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In this paper, a method for measuring radiance in situ in living tissue is described. This work includes details of the construction of micro-scale probes for different measurements of radiance and irradiance, provides guidance for mounting tissue for the characterization of radiance, and outlines computational methods for analyzing the resulting data.
Organisms appear opaque largely because their outer tissue layers are strongly scattering to incident light; strongly absorbing pigments, such as blood, typically have narrow absorbances, such that the mean free path of light outside the absorbance peaks can be quite long. As people cannot see through tissue, they generally imagine that tissues like the brain, fat, and bone contain little or no light. However, photoresponsive opsin proteins are expressed within many of these tissues, and their functions are poorly understood. Radiance internal to tissue is also important for understanding photosynthesis. For example, giant clams are strongly absorbing yet maintain a dense population of algae deep in the tissue. Light propagation through systems like sediments and biofilms can be complex, and these communities can be major contributors to ecosystem productivity. Therefore, a method for constructing optical micro-probes for measuring scalar irradiance (photon flux intersecting a point) and downwelling irradiance (photon flux crossing a plane perpendicularly) to better understand these phenomena inside living tissue has been developed. This technique is also tractable in field laboratories. These micro-probes are made from heat-pulled optical fibers that are then secured in pulled glass pipettes. To change the angular acceptance of the probe, a 10-100 µm sized sphere of UV-curable epoxy mixed with titanium dioxide is then secured to the end of a pulled, trimmed fiber. The probe is inserted into living tissue, and its position is controlled using a micromanipulator. These probes are capable of measuring in situ tissue radiance at spatial resolutions of 10-100 µm or on the scale of single cells. These probes were used to characterize the light reaching the adipose and brain cells 4 mm below the skin of a living mouse and to characterize the light reaching similar depths within living algae-rich giant clam tissue.
Surprisingly, land animals and shallow ocean dwellers have enough light within their body for visual physiology and even photosynthesis. For example, the light levels in the center of a mouse's head (outside of the strong hemoglobin absorbance bands) are attenuated by three or four orders of magnitude relative to the outside world. This is roughly the difference between the light levels indoors and outside. So, the opacity of a tissue or material due to strong scattering is not the same as opacity due to strong light absorption. Light can keep propagating over long distances in a strongly forward-scattering system, similar to light propagating through aquatic syst....
This study is compliant with all the relevant ethical regulations of Yale University regarding vertebrate and invertebrate animal research.
1. Building the optical micro-probe
This protocol describes the procedure for constructing a micro-optical probe that can be used to measure the downwelling irradiance (the light reaching a point from one direction) or, with the addition of a light-scattering spherical tip, to measure the scalar irradiance (the light reaching a point from all directions). These probes can measure irradiance at spatial resolutions approaching the length scales of single cells inside living tissue. This protocol also describes a representative method for preparing a tissue s.......
This protocol describes a technique for systematically characterizing the optical environment through a large volume of living tissue with a spatial resolution approximately on the scale of single cells. This inexpensive, flexible, and field-tractable method could be useful to any researchers studying the propagation of light within living systems. From experience, compared to existing methods7, these probes require a little more practice and skill to build but result in less tissue damage and the.......
The authors thank Sanaz Vahidinia for introducing us to Dr. Jorgensen's colleagues and his work. This research was supported by grants from the Army Research Office (no. W911NF-10-0139), the Office of Naval Research (through MURI award no. N00014-09-1-1053), and NSF-INSPIRE award NSF-1343158.
....Name | Company | Catalog Number | Comments |
1" travel ball bearing center+D11+A2:D31+A2:A2:D31 | Edmond Optics | 37-935 | Part 2 of manipulator for lowering sample |
1/4" thick acrylic sheet | McMaster-Carr | 8505K754 | For making Petri dish holder |
3/4" mini spring clamp | Anvil | 99693 | Use as weight for pulling optical fiber |
8 mm biopsy punch | Fisher Scientific | NC9324386 | For tissue sample |
Butane Torch | McMaster-Carr | MT-51 | Heat source for pulling fiber and pipette |
Collimating lens | Thorlabs | LLG5A1-A | To collimate light source through liquid light guide |
Compressed air | McMaster-Carr | 7437K35 | For drying pulled fiber and pipette |
Cyanoacrylate glue - liquid | McMaster-Carr | 66635A31 | For securing tapered fiber end at top of pulled pipette |
Electrical tape | McMaster-Carr | 76455A21 | For securing fiber in pipette and for adding grip to clamps |
Fine grade carborundum paper | McMaster-Carr | 4649A24 | Small triangle on exacto knife holder works well |
Gelatin | Knox | 10043000048679 | For securing the tissue biopsy in the petri dish |
Glass Pasteur Pipete | Fisher Scientific | 13-678-20B | Disposable glass pipette 5.75" in length |
Insulin syringes, 31G needle | BD | 320440 | For applying glue |
Isopropanol | McMaster-Carr | 54845T42 | For cleaning pulled fiber and pipette |
Kimwipe | Cole-Parmer | SKU 33670-04 | For wiping optical fiber and glass pipette clean |
LED driver | Thorlabs | LEDD1B | For powering the UV LEDs |
Light source for measurements | Cole-Parmer | UX-78905-05 | Low heat white light source for measurements |
Linear metric X-Y-Z axis rack and pinion stage | Edmond Optics | 55-023 | Part 1 of manipulator for lowering sample |
Liquid light guide | Thorlabs | LLG5-4T | For light source in measurements |
Magnetic feet | Siskiyou | MGB 8-32 | For use with magnetic strips |
Magnetic strips | Siskiyou | MS-6.0 | For mounting magnetically to breadboard |
Manipulator #1 | Siskiyou | MX10R | 4-axis manipulator with pipette holder |
Opaquer pen, small | WindowTint | TOP01 | For opaquing side of optical fiber to prevent stray light from enter probe |
Optical breadboard | Edmond Optics | 03-640 | For stable affixation of probe holder, sample, microscope and light source |
Optical fibers | Ocean Optics | P-100-2-UV-VIS | About 4 fibers are good to have |
Plasma light source | Thorlabs | HPLS345 | For tissue radiometry measurements |
Plastic plier clamp | McMaster-Carr | 5070A11 | Plier clamp used for weight in pulling pipette |
Polystyrene Petri dishes | Thomas scientific | 3488N10 | Sample holders, enough volume to hold sample thickness plus ~10 mm of gelatin on top |
Razor blades | McMaster-Carr | 3962A3 | For stripping jacketing from optical fiber |
Silicone oil lubricant | Thomas scientific | 1232E30 | For reducing friction between probe and tissue |
Software for analyzing data | Matlab | Chosen software for data analysis | |
Spectrometer + spectrometer software | Avantes | AvaSpec-2048L | Spectrometer can be any brand, this one is compatible with sma-terminated optical fibers and comes with its own software for running the spectrometer |
Titanium dioxide powder | Sigma Aldrich | 718467-100G | For making scattering sphere |
Toolour tabletop clip | Toolour | Toolour0004 | For holding pipette while pulling and for holding finished probes |
Trigger-action bar clamps | mcMaster-Carr | 51755A2 | Good for holding optical fibers while pulling or curing |
UV curable adhesive | Delo Photobond | GB368 | For making scattering sphere |
UV light source | Thorlabs | M365FP1 | Light source for curing adhesive in scattering ball, this one is sma-fiber compatible, higher intensity = less cure time |
White LED light source | Thorlabs | MCWHF2 | For characterizing pulled fiber and scattering sphere |
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