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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

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.

Abstract

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.

Introduction

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....

Protocol

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

  1. Building the glass sleeve, material: Pasteur pipette, 5.75 in(see Table of Materials)
    1. Using a mountable alligator clip (Table of Materials), mount the glass pipette by the wide end such that the tapered end is facing down toward the floor and the .......

Representative Results

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.......

Discussion

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.......

Acknowledgements

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.

....

Materials

NameCompanyCatalog NumberComments
1" travel ball bearing center+D11+A2:D31+A2:A2:D31Edmond Optics37-935Part 2 of manipulator for lowering sample
1/4" thick acrylic sheetMcMaster-Carr8505K754For making Petri dish holder
3/4" mini spring clampAnvil99693Use as weight for pulling optical fiber
8 mm biopsy punchFisher ScientificNC9324386For tissue sample
Butane TorchMcMaster-CarrMT-51Heat source for pulling fiber and pipette
Collimating lensThorlabsLLG5A1-ATo collimate light source through liquid light guide
Compressed airMcMaster-Carr7437K35For drying pulled fiber and pipette
Cyanoacrylate glue - liquidMcMaster-Carr66635A31For securing tapered fiber end at top of pulled pipette
Electrical tapeMcMaster-Carr76455A21For securing fiber in pipette and for adding grip to clamps
Fine grade carborundum paperMcMaster-Carr4649A24Small triangle on exacto knife holder works well
GelatinKnox10043000048679For securing the tissue biopsy in the petri dish
Glass Pasteur PipeteFisher Scientific13-678-20BDisposable glass pipette 5.75" in length
Insulin syringes, 31G needleBD320440For applying glue
IsopropanolMcMaster-Carr54845T42For cleaning pulled fiber and pipette
KimwipeCole-ParmerSKU 33670-04For wiping optical fiber and glass pipette clean
LED driverThorlabsLEDD1BFor powering the UV LEDs
Light source for measurementsCole-ParmerUX-78905-05Low heat white light source for measurements
Linear metric X-Y-Z axis rack and pinion stageEdmond Optics55-023Part 1 of manipulator for lowering sample
Liquid light guideThorlabsLLG5-4TFor light source in measurements
Magnetic feetSiskiyouMGB 8-32For use with magnetic strips
Magnetic stripsSiskiyouMS-6.0For mounting magnetically to breadboard
Manipulator #1SiskiyouMX10R4-axis manipulator with pipette holder
Opaquer pen, smallWindowTintTOP01For opaquing side of optical fiber to prevent stray light from enter probe
Optical breadboardEdmond Optics03-640For stable affixation of probe holder, sample, microscope and light source
Optical fibersOcean OpticsP-100-2-UV-VISAbout 4 fibers are good to have
Plasma light sourceThorlabsHPLS345For tissue radiometry measurements
Plastic plier clampMcMaster-Carr5070A11Plier clamp used for weight in pulling pipette
Polystyrene Petri dishesThomas scientific3488N10Sample holders, enough volume to hold sample thickness plus ~10 mm of gelatin on top
Razor bladesMcMaster-Carr3962A3For stripping jacketing from optical fiber
Silicone oil lubricantThomas scientific1232E30For reducing friction between probe and tissue
Software for analyzing dataMatlabChosen software for data analysis
Spectrometer + spectrometer softwareAvantesAvaSpec-2048LSpectrometer 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 powderSigma Aldrich718467-100GFor making scattering sphere
Toolour tabletop clipToolourToolour0004For holding pipette while pulling and for holding finished probes
Trigger-action bar clampsmcMaster-Carr51755A2Good for holding optical fibers while pulling or curing
UV curable adhesiveDelo PhotobondGB368For making scattering sphere
UV light sourceThorlabsM365FP1Light source for curing adhesive in scattering ball, this one is sma-fiber compatible, higher intensity = less cure time
White LED light sourceThorlabsMCWHF2For characterizing pulled fiber and scattering sphere

References

  1. Williams, J. Optical properties of the ocean. Reports on Progress in Physics. 36 (12), 1567-1608 (2001).
  2. Solonenko, M., et al. In vivo reflectance measurement of optical pr....

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