–3
For years, this type of analysis has relied on histological preparations which involve producing thin sections followed by preferential staining. The stained tissue is then examined with an optical microscope. Several limitations associated with histopathology are that the staining procedure involves multiple steps during which the material of interest may be lost or destroyed. In addition, this type of analysis relies on trained visual observation; therefore the rate of misdiagnosis can be as high as ~30%.4
Infrared imaging methods can be employed to gain similar information as histopathology, but is beneficial in that it also provides the molecular information required for objective disease detection.5,6
In 1996, Estepa-Maurice et al. described a method for the analysis of crystal deposits in kidney tissue biopsies using FTIR microscopy.7
Their investigation included two different sample preparations for transmission FTIR microscopy analysis. The first sampling method required placing kidney biopsies with mineral inclusions directly on a CaF2
window. Because CaF2
windows are expensive and pathologists typically do not have them readily available, a second approach was investigated. This latter approach involved excising individual inclusions with a fine pointed probe and transferring them to a NaCl window for subsequent analysis. Analyzing the mineral inclusions ex-situ
does not allow the investigator to gain a full understanding of the inclusion in its native environment. In addition, this type of analysis only permits particles in the range of 5–10 μm to be routinely studied.
In 2005, Anderson et al. developed a protocol utilizing IR microspectroscopy with a transflection approach to study mineral inclusions in tissue biopsies.8
Unlike the previous studies of Estepa-Maurice et al., this protocol allowed direct analysis of the biopsy with the use of a low-E glass slide. A low-E slide is a glass slide with a thin reflective coating that reflects nearly all mid-IR radiation and is transparent to visible light. Therefore, the low-E glass slide permits both visual and IR images to be collected from the same sample. The transflection sampling method provides direct molecular identification of the species being studied on sample areas as small as 12 μm using a probe wavelength of 3μm. Anderson et al. showed that within an IR image of a biopsy, many spectra will show a mixture of two or more components (e.g. tissue and hydroxylapatite). With the prevalence of kidney stone disease progressing specifically in industrialized countries, there is a need to obtain quantitative information from small mineral inclusions. This quantitative information may allow a better understanding of stone chemistry at the very initial stages of formation. However, spectral artifacts typically present in transflection spectra of tissues not only make sample identification difficult, but also do not provide the photometrically accurate spectra necessary for quantitative purposes.
Combining the disparate disciplines of histology and infrared microspectroscopy has proven to be beneficial for many biological applications. Many groups have used FT-IR microspectroscopic imaging for biomedical applications. Recently, Mendelsohn, Diem and Bhargava have used infrared microscopy for the analysis of tissues for various applications including wound healing, cell analysis, and cancer diagnosis. These studies have typically relied on a transflection or transmission sampling method for analysis.9–11
Recently, efforts to incorporate infrared microanalysis into a protocol for disease detection have settled on the use of reflective substrates, most commonly low-E glass slides, in conjunction with transflection spectroscopy. Although this approach is a useful method for tissue analysis, it can pose several limitations. Transflection is typically a straightforward analysis provided the sample is a continuous film with low contrast interfaces. Low contrast interfaces are characterized as those having optically similar materials present on either side of the interface where the refractive index of both materials becomes the most important parameter. However, many tissue sections do not meet these criteria and contain discontinuities such as blood vessels and in the current case, mineral inclusions, which can present high contrast edges promoting scattering, diffraction, reflection and dispersion. All of these optical effects can manifest in the spectrum in a variety of ways. Further, these effects are amplified due to the size and shape of the sample and the high convergence of the impinging infrared radiation. In the current case where small mineral inclusions are surrounded with tissue, not only does the mineral present a high contrast edge, but it also acts as a highly scattering point defect. This phenomenon is known as the Christiansen effect where the sample exhibits anomalous dispersion near an absorption band. Mineral inclusions can also exhibit the reststrahlen effect, where the sample becomes a strong reflector near an absorption band.12,13
Reststrahlen bands are usually associated with the reflectance that occurs at the strongest bands of ionic materials and metals, but it has been shown that this effect is not limited to these substances.14
When collecting infrared spectra from a kidney biopsy with transflection spectroscopy, any or all of these effects can occur simultaneously. Therefore it is difficult to determine which effect dominates over another especially when the spectra usually possess several distortions from a combination of effects. From a quantitative perspective, the adherence of the Beer Lambert law dictates that the sole mechanism for the attenuation of light must be absorption and the optical path length through the sample must be well known. With transflection and transmission approaches, scattering and diffraction will prominently affect the former and changes in refractive index affect the latter.
In 1991, Sommer reported on dispersive band shapes in IR microspectroscopy and later Stewart and Sommer demonstrated that these optical nonlinearities increase with a greater difference in refractive index between the two materials. Further, the magnitude of the effect increases with a decreasing spatial domain of one material embedded in the other.15–17
Consider the simple case of an air/tissue interface within a tissue biopsy. The difference in refractive index between these two materials is ~0.40. When an IR spectrum is obtained at such an interface, portions of the light will undergo specular (Fresnel) reflection and anomalous dispersion which are manifested in the spectrum as derivative-shaped peaks. Later in 1998, Bhargava demonstrated that spectral artifacts are observed when imaging the interface of polymer-dispersed liquid crystals.18
Bhargava described the optical effects that occur when imaging multicomponent systems and how these effects can complicate quantitative analyses. More recently in 2005, Romeo and Mohlenhoff reported the observation of dispersive line shapes from scattering and/or diffraction when analyzing tissue samples with a transflection or diffuse reflection approach.19–21
Romeo proposed a computational method to reduce the dispersive artifact which causes an abnormal ratio of the amide I and II bands, a lower wavenumber shift of bands, and a sloping baseline. Their approach is very similar to the conversion of a Fresnel reflection spectrum to the optical constant (n
) spectra using Fourier transform. The authors explained that these spectral artifacts make correct identification of disease states difficult and have a significant influence on statistical analyses, but also state that their computational correction is not completely ideal. In 2005, Mohlenhoff stated that these artifacts need to be firmly understood before IR microspectroscopy can be utilized as a diagnostic indicator of disease.
A method which overcomes these limitations is attenuated total internal reflection (ATR) infrared microspectroscopy.22
ATR with a germanium internal reflection element (IRE) can eliminate all of the previously mentioned spectral artifacts.8,22,23
Although, Bhargava has shown that other IRE types such as diamond do not eliminate all spectral artifacts due to the increased penetration depth.14
ATR also provides a decreased focus beam size by immersing the sample in a high index medium, similar to immersion microscopy.24
Unlike immersion microscopy, which employs a high index liquid, ATR infrared microspectroscopy employs a solid germanium hemisphere commonly known as an IRE. The IRE is placed into intimate contact with the sample and infrared light is internally reflected producing an evanescent wave which probes the sample to a depth of 0.063λ (assuming the angle of incidence is 45° and the refractive index of the sample is 1.5). The diffraction limited diameter for light focused to a point is defined as the bright central maximum of the Airy disk and is given by:
where λ is the wavelength of light employed and NA is the numerical aperture of the system. The NA is defined as the product of the refractive index of the medium in which the sample is immersed (n1
) and sinθ
, where θ
is the half angle acceptance of the optic. This equation is only valid for a circular aperture that is uniformly filled.25
Because transflection typically uses half the objective to excite the sample and the other half to collect the reflected light, the aperture is no longer circular. Although the half angle acceptance does not change, the above equation may not give a good estimate of the diffraction limited diameter for a reflection measurement. However, for the current system and those investigated by Tisinger, the focused beam diameter measured in reflection is twice that of the focused beam diameter measured in transmission (essentially reducing the numerical aperture by a factor of two.)26
No matter what perspective is taken, the focused beam diameter in transflection (n1
= 1.0) will be 4 times larger than that for ATR when using a germanium hemisphere (n1
= 4.0). The diffraction-limited spot size at the microscope focus is reduced by the refractive index of the IRE, thereby increasing the spatial resolution. In addition to increasing the spatial resolution of the measurement, ATR microspectroscopy eliminates significant sample preparation associated with transmission measurements and spectral artifacts associated with transmission and transflection measurements.
The development of ATR occurred over forty years ago when Harrick and Fahrenfort employed the method to study the infrared spectra of organic materials.27,28
Currently, there are two approaches for ATR imaging: on and off-axis imaging. With on-axis ATR imaging, the hemisphere/sample composite is centered at the microscope’s focus and illuminated globally. Radiation that is internally reflected is then imaged onto a two dimensional array detector. On the other hand, off-axis ATR imaging requires the hemisphere/sample composite to be initially centered at the microscope’s focus and then imaging is conducted by moving the composite off-axis. The development and commercialization of off-axis ATR imaging has taken place over the last 15 years. In 1992 and 1994, Nakano and Kawata performed off-axis ATR measurements and reported an improvement in spatial resolution equal to the refractive index of the germanium IRE.29,30
Although Nakano and Kawata were the first to report this improvement for ATR, the concept of immersion has long been known and employed by optical designers to collect and focus more available light onto a small area detector. The size of the detector could be reduced by a factor equal to the refractive index if the detector was placed in optical contact with the plano surface of the hemisphere.31
Later, Lewis and Sommer reported on the approach taken by Nakano et al., but on a commercial PerkinElmer i-series microscope.32,33
The next significant development of off-axis ATR imaging came in 2006, when Patterson and Havrilla employed a 12.5 mm diameter hemisphere and demonstrated that spherical aberrations limiting the total sampled area was directly related to the radius of the hemisphere.34
Later in 2006, PerkinElmer developed and introduced an ATR accessory based on the work of the off-axis imaging concept of Nakano et al. and Lewis et al.35
The goal of this study is to demonstrate the capabilities and benefits of an off-axis ATR imaging approach over existing methodologies, principally transflection spectroscopy, for examining small mineral inclusions within kidney tissue. The future goal is to extend this type of analysis to allow quantitative analysis of small mineral inclusions which may be of mixed composition at the localized mineral/tissue interface.36