Biological Thermal Detection in Infrared Imaging ... - ACS Publications

Biomacromolecules 2001, 2, 757-764

757

Biological Thermal Detection in Infrared Imaging Snakes. 1. Ultramicrostructure of Pit Receptor Organs N. Fuchigami,†,# J. Hazel,‡ V. V. Gorbunov,†,⊥ M. Stone,§ M. Grace,| and V. V. Tsukruk†,* Department of Material Science & Engineering, Iowa State University, Ames, Iowa 50011; Materials Science & Engineering Program, Western Michigan University, Kalamazoo, Michigan 49008; Materials and Manufacturing Directorate, Air Force Research Laboratory/MLPJ, Wright-Patterson AFB, Ohio 45433; and Department of Biological Sciences, Florida Institute of Technology, Melbourne, Florida 32901 Received March 17, 2001; Revised Manuscript Received May 30, 2001

The receptor organs of snakes with “thermal vision” were studied with ultra-high-resolution scanning probe microscopy (SPM) at close to in vivo conditions to elucidate their surface morphology and materials properties critical for prospective biomimetic design of “soft matter”-based infrared (IR) sensors. The surfaces of living tissues were scanned under wet ambient conditions in physiological solution, and the resulting parameters were compared with SPM data obtained for chemically treated (formaldehyde-fixed) tissue in ambient air and TEM studies in high vacuum. We found that the microstructural parameters for the living tissue are similar to ones observed for the formaldehyde-fixed snake tissues. However, previous data obtained from TEM analysis in high vacuum underestimated actual dimensions of surface microstructures. The average spacing of the nanopit array observed within receptor surface areas, which was suggested to play a critical role in selective IR adsorption, was determined to be 520 nm. This value is close to the grating spacing required for efficient reflection of electromagnetic radiation characteristic for sunlight without affecting IR adsorbance. Introduction Biological inspiration in the design of prospective artificial sensors composed of “soft” matter such as macromolecular and organic materials is one of the milestones in biomimetics. In such designs, unique biological material microstructures and properties are targeted to be replicated with synthetic macromolecular-based materials to replace inorganic, solidstate devices. A key issue in such an approach is understanding how biomaterial properties and microstructures can define a specific targeted performance. In the spirit of this approach, we focused on finding the microstructural design and a combination of materials properties promising for artificial thermal sensors from organic/polymeric materials. The first challenging task is understanding the biomaterial organization and properties under conditions close to that of those occurring in the functional living system. In our previous publication, we studied the multilayered microstructure of IR receptors in Melanophila acuminata beetles.1 Here, we discuss results obtained from snakes with the ability for highly sensitive “infrared vision”. In recent publications, we demonstrated the nanopit microstructure of snake skins and their unique * To whom correspondence should be addressed, [email protected]. † Iowa State University. ‡ Western Michigan University. § Air Force Research Laboratory. | Florida Institute of Technology. # Current address: Angstroms Systems Inc., San Jose, CA. ⊥ Current address: Thermomicroscopes, Sunnyvale, CA.

surface morphology.2,3 In the course of this study, we focus on comparative studies of surface morphology, on micromechanical and microthermal properties of several snakes with thermal imaging capabilities, and on unique results for the live IR receptor organs in comparison with previous results on formaldehyde-fixed specimens. In part 1 of this paper, we will discuss snake skin microstructure, and the following publication will be devoted to the analysis of micromechanical and microthermal properties of the receptor organs. For a long time, it has been known that several types of snakes (pythons, boas, and pit vipers) are capable of distant hunting via heat detection of prey under a bright sun and complete darkness.4-10 Physiological studies have shown that the snake pit organs located on the upper and/or lower jaw (Figure 1) can function as highly sensitive infrared receptors capable of detecting minute and distant temperature variations and can provide an additional imaging channel independent from ocular vision. Photophysical studies confirmed very high sensitivity of snake receptors reported before and established 0.003 °C as a lower limit for their temperature sensitivity.10 Recently, it has been pointed out that this sensitivity is related to local variation of surface temperature and should be carefully considered when short-range distant imaging of snake receptors is discussed.11 Detailed histological studies established that the snake pit organs possess several common features such as high concentration of terminal nerve masses and a capillary net in close proximity to the pit organ (receptor) surface.5 Recent

10.1021/bm015537z CCC: $20.00 © 2001 American Chemical Society Published on Web 07/19/2001

758

Biomacromolecules, Vol. 2, No. 3, 2001

Fuchigami et al.

Figure 2. Mounting apparatus for snake skin specimens, which allows SPM scanning under constant wet conditions. Glass support with tissue is mounted on a two-directional microstage.

Figure 1. Top: head of an amelanistic Burmese python showing infrared-imaging pit organs (arrows) on the upper jaw; nostril indicated by arrowhead. Bottom: head of a carpet python showing three pit organs on upper jaw (arrowheads) and an array of pit organs on lower jaw (arrows).

high-resolution transmission electron microscopy (TEM) and scanning probe microscopy (SPM) studies of snake heat receptors also have shown a well-developed array of surface nanopits of several tens of nanometers in diameter and with several hundred nanometers spacing.2,3,5,7 It was speculated that a combination of nerve mass proximity to the surface and highly reflective nanopit array acting as a selective IR filter, which cuts nonspecific radiation, can play a decisive role in high sensitivity of biological IR receptors.5,7,11 A photothermal mechanism of heat detection was discussed in very recent studies based upon wavelength/power behavior of the physiological response.11 However, specific materials properties that can make this photothermal mechanism functional have not been identified and evaluated. Very special surface microstructure in the form of pit array with periodicity in a nanoscale range was observed only for a dried snake skin and stained receptor slices under high vacuum. The high-vacuum conditions of analysis used for TEM studies were not favorable for biological materials. Therefore, the questions if the observed surface microstructure really exists in live IR receptors and if it possesses characteristics suitable for their selective grating performance are unresolved to date. The quantitative parameters of surface morphology and microstructure of snake skin close to in vivo conditions are still unknown and actual morphology of receptor organs is still an open issue. In this publication, we report on surface morphology and microstructure of liVing and chemically treated snake IR receptor organs deduced by the application of ultra-high-

resolution SPM technique under wet conditions. We discuss our findings from the point of view of materials science focusing on significance of their surface microstructures for their functioning as a live highly sensitive IR sensor, which can be useful for prospective artificial sensors. SPM technique is known as a high-resolution tool for surface topography characterization of various macromolecular materials including biomaterials.12-15 Several major questions were addressed in the course of this study: • What is the surface morphology of the snake IR receptor organs under physiological conditions? • Is this morphology relevant to the surface morphology of chemically treated snake tissue and tissue studied under high vacuum? • Does microstructural organization of IR receptor organs support its function as a high-sensitivity photothermal receptor organ? Special attention will be paid to the estimation of actual dimensions of the surface microstructures of the receptor organs in their close to liVing conditions. This is a crucial issue in any future biomimetics-based design of surfaces of artificial IR sensors. As was mentioned above, these parameters were suggested to play a critical role in hypothetical performance of the surface receptor structures as a highly selective IR filter. Such performance critically depends on the average spacing of surface gratings represented by the nanopit array. This defines character of interaction (absorption and reflection) of incoming radiation of a particular wavelength with a surface grating of a particular spacing. Any future models of artificial IR sensors based upon this design should include surface gratings with spacings, which provide for the most effective selectivity in a specific spectral range. Experimental Section SPM studies of surface topography were performed in tapping and contact modes on a Dimension 3000 (Digital

Biological Thermal Detection

Biomacromolecules, Vol. 2, No. 3, 2001 759

Figure 3. Surface morphology of living snake skin of Burmese python: for nonspecific skin areas between snake pits (left) and within pit organ receptor areas (right); top view (top) and 3D view (bottom). Lateral sizes are 10 µm × 10 µm; z scale is 500 and 200 nm for left and right images, respectively. Table 1. Microscopic Dimensions of Surface Morphology of Snake Skin in Pit Organ and Nonpit Organ Areasa

diameter of nanopits (nm) Burmese python (living tissue, wet) ball python (formaldehyde-treated sample, wet) carpet python (formaldehyde-treated sample, dry, air, ref 2) ball python (formaldehyde-treated sample, dry, vacuum, ref 7) depth of nanopits (nm) Burmese python (living tissue, wet) ball python (formaldehyde-treated sample, wet) carpet python (formaldehyde-treated sample, dry, air, ref 2) ball python (formaldehyde-treated sample, dry, vacuum, ref 7) distance between nanopits (nm) Burmese python (living tissue, wet) ball python (formaldehyde-treated sample, wet) carpet python (formaldehyde-treated sample, dry, air, ref 2) ball python (formaldehyde-treated sample, dry, vacuum, ref 7) periodicity of terraces (µm) Burmese python (living tissue, wet) ball python (formaldehyde-treated sample, wet) carpet python (formaldehyde-treated sample, dry, air, ref 2) ball python (formaldehyde-treated sample, dry, vacuum, ref 7) a

pit organ

nonpit organ

390 ( 65 440 ( 99 320 ( 92 125 ( 25

130 ( 20 120 ( 21 250 ( 72 NA

120 ( 25 90 ( 23 46 ( 23 55 ( 45

23 ( 6 24 ( 5 16 ( 10 NA

520 ( 78 670 ( 134 810 ( 284 300 ( 150

330 ( 71 450 ( 96 680 ( 290 NA

5.8 ( 1 5.5 ( 1 3.5 ( 1 3.5 ( 1

5.3 ( 1.4 2.0 ( 0.5 1.9 ( 0.5 2.5 ( 1

Data are presented with standard deviations obtained from random sampling, whenever possible.

Instruments (DI), Inc.) microscope according to the procedure described in detail earlier.14,15 Silicon cantilevers had spring constants about 50 N/m for the tapping mode and 0.25 N/m for the contact mode, and the tip radius was within 20-40

nm. Imaging was done at scan rates in the range 1-2 Hz for the tapping mode and 2-5 Hz for the contact mode. For comparative and preliminary studies, we used formaldehyde-fixed tissue from ball pythons.16 The fixed snake

760


Figure 4. Histograms of surface distribution of microscopic dimensions of surface morphological features. Periodicity of terracelike structures or the width of the layers (top) and the periodicity of the nanopit array (bottom) for living snake material from within the pit organ (gray, here and below) and from areas outside pit organs (dashed, here and below). Vertical dashed and solid lines represent means of data sets.

head was stored in a refrigerator until used. For studies, pit organs were isolated with a razor blade and placed in a wet holder to prevent skin drying (for comparison, see preparation procedure for usual TEM studies in ref 8). Living tissue was obtained from Burmese pythons. Snakes were deeply anesthetized by inhalation anesthesia (Metofane), and were then euthanized by cervical dislocation. Pit organ and other tissues were quickly dissected and held in oxygenated reptilian physiological Ringer’s solution.17 Under these conditions, tissues were able to survive for up to several days. This allowed us to obtain images of several tissue slices with SPM during their estimated lifetime. Multiple scannings of a single specimen took several hours with an instant supply of fresh physiological Ringer’s solution. This was followed by immediate specimen replacement with a fresh one. No significant differences were observed among specimens studied instantly within 3 days after the beginning of the experiment. Samples for investigation were placed on a concave glass support covered by a wet towel with its edges submerged in an aqueous bath to provide a local 100% humid environment during measurements (Figure 2). Liquid absorbed by the towel formed a thin water layer on an open surface area available for SPM scanning. This unique technique was used

Fuchigami et al.

Figure 5. Histograms of the depth (top) and the diameter (bottom) of nanopits on living snake pit organs and from areas outside pit organs. Vertical dashed and solid lines represent means of data sets.

for both living tissue and formaldehyde-fixed tissue to prevent drying and shrinkage. Fixed tissues were studied in a water bath; living tissues were analyzed in a bath of physiological Ringer’s solution. SPM scanning was performed on living tissue specimens within several hours after mounting to ensure the nondegraded state of the surface. The SPM tip has a height of 3.5 µm, and the fluid layer, estimated to be less than 0.2 µm thickness, does not affect tip response in contact and tapping modes. We should mention that the presence of an aqueous surface layer and instant supply of physiological solution do not affect significantly SPM measurements although this makes them very cumbersome, time-consuming, and prone to instabilities. Only a few high quality and high resolution images can be collected under these circumstances over long periods of time. To the best of our knowledge, such highresolution SPM measurements of living biological tissues are extremely rare, and the same fact of successful implementation of this approach is unique. Results and Discussion Surface Morphology. In this study, we focused on surface morphology observed for living Burmese python tissue cut



Figure 6. Comparison of the periodicity of terracelike structures (top) and the periodicity of the nanopit array (bottom) from living tissue, formaldehyde-treated skin of ball python, carpet python (ref 2), and ball python (TEM, ref 7). Data for pit organ receptor areas (gray) and areas outside pit organs (hatched). Error bars represent standard deviation.

Figure 7. Comparison of the diameter (top) and the depth (bottom) of the nanopits on living tissue, formaldehyde-treated skin of ball python, carpet python (ref 2), and ball python (TEM, ref 7). Data for pit organ receptor areas (gray) and areas outside pit organs (hatched). Error bars represent standard deviation.

from receptor and nonspecific skin areas close to the receptor organs. We compared data collected from these specimens with concurrent data collected for formaldehyde-fixed skin areas from ball pythons. We also considered previous SPM studies of carpet python skin under ambient conditions (ref 2) and TEM studies of ball python snake skin under high vacuum conditions (ref 7). Living Burmese python skin samples from outside the pit organ receptor area under wet conditions display arrays of uniformly aligned terraces and randomly distributed nanopits with virtually uniform surface density over the entire skin surface (Figure 3). The receptor areas (pit organs) show very similar morphology with familiar terracelike microstructure and nanopit arrays (Figure 3). The SPM data from living tissue confirms previous SPM and TEM observations of nanopit and terracelike surface morphology for chemically treated skin.2,5,7 This type of surface morphology is common among very different types of snakes, which possess infrared vision despite differences in species and age (five different species were studied in refs 2, 3, 5, 7, and 17). In our current studies of two different samples of formaldehyde-fixed ball python tissue from snakes of different ages, we observed virtually identical surface morphology with indistinguishable

geometrical parameters. On the basis of these studies, we confirmed that the observed surface morphology exists in all snakes with an IR imaging capability. Therefore, we conducted comparative studies of the surface morphology of receptor and nonreceptor areas from two types of snakes available for our studies and compared these results with recently published observations from other snakes (Table 1). For the detailed analysis of surface morphology, we selected several “geometrical” parameters used before: the periodicity or the average distance between centers of adjacent elements of the terracelike microstructure and the nanopit array, and the diameter and the depth of nanopits themselves (Table 1). All these parameters were obtained from the analysis of topographical cross sections from several independent SPM images obtained at different magnifications and at different, randomly selected locations within and outside of receptor areas. The histograms of surface distribution were built based on at least 40-50 independent measurements of specific parameter for surface features from at three to four different locations. Corresponding statistics along with average values and specific numbers of measurements within a particular interval are presented in Figures 4 and 5.

762


Fuchigami et al.

Figure 8. Topographical images of inner tissue of ball python at higher depth demonstrating fibrillar network microstructure with highly developed microporous organization from 10 × 10 µm to 2 × 2 µm scan sizes. Left is topography and right is phase imaging.

Histograms of the distribution of skin morphological parameters obtained from living tissues in receptor and nonspecific skin areas clearly showed that the average periodicity of the nanopit array is more than 1 order of magnitude smaller than the periodicity of the terracelike structure: 300-500 nm vs 5-6 µm (Table 1). Variations of both parameters over widely separated and randomly selected surface areas are within 25%. Within this variation,

the periodicity of the terracelike structure is indistinguishable for nonspecific skin and receptor areas giving an average value of approximately 5.5 µm. The average periodicity of the nanopit array is much larger for the receptor areas (Figure 4b). This value is 520 nm within pit organs and 330 nm for nonspecific skin areas between pit organs. This difference is significantly higher that the standard deviation of the surface distributions (about 20%). Similar differences are also



Figure 9. Topography of the inner tissue of a ball python at shallow depths demonstrating the cellular network microstructure at different scales: 10 × 10 µm (left) and 2 × 2 µm (right).

observed for the diameter and the depth of the nanopits: the nanopits are much wider (390 nm vs 130 nm) and deeper (120 nm vs 20 nm) in the receptor areas than in nonspecific areas (Figure 5). Standard deviation is comparable in both areas. Similar trends are observed for formaldehyde-treated skin samples of ball python from this study and for carpet python from previous studies (Table 1, Figures 6 and 7). The periodicity of the terracelike structures and nanopit arrays and the diameter and the depth of nanopits are all higher in the pit organ relative to nonspecific skin areas. The absolute value of these parameters varies, but is usually close to the data obtained from living tissue (within standard deviation). The only exception is from TEM studies on ball python samples (Table 1). In this snake, geometric parameters are generally much (from two to four times) lower than for skin specimens scanned under ambient conditions. These ball python data were obtained from specimens not only stained and microtomed but also dried in high vacuum. We suggest that the dramatic difference in geometry of ball python morphological features is due to the preparation procedure, which may have caused significant shrinkage of the skin surface after drying in air and high vacuum. Tissue Microstructure. Our previous studies suggested that a subsurface network of interconnected micropores may allow permeability of viscous fluids.3 TEM data supported this conclusion as well and indicated that microscopic pores exist in the proximity to the skin surface along with an extensive capillary network.7 In addition, high-resolution studies of tissue cross sections revealed a fine-layered microstructure of the topmost cornified skin layer with cracklike features. We expected that the number of microvoids and micropores visible on TEM micrographs was greatly underestimated due to the fact that the drying procedure and ultrahigh vacuum studies could lead to the collapse of the compliant micropore structures. To gain more insight on possible tissue microstructure within IR receptors, we conducted additional studies of underlying tissue, which was exposed by removing the outermost skin layers. In this way, we exposed tissue microstructure at various depths from a micrometer to several

tens of micrometers (Figures 8 and 9). These sections showed two primary types of tissue organization: highly branched and random network of fibrillar structures and cell-like microstructure formed by short-branched morphological features. The first type of microstructure observed at deeper levels is likely composed of collagen-based microfibrills of about 100 nm in diameter and with very regular periodicity of 68 nm along the fibril axis (Figure 8). These parameters are very close to ones well-known for collagen microfibrils.18 Highly developed cellular morphology observed in proximity to the surface may represent the endings of nerve fibrils (Figure 9). The level of porosity estimated from the SPM images is within 30-60% for various locations and the pores possess a wide distribution of sizes with the average value about or below 1 µm. The nature of these microstructures will be a subject of further investigations; here we only point out that the highly porous character of both organizations confirms our suggestion of a microporous internal microstructure of subsurface tissue within receptor areas. Conclusions In conclusion, we characterized the surface morphology of IR receptor areas obtained from both living tissue and chemically treated tissue using SPM techniques under humid environmental conditions. For the first time, the surfaces of living tissues were scanned under wet ambient conditions and the actual dimensions of surface microstructure of IR receptor areas were collected with a nanometer scale resolution. The resulting geometrical parameters were compared with data obtained for chemically treated (formaldehydefixed) tissue obtained in ambient air and TEM data in high vacuum. We concluded that the microstructural parameters for the formaldehyde-fixed snake tissues are similar to ones observed for living skin. However, previous data obtained from TEM analysis in high vacuum underestimated actual dimensions of surface microstructures. A highly developed porous microstructure was observed for snake tissue beneath the pit receptor areas. Micromechanical and microthermal properties of snake receptors will be discussed in the second part of this study (ref 19).

764


Fuchigami et al.

close to the maximum emmitance of natural sunlight. This can effectively reduce overheating of these receptor areas and make them function under conditions of a very strong thermal background. Any future models of artificial IR sensors based upon this design should include this surface feature with dimensions that provide the most effective selectivity in the specific spectral range. An alternative role of these surface structures as a passive array for lowering surface thermal conductivity and creating a “thermal cavity” will be discussed in the second part of this study (ref 19). Acknowledgment. This work is supported by AFOSR, F49620-98-1-0480 Contract. References and Notes

Figure 10. Energy spectrum of sunlight (after ref 20) and a specific area of the highest efficiency of the surface reflectivity for the surface nanopit array that blocks the most intensive spectrum range and the wavelength range where the biological IR reception is the most efficient.

The actual average spacing of the nanopit array within receptor surface areas, which was suggested to play a critical role in selective IR adsorption, was determined to be 520 nm that was much larger than 330 nm spacing observed outside of the receptor organ areas. This value is close to the value of the grating spacing required for efficient filtration of nonspecific radiation of both UV and visible spectral ranges below the actual IR range. The surface array with the average periodicity of 520 nm should possess a broad distribution of reflective ability that covers, depending upon specific symmetry of gratings and local spacing, a range from 400 to 700 nm. This is very close to the position of the maximum (550 nm) on the bell-shaped distribution of the sunlight radiation20 and, in fact, blocks the most intensive range almost completely without affecting the IR-sensitive range (Figure 10). Indeed, as was attested to by Goris,21 special prevention should be taken during filming of snakes due to the strong reflection of visible light in the receptor areas. Therefore, the efficiency of reflection of incoming radiation with such surface grating, as we revealed on the biological IR receptors, should be the highest in the range

(1) Hazel, J.; Fuchigami, N.; Gorbunov, V.; Schmitz, H.; Stone, M.; Tsukruk, V. V. Biomacromolecules 2001, 2, 304. (2) Campbell, A. L.; Bunning, T.; Stone, M. O.; Church, D.; Grace, M. J. Struct. Biol. 1999, 126, 105. (3) Hazel, J.; Stone, M.; Grace, M. S.; Tsukruk, V. V. J. Biomech. 1999, 32, 477. (4) Terashima, S.; Zhu, A.-Q. Cell. Mol. Neurobiol. 1997, 17, 195. (5) Amemiya, F.; Goris, R. C.; Atobe, Y.; Ishii, N.; Kusunoki, T. Animal Eye Res. 1996, 15, 13. (6) Amemiya, F.; Ushuki, T.; Goris, R. C.; Kusunoki, T. Anat. Rec. 1996, 246, 135. (7) Amemiya, F.; Goris, R. C.; Masuda, Y.; Kishida, R.; Atobe, Y.; Ishii, N.; Kusunoki, T. Biomed. Res. 1996, 16, 411. (8) Terashima, S.; Goris, R. C., Eds. Infrared Receptors and the Trigeminal Sensory System; Harwood Acad. Publ.: Amsterdam, 1998. (9) Grace, M. S.; Woodward, O. M.; Church, D. R.; Calisch, G BehaV. Brain Res. 2001, 119, 23. (10) Bullock, T. H.; Diecke, P. J. J. Physiol. 1956, 134, 47. (11) Jones, B. S.; Lynn, W. F.; Stone, M. O. J. Theor. Biol. 2001, in press. (12) Sarid, D. Scanning Force Microscopy; Oxford University Press: New York, 1991. (13) Magonov, S.; Whangbo, M.-H. Surface Analysis with STM and SPM; VCH: Weinheim, Germany, 1996. (14) Ratner, B.; Tsukruk, V. V. Eds. Scanning Probe Microscopy in Polymers; ACS Symposium Series 694; American Chemical Society: Washington, DC, 1998. (15) Tsukruk, V. V. Rubber Chem. Technol. 1997, 70, 430. (16) Grace, M.; Church, D.; Kelly, C. T.; Lynn, W. L.; Cooper, T. M. Biosensors Bioelectr. 1999, 14, 53. (17) Guillette, L. J. Herpetological ReV. 1982, 13, 37. (18) Nimni, M. E., Ed. Collagen; CRC Press: Boca Raton, FL, 1988, Vol. 1. (19) Gorbunov, V.; Fuchigami, N.; Stone, M.; Grace, M.; Tsukruk, V. V. Biomacromolecules, submitted for publication. (20) http://grove.ufl.edu/∼abuenfil/lecture-8/sld030.htm. (21) Goris, R. C. AFOSR Workshop on Biomimetics, University of Texas, Austin, TX, Feb. 1999.

BM015537Z

Biological Thermal Detection in Infrared Imaging ... - ACS Publications

Recommend Documents