Abstract

Confocal reflectance microscopy and confocal Raman spectroscopy have shown potential for non-destructive analysis of samples at micron-scale resolutions. Current studies utilizing these techniques often employ large bench-top microscopes, and are not suited for use outside of laboratory settings. We have developed a microscope which combines laser scanning confocal reflectance imaging and confocal Raman spectroscopy into a compact handheld probe that is capable of high-resolution imaging and spectroscopy in a variety of settings. The compact size of the probe is largely due to the use of a MEMS mirror for beam scanning. The probe is capable of axial resolutions of up to 4 μm for the confocal imaging channel and 10 μm for the confocal Raman spectroscopy channel. Here, we report instrument design, characterize optical performance, and provide images and spectra from normal skin to demonstrate the instrument’s capabilities for clinical diagnostics.

© 2012 OSA

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  1. D. B. Hovis and A. H. Heuer, “The use of laser scanning confocal microscopy (LSCM) in materials science,” J. Microsc.240(3), 173–180 (2010).
    [CrossRef] [PubMed]
  2. A. Gupper, P. Wilhelm, M. Schmied, S. G. Kazarian, K. L. A. Chan, and J. Reußner, “Combined application of imaging methods for the characterization of a polymer blend,” Appl. Spectrosc.56(12), 1515–1523 (2002).
    [CrossRef]
  3. J. T. Fredrich, “3D imaging of porous media using laser scanning confocal microscopy with application to microscale transport processes,” Phys. Chem. Earth, Part A Solid Earth Geod.24(7), 551–561 (1999).
    [CrossRef]
  4. K. D. McKeegan, A. B. Kudryavtsev, and J. W. Schopf, “Raman and ion microscopic imagery of graphitic inclusions in apatite from older than 3830 Ma Akilia supracrustal rocks, west Greenland,” Geology35(7), 591–594 (2007).
    [CrossRef]
  5. M. Claybourn and M. Ansell, “Using Raman spectroscopy to solve crime: inks, questioned documents and fraud,” Sci. Justice40(4), 261–271 (2000).
    [CrossRef] [PubMed]
  6. R. S. Scott, P. S. Ungar, T. S. Bergstrom, C. A. Brown, F. E. Grine, M. F. Teaford, and A. Walker, “Dental microwear texture analysis shows within-species diet variability in fossil hominins,” Nature436(7051), 693–695 (2005).
    [CrossRef] [PubMed]
  7. M. A. Pabst, I. Letofsky-Papst, M. Moser, K. Spindler, E. Bock, P. Wilhelm, M. D. Leopold Dorfer, J. B. Geigl, M. Auer, M. R. Speicher, and F. Hofer, “Different staining substances were used in decorative and therapeutic tattoos in a 1000-year-old Peruvian mummy,” J. Archaeol. Sci.37(12), 3256–3262 (2010).
    [CrossRef]
  8. K.-J. Halbhuber and K. König, “Modern laser scanning microscopy in biology, biotechnology and medicine,” Ann. Anat.185(1), 1–20 (2003).
    [CrossRef] [PubMed]
  9. R. Petry, M. Schmitt, and J. Popp, “Raman spectroscopy--a prospective tool in the life sciences,” ChemPhysChem4(1), 14–30 (2003).
    [CrossRef] [PubMed]
  10. P. J. Caspers, G. W. Lucassen, and G. J. Puppels, “Combined in vivo confocal Raman spectroscopy and confocal microscopy of human skin,” Biophys. J.85(1), 572–580 (2003).
    [CrossRef] [PubMed]
  11. C. L. Arrasmith, D. L. Dickensheets, and A. Mahadevan-Jansen, “MEMS-based handheld confocal microscope for in-vivo skin imaging,” Opt. Express18(4), 3805–3819 (2010).
    [CrossRef] [PubMed]
  12. M. Rajadhyaksha, R. R. Anderson, and R. H. Webb, “Video-rate confocal scanning laser microscope for imaging human tissues in vivo,” Appl. Opt.38(10), 2105–2115 (1999).
    [CrossRef] [PubMed]
  13. P. J. Caspers, G. W. Lucassen, R. Wolthuis, H. A. Bruining, and G. J. Puppels, “In vitro and in vivo Raman spectroscopy of human skin,” Biospectroscopy4(S5), S31–S39 (1998).
    [CrossRef] [PubMed]
  14. A. Palm, “Raman spectrum of polystyrene,” J. Phys. Chem.55(8), 1320–1324 (1951).
    [CrossRef]
  15. C. A. Lieber and A. Mahadevan-Jansen, “Automated method for subtraction of fluorescence from biological Raman spectra,” Appl. Spectrosc.57(11), 1363–1367 (2003).
    [CrossRef] [PubMed]
  16. P. J. Caspers, G. W. Lucassen, E. A. Carter, H. A. Bruining, and G. J. Puppels, “In vivo confocal Raman microspectroscopy of the skin: noninvasive determination of molecular concentration profiles,” J. Invest. Dermatol.116(3), 434–442 (2001).
    [CrossRef] [PubMed]
  17. A. Nijssen, T. C. Bakker Schut, F. Heule, P. J. Caspers, D. P. Hayes, M. H. A. Neumann, and G. J. Puppels, “Discriminating basal cell carcinoma from its surrounding tissue by Raman spectroscopy,” J. Invest. Dermatol.119(1), 64–69 (2002).
    [CrossRef] [PubMed]
  18. C. A. Lieber, S. K. Majumder, D. L. Ellis, D. D. Billheimer, and A. Mahadevan-Jansen, “In vivo nonmelanoma skin cancer diagnosis using Raman microspectroscopy,” Lasers Surg. Med.40(7), 461–467 (2008).
    [CrossRef] [PubMed]
  19. M. Rajadhyaksha, S. González, J. M. Zavislan, R. R. Anderson, and R. H. Webb, “In vivo confocal scanning laser microscopy of human skin II: advances in instrumentation and comparison with histology,” J. Invest. Dermatol.113(3), 293–303 (1999).
    [CrossRef] [PubMed]
  20. M. Rajadhyaksha, G. Menaker, T. Flotte, P. J. Dwyer, and S. González, “Confocal examination of nonmelanoma cancers in thick skin excisions to potentially guide mohs micrographic surgery without frozen histopathology,” J. Invest. Dermatol.117(5), 1137–1143 (2001).
    [CrossRef] [PubMed]

2010 (3)

D. B. Hovis and A. H. Heuer, “The use of laser scanning confocal microscopy (LSCM) in materials science,” J. Microsc.240(3), 173–180 (2010).
[CrossRef] [PubMed]

M. A. Pabst, I. Letofsky-Papst, M. Moser, K. Spindler, E. Bock, P. Wilhelm, M. D. Leopold Dorfer, J. B. Geigl, M. Auer, M. R. Speicher, and F. Hofer, “Different staining substances were used in decorative and therapeutic tattoos in a 1000-year-old Peruvian mummy,” J. Archaeol. Sci.37(12), 3256–3262 (2010).
[CrossRef]

C. L. Arrasmith, D. L. Dickensheets, and A. Mahadevan-Jansen, “MEMS-based handheld confocal microscope for in-vivo skin imaging,” Opt. Express18(4), 3805–3819 (2010).
[CrossRef] [PubMed]

2008 (1)

C. A. Lieber, S. K. Majumder, D. L. Ellis, D. D. Billheimer, and A. Mahadevan-Jansen, “In vivo nonmelanoma skin cancer diagnosis using Raman microspectroscopy,” Lasers Surg. Med.40(7), 461–467 (2008).
[CrossRef] [PubMed]

2007 (1)

K. D. McKeegan, A. B. Kudryavtsev, and J. W. Schopf, “Raman and ion microscopic imagery of graphitic inclusions in apatite from older than 3830 Ma Akilia supracrustal rocks, west Greenland,” Geology35(7), 591–594 (2007).
[CrossRef]

2005 (1)

R. S. Scott, P. S. Ungar, T. S. Bergstrom, C. A. Brown, F. E. Grine, M. F. Teaford, and A. Walker, “Dental microwear texture analysis shows within-species diet variability in fossil hominins,” Nature436(7051), 693–695 (2005).
[CrossRef] [PubMed]

2003 (4)

C. A. Lieber and A. Mahadevan-Jansen, “Automated method for subtraction of fluorescence from biological Raman spectra,” Appl. Spectrosc.57(11), 1363–1367 (2003).
[CrossRef] [PubMed]

K.-J. Halbhuber and K. König, “Modern laser scanning microscopy in biology, biotechnology and medicine,” Ann. Anat.185(1), 1–20 (2003).
[CrossRef] [PubMed]

R. Petry, M. Schmitt, and J. Popp, “Raman spectroscopy--a prospective tool in the life sciences,” ChemPhysChem4(1), 14–30 (2003).
[CrossRef] [PubMed]

P. J. Caspers, G. W. Lucassen, and G. J. Puppels, “Combined in vivo confocal Raman spectroscopy and confocal microscopy of human skin,” Biophys. J.85(1), 572–580 (2003).
[CrossRef] [PubMed]

2002 (2)

A. Gupper, P. Wilhelm, M. Schmied, S. G. Kazarian, K. L. A. Chan, and J. Reußner, “Combined application of imaging methods for the characterization of a polymer blend,” Appl. Spectrosc.56(12), 1515–1523 (2002).
[CrossRef]

A. Nijssen, T. C. Bakker Schut, F. Heule, P. J. Caspers, D. P. Hayes, M. H. A. Neumann, and G. J. Puppels, “Discriminating basal cell carcinoma from its surrounding tissue by Raman spectroscopy,” J. Invest. Dermatol.119(1), 64–69 (2002).
[CrossRef] [PubMed]

2001 (2)

P. J. Caspers, G. W. Lucassen, E. A. Carter, H. A. Bruining, and G. J. Puppels, “In vivo confocal Raman microspectroscopy of the skin: noninvasive determination of molecular concentration profiles,” J. Invest. Dermatol.116(3), 434–442 (2001).
[CrossRef] [PubMed]

M. Rajadhyaksha, G. Menaker, T. Flotte, P. J. Dwyer, and S. González, “Confocal examination of nonmelanoma cancers in thick skin excisions to potentially guide mohs micrographic surgery without frozen histopathology,” J. Invest. Dermatol.117(5), 1137–1143 (2001).
[CrossRef] [PubMed]

2000 (1)

M. Claybourn and M. Ansell, “Using Raman spectroscopy to solve crime: inks, questioned documents and fraud,” Sci. Justice40(4), 261–271 (2000).
[CrossRef] [PubMed]

1999 (3)

J. T. Fredrich, “3D imaging of porous media using laser scanning confocal microscopy with application to microscale transport processes,” Phys. Chem. Earth, Part A Solid Earth Geod.24(7), 551–561 (1999).
[CrossRef]

M. Rajadhyaksha, R. R. Anderson, and R. H. Webb, “Video-rate confocal scanning laser microscope for imaging human tissues in vivo,” Appl. Opt.38(10), 2105–2115 (1999).
[CrossRef] [PubMed]

M. Rajadhyaksha, S. González, J. M. Zavislan, R. R. Anderson, and R. H. Webb, “In vivo confocal scanning laser microscopy of human skin II: advances in instrumentation and comparison with histology,” J. Invest. Dermatol.113(3), 293–303 (1999).
[CrossRef] [PubMed]

1998 (1)

P. J. Caspers, G. W. Lucassen, R. Wolthuis, H. A. Bruining, and G. J. Puppels, “In vitro and in vivo Raman spectroscopy of human skin,” Biospectroscopy4(S5), S31–S39 (1998).
[CrossRef] [PubMed]

1951 (1)

A. Palm, “Raman spectrum of polystyrene,” J. Phys. Chem.55(8), 1320–1324 (1951).
[CrossRef]

Anderson, R. R.

M. Rajadhyaksha, S. González, J. M. Zavislan, R. R. Anderson, and R. H. Webb, “In vivo confocal scanning laser microscopy of human skin II: advances in instrumentation and comparison with histology,” J. Invest. Dermatol.113(3), 293–303 (1999).
[CrossRef] [PubMed]

M. Rajadhyaksha, R. R. Anderson, and R. H. Webb, “Video-rate confocal scanning laser microscope for imaging human tissues in vivo,” Appl. Opt.38(10), 2105–2115 (1999).
[CrossRef] [PubMed]

Ansell, M.

M. Claybourn and M. Ansell, “Using Raman spectroscopy to solve crime: inks, questioned documents and fraud,” Sci. Justice40(4), 261–271 (2000).
[CrossRef] [PubMed]

Arrasmith, C. L.

Auer, M.

M. A. Pabst, I. Letofsky-Papst, M. Moser, K. Spindler, E. Bock, P. Wilhelm, M. D. Leopold Dorfer, J. B. Geigl, M. Auer, M. R. Speicher, and F. Hofer, “Different staining substances were used in decorative and therapeutic tattoos in a 1000-year-old Peruvian mummy,” J. Archaeol. Sci.37(12), 3256–3262 (2010).
[CrossRef]

Bakker Schut, T. C.

A. Nijssen, T. C. Bakker Schut, F. Heule, P. J. Caspers, D. P. Hayes, M. H. A. Neumann, and G. J. Puppels, “Discriminating basal cell carcinoma from its surrounding tissue by Raman spectroscopy,” J. Invest. Dermatol.119(1), 64–69 (2002).
[CrossRef] [PubMed]

Bergstrom, T. S.

R. S. Scott, P. S. Ungar, T. S. Bergstrom, C. A. Brown, F. E. Grine, M. F. Teaford, and A. Walker, “Dental microwear texture analysis shows within-species diet variability in fossil hominins,” Nature436(7051), 693–695 (2005).
[CrossRef] [PubMed]

Billheimer, D. D.

C. A. Lieber, S. K. Majumder, D. L. Ellis, D. D. Billheimer, and A. Mahadevan-Jansen, “In vivo nonmelanoma skin cancer diagnosis using Raman microspectroscopy,” Lasers Surg. Med.40(7), 461–467 (2008).
[CrossRef] [PubMed]

Bock, E.

M. A. Pabst, I. Letofsky-Papst, M. Moser, K. Spindler, E. Bock, P. Wilhelm, M. D. Leopold Dorfer, J. B. Geigl, M. Auer, M. R. Speicher, and F. Hofer, “Different staining substances were used in decorative and therapeutic tattoos in a 1000-year-old Peruvian mummy,” J. Archaeol. Sci.37(12), 3256–3262 (2010).
[CrossRef]

Brown, C. A.

R. S. Scott, P. S. Ungar, T. S. Bergstrom, C. A. Brown, F. E. Grine, M. F. Teaford, and A. Walker, “Dental microwear texture analysis shows within-species diet variability in fossil hominins,” Nature436(7051), 693–695 (2005).
[CrossRef] [PubMed]

Bruining, H. A.

P. J. Caspers, G. W. Lucassen, E. A. Carter, H. A. Bruining, and G. J. Puppels, “In vivo confocal Raman microspectroscopy of the skin: noninvasive determination of molecular concentration profiles,” J. Invest. Dermatol.116(3), 434–442 (2001).
[CrossRef] [PubMed]

P. J. Caspers, G. W. Lucassen, R. Wolthuis, H. A. Bruining, and G. J. Puppels, “In vitro and in vivo Raman spectroscopy of human skin,” Biospectroscopy4(S5), S31–S39 (1998).
[CrossRef] [PubMed]

Carter, E. A.

P. J. Caspers, G. W. Lucassen, E. A. Carter, H. A. Bruining, and G. J. Puppels, “In vivo confocal Raman microspectroscopy of the skin: noninvasive determination of molecular concentration profiles,” J. Invest. Dermatol.116(3), 434–442 (2001).
[CrossRef] [PubMed]

Caspers, P. J.

P. J. Caspers, G. W. Lucassen, and G. J. Puppels, “Combined in vivo confocal Raman spectroscopy and confocal microscopy of human skin,” Biophys. J.85(1), 572–580 (2003).
[CrossRef] [PubMed]

A. Nijssen, T. C. Bakker Schut, F. Heule, P. J. Caspers, D. P. Hayes, M. H. A. Neumann, and G. J. Puppels, “Discriminating basal cell carcinoma from its surrounding tissue by Raman spectroscopy,” J. Invest. Dermatol.119(1), 64–69 (2002).
[CrossRef] [PubMed]

P. J. Caspers, G. W. Lucassen, E. A. Carter, H. A. Bruining, and G. J. Puppels, “In vivo confocal Raman microspectroscopy of the skin: noninvasive determination of molecular concentration profiles,” J. Invest. Dermatol.116(3), 434–442 (2001).
[CrossRef] [PubMed]

P. J. Caspers, G. W. Lucassen, R. Wolthuis, H. A. Bruining, and G. J. Puppels, “In vitro and in vivo Raman spectroscopy of human skin,” Biospectroscopy4(S5), S31–S39 (1998).
[CrossRef] [PubMed]

Chan, K. L. A.

Claybourn, M.

M. Claybourn and M. Ansell, “Using Raman spectroscopy to solve crime: inks, questioned documents and fraud,” Sci. Justice40(4), 261–271 (2000).
[CrossRef] [PubMed]

Dickensheets, D. L.

Dwyer, P. J.

M. Rajadhyaksha, G. Menaker, T. Flotte, P. J. Dwyer, and S. González, “Confocal examination of nonmelanoma cancers in thick skin excisions to potentially guide mohs micrographic surgery without frozen histopathology,” J. Invest. Dermatol.117(5), 1137–1143 (2001).
[CrossRef] [PubMed]

Ellis, D. L.

C. A. Lieber, S. K. Majumder, D. L. Ellis, D. D. Billheimer, and A. Mahadevan-Jansen, “In vivo nonmelanoma skin cancer diagnosis using Raman microspectroscopy,” Lasers Surg. Med.40(7), 461–467 (2008).
[CrossRef] [PubMed]

Flotte, T.

M. Rajadhyaksha, G. Menaker, T. Flotte, P. J. Dwyer, and S. González, “Confocal examination of nonmelanoma cancers in thick skin excisions to potentially guide mohs micrographic surgery without frozen histopathology,” J. Invest. Dermatol.117(5), 1137–1143 (2001).
[CrossRef] [PubMed]

Fredrich, J. T.

J. T. Fredrich, “3D imaging of porous media using laser scanning confocal microscopy with application to microscale transport processes,” Phys. Chem. Earth, Part A Solid Earth Geod.24(7), 551–561 (1999).
[CrossRef]

Geigl, J. B.

M. A. Pabst, I. Letofsky-Papst, M. Moser, K. Spindler, E. Bock, P. Wilhelm, M. D. Leopold Dorfer, J. B. Geigl, M. Auer, M. R. Speicher, and F. Hofer, “Different staining substances were used in decorative and therapeutic tattoos in a 1000-year-old Peruvian mummy,” J. Archaeol. Sci.37(12), 3256–3262 (2010).
[CrossRef]

González, S.

M. Rajadhyaksha, G. Menaker, T. Flotte, P. J. Dwyer, and S. González, “Confocal examination of nonmelanoma cancers in thick skin excisions to potentially guide mohs micrographic surgery without frozen histopathology,” J. Invest. Dermatol.117(5), 1137–1143 (2001).
[CrossRef] [PubMed]

M. Rajadhyaksha, S. González, J. M. Zavislan, R. R. Anderson, and R. H. Webb, “In vivo confocal scanning laser microscopy of human skin II: advances in instrumentation and comparison with histology,” J. Invest. Dermatol.113(3), 293–303 (1999).
[CrossRef] [PubMed]

Grine, F. E.

R. S. Scott, P. S. Ungar, T. S. Bergstrom, C. A. Brown, F. E. Grine, M. F. Teaford, and A. Walker, “Dental microwear texture analysis shows within-species diet variability in fossil hominins,” Nature436(7051), 693–695 (2005).
[CrossRef] [PubMed]

Gupper, A.

Halbhuber, K.-J.

K.-J. Halbhuber and K. König, “Modern laser scanning microscopy in biology, biotechnology and medicine,” Ann. Anat.185(1), 1–20 (2003).
[CrossRef] [PubMed]

Hayes, D. P.

A. Nijssen, T. C. Bakker Schut, F. Heule, P. J. Caspers, D. P. Hayes, M. H. A. Neumann, and G. J. Puppels, “Discriminating basal cell carcinoma from its surrounding tissue by Raman spectroscopy,” J. Invest. Dermatol.119(1), 64–69 (2002).
[CrossRef] [PubMed]

Heuer, A. H.

D. B. Hovis and A. H. Heuer, “The use of laser scanning confocal microscopy (LSCM) in materials science,” J. Microsc.240(3), 173–180 (2010).
[CrossRef] [PubMed]

Heule, F.

A. Nijssen, T. C. Bakker Schut, F. Heule, P. J. Caspers, D. P. Hayes, M. H. A. Neumann, and G. J. Puppels, “Discriminating basal cell carcinoma from its surrounding tissue by Raman spectroscopy,” J. Invest. Dermatol.119(1), 64–69 (2002).
[CrossRef] [PubMed]

Hofer, F.

M. A. Pabst, I. Letofsky-Papst, M. Moser, K. Spindler, E. Bock, P. Wilhelm, M. D. Leopold Dorfer, J. B. Geigl, M. Auer, M. R. Speicher, and F. Hofer, “Different staining substances were used in decorative and therapeutic tattoos in a 1000-year-old Peruvian mummy,” J. Archaeol. Sci.37(12), 3256–3262 (2010).
[CrossRef]

Hovis, D. B.

D. B. Hovis and A. H. Heuer, “The use of laser scanning confocal microscopy (LSCM) in materials science,” J. Microsc.240(3), 173–180 (2010).
[CrossRef] [PubMed]

Kazarian, S. G.

König, K.

K.-J. Halbhuber and K. König, “Modern laser scanning microscopy in biology, biotechnology and medicine,” Ann. Anat.185(1), 1–20 (2003).
[CrossRef] [PubMed]

Kudryavtsev, A. B.

K. D. McKeegan, A. B. Kudryavtsev, and J. W. Schopf, “Raman and ion microscopic imagery of graphitic inclusions in apatite from older than 3830 Ma Akilia supracrustal rocks, west Greenland,” Geology35(7), 591–594 (2007).
[CrossRef]

Leopold Dorfer, M. D.

M. A. Pabst, I. Letofsky-Papst, M. Moser, K. Spindler, E. Bock, P. Wilhelm, M. D. Leopold Dorfer, J. B. Geigl, M. Auer, M. R. Speicher, and F. Hofer, “Different staining substances were used in decorative and therapeutic tattoos in a 1000-year-old Peruvian mummy,” J. Archaeol. Sci.37(12), 3256–3262 (2010).
[CrossRef]

Letofsky-Papst, I.

M. A. Pabst, I. Letofsky-Papst, M. Moser, K. Spindler, E. Bock, P. Wilhelm, M. D. Leopold Dorfer, J. B. Geigl, M. Auer, M. R. Speicher, and F. Hofer, “Different staining substances were used in decorative and therapeutic tattoos in a 1000-year-old Peruvian mummy,” J. Archaeol. Sci.37(12), 3256–3262 (2010).
[CrossRef]

Lieber, C. A.

C. A. Lieber, S. K. Majumder, D. L. Ellis, D. D. Billheimer, and A. Mahadevan-Jansen, “In vivo nonmelanoma skin cancer diagnosis using Raman microspectroscopy,” Lasers Surg. Med.40(7), 461–467 (2008).
[CrossRef] [PubMed]

C. A. Lieber and A. Mahadevan-Jansen, “Automated method for subtraction of fluorescence from biological Raman spectra,” Appl. Spectrosc.57(11), 1363–1367 (2003).
[CrossRef] [PubMed]

Lucassen, G. W.

P. J. Caspers, G. W. Lucassen, and G. J. Puppels, “Combined in vivo confocal Raman spectroscopy and confocal microscopy of human skin,” Biophys. J.85(1), 572–580 (2003).
[CrossRef] [PubMed]

P. J. Caspers, G. W. Lucassen, E. A. Carter, H. A. Bruining, and G. J. Puppels, “In vivo confocal Raman microspectroscopy of the skin: noninvasive determination of molecular concentration profiles,” J. Invest. Dermatol.116(3), 434–442 (2001).
[CrossRef] [PubMed]

P. J. Caspers, G. W. Lucassen, R. Wolthuis, H. A. Bruining, and G. J. Puppels, “In vitro and in vivo Raman spectroscopy of human skin,” Biospectroscopy4(S5), S31–S39 (1998).
[CrossRef] [PubMed]

Mahadevan-Jansen, A.

Majumder, S. K.

C. A. Lieber, S. K. Majumder, D. L. Ellis, D. D. Billheimer, and A. Mahadevan-Jansen, “In vivo nonmelanoma skin cancer diagnosis using Raman microspectroscopy,” Lasers Surg. Med.40(7), 461–467 (2008).
[CrossRef] [PubMed]

McKeegan, K. D.

K. D. McKeegan, A. B. Kudryavtsev, and J. W. Schopf, “Raman and ion microscopic imagery of graphitic inclusions in apatite from older than 3830 Ma Akilia supracrustal rocks, west Greenland,” Geology35(7), 591–594 (2007).
[CrossRef]

Menaker, G.

M. Rajadhyaksha, G. Menaker, T. Flotte, P. J. Dwyer, and S. González, “Confocal examination of nonmelanoma cancers in thick skin excisions to potentially guide mohs micrographic surgery without frozen histopathology,” J. Invest. Dermatol.117(5), 1137–1143 (2001).
[CrossRef] [PubMed]

Moser, M.

M. A. Pabst, I. Letofsky-Papst, M. Moser, K. Spindler, E. Bock, P. Wilhelm, M. D. Leopold Dorfer, J. B. Geigl, M. Auer, M. R. Speicher, and F. Hofer, “Different staining substances were used in decorative and therapeutic tattoos in a 1000-year-old Peruvian mummy,” J. Archaeol. Sci.37(12), 3256–3262 (2010).
[CrossRef]

Neumann, M. H. A.

A. Nijssen, T. C. Bakker Schut, F. Heule, P. J. Caspers, D. P. Hayes, M. H. A. Neumann, and G. J. Puppels, “Discriminating basal cell carcinoma from its surrounding tissue by Raman spectroscopy,” J. Invest. Dermatol.119(1), 64–69 (2002).
[CrossRef] [PubMed]

Nijssen, A.

A. Nijssen, T. C. Bakker Schut, F. Heule, P. J. Caspers, D. P. Hayes, M. H. A. Neumann, and G. J. Puppels, “Discriminating basal cell carcinoma from its surrounding tissue by Raman spectroscopy,” J. Invest. Dermatol.119(1), 64–69 (2002).
[CrossRef] [PubMed]

Pabst, M. A.

M. A. Pabst, I. Letofsky-Papst, M. Moser, K. Spindler, E. Bock, P. Wilhelm, M. D. Leopold Dorfer, J. B. Geigl, M. Auer, M. R. Speicher, and F. Hofer, “Different staining substances were used in decorative and therapeutic tattoos in a 1000-year-old Peruvian mummy,” J. Archaeol. Sci.37(12), 3256–3262 (2010).
[CrossRef]

Palm, A.

A. Palm, “Raman spectrum of polystyrene,” J. Phys. Chem.55(8), 1320–1324 (1951).
[CrossRef]

Petry, R.

R. Petry, M. Schmitt, and J. Popp, “Raman spectroscopy--a prospective tool in the life sciences,” ChemPhysChem4(1), 14–30 (2003).
[CrossRef] [PubMed]

Popp, J.

R. Petry, M. Schmitt, and J. Popp, “Raman spectroscopy--a prospective tool in the life sciences,” ChemPhysChem4(1), 14–30 (2003).
[CrossRef] [PubMed]

Puppels, G. J.

P. J. Caspers, G. W. Lucassen, and G. J. Puppels, “Combined in vivo confocal Raman spectroscopy and confocal microscopy of human skin,” Biophys. J.85(1), 572–580 (2003).
[CrossRef] [PubMed]

A. Nijssen, T. C. Bakker Schut, F. Heule, P. J. Caspers, D. P. Hayes, M. H. A. Neumann, and G. J. Puppels, “Discriminating basal cell carcinoma from its surrounding tissue by Raman spectroscopy,” J. Invest. Dermatol.119(1), 64–69 (2002).
[CrossRef] [PubMed]

P. J. Caspers, G. W. Lucassen, E. A. Carter, H. A. Bruining, and G. J. Puppels, “In vivo confocal Raman microspectroscopy of the skin: noninvasive determination of molecular concentration profiles,” J. Invest. Dermatol.116(3), 434–442 (2001).
[CrossRef] [PubMed]

P. J. Caspers, G. W. Lucassen, R. Wolthuis, H. A. Bruining, and G. J. Puppels, “In vitro and in vivo Raman spectroscopy of human skin,” Biospectroscopy4(S5), S31–S39 (1998).
[CrossRef] [PubMed]

Rajadhyaksha, M.

M. Rajadhyaksha, G. Menaker, T. Flotte, P. J. Dwyer, and S. González, “Confocal examination of nonmelanoma cancers in thick skin excisions to potentially guide mohs micrographic surgery without frozen histopathology,” J. Invest. Dermatol.117(5), 1137–1143 (2001).
[CrossRef] [PubMed]

M. Rajadhyaksha, R. R. Anderson, and R. H. Webb, “Video-rate confocal scanning laser microscope for imaging human tissues in vivo,” Appl. Opt.38(10), 2105–2115 (1999).
[CrossRef] [PubMed]

M. Rajadhyaksha, S. González, J. M. Zavislan, R. R. Anderson, and R. H. Webb, “In vivo confocal scanning laser microscopy of human skin II: advances in instrumentation and comparison with histology,” J. Invest. Dermatol.113(3), 293–303 (1999).
[CrossRef] [PubMed]

Reußner, J.

Schmied, M.

Schmitt, M.

R. Petry, M. Schmitt, and J. Popp, “Raman spectroscopy--a prospective tool in the life sciences,” ChemPhysChem4(1), 14–30 (2003).
[CrossRef] [PubMed]

Schopf, J. W.

K. D. McKeegan, A. B. Kudryavtsev, and J. W. Schopf, “Raman and ion microscopic imagery of graphitic inclusions in apatite from older than 3830 Ma Akilia supracrustal rocks, west Greenland,” Geology35(7), 591–594 (2007).
[CrossRef]

Scott, R. S.

R. S. Scott, P. S. Ungar, T. S. Bergstrom, C. A. Brown, F. E. Grine, M. F. Teaford, and A. Walker, “Dental microwear texture analysis shows within-species diet variability in fossil hominins,” Nature436(7051), 693–695 (2005).
[CrossRef] [PubMed]

Speicher, M. R.

M. A. Pabst, I. Letofsky-Papst, M. Moser, K. Spindler, E. Bock, P. Wilhelm, M. D. Leopold Dorfer, J. B. Geigl, M. Auer, M. R. Speicher, and F. Hofer, “Different staining substances were used in decorative and therapeutic tattoos in a 1000-year-old Peruvian mummy,” J. Archaeol. Sci.37(12), 3256–3262 (2010).
[CrossRef]

Spindler, K.

M. A. Pabst, I. Letofsky-Papst, M. Moser, K. Spindler, E. Bock, P. Wilhelm, M. D. Leopold Dorfer, J. B. Geigl, M. Auer, M. R. Speicher, and F. Hofer, “Different staining substances were used in decorative and therapeutic tattoos in a 1000-year-old Peruvian mummy,” J. Archaeol. Sci.37(12), 3256–3262 (2010).
[CrossRef]

Teaford, M. F.

R. S. Scott, P. S. Ungar, T. S. Bergstrom, C. A. Brown, F. E. Grine, M. F. Teaford, and A. Walker, “Dental microwear texture analysis shows within-species diet variability in fossil hominins,” Nature436(7051), 693–695 (2005).
[CrossRef] [PubMed]

Ungar, P. S.

R. S. Scott, P. S. Ungar, T. S. Bergstrom, C. A. Brown, F. E. Grine, M. F. Teaford, and A. Walker, “Dental microwear texture analysis shows within-species diet variability in fossil hominins,” Nature436(7051), 693–695 (2005).
[CrossRef] [PubMed]

Walker, A.

R. S. Scott, P. S. Ungar, T. S. Bergstrom, C. A. Brown, F. E. Grine, M. F. Teaford, and A. Walker, “Dental microwear texture analysis shows within-species diet variability in fossil hominins,” Nature436(7051), 693–695 (2005).
[CrossRef] [PubMed]

Webb, R. H.

M. Rajadhyaksha, S. González, J. M. Zavislan, R. R. Anderson, and R. H. Webb, “In vivo confocal scanning laser microscopy of human skin II: advances in instrumentation and comparison with histology,” J. Invest. Dermatol.113(3), 293–303 (1999).
[CrossRef] [PubMed]

M. Rajadhyaksha, R. R. Anderson, and R. H. Webb, “Video-rate confocal scanning laser microscope for imaging human tissues in vivo,” Appl. Opt.38(10), 2105–2115 (1999).
[CrossRef] [PubMed]

Wilhelm, P.

M. A. Pabst, I. Letofsky-Papst, M. Moser, K. Spindler, E. Bock, P. Wilhelm, M. D. Leopold Dorfer, J. B. Geigl, M. Auer, M. R. Speicher, and F. Hofer, “Different staining substances were used in decorative and therapeutic tattoos in a 1000-year-old Peruvian mummy,” J. Archaeol. Sci.37(12), 3256–3262 (2010).
[CrossRef]

A. Gupper, P. Wilhelm, M. Schmied, S. G. Kazarian, K. L. A. Chan, and J. Reußner, “Combined application of imaging methods for the characterization of a polymer blend,” Appl. Spectrosc.56(12), 1515–1523 (2002).
[CrossRef]

Wolthuis, R.

P. J. Caspers, G. W. Lucassen, R. Wolthuis, H. A. Bruining, and G. J. Puppels, “In vitro and in vivo Raman spectroscopy of human skin,” Biospectroscopy4(S5), S31–S39 (1998).
[CrossRef] [PubMed]

Zavislan, J. M.

M. Rajadhyaksha, S. González, J. M. Zavislan, R. R. Anderson, and R. H. Webb, “In vivo confocal scanning laser microscopy of human skin II: advances in instrumentation and comparison with histology,” J. Invest. Dermatol.113(3), 293–303 (1999).
[CrossRef] [PubMed]

Ann. Anat. (1)

K.-J. Halbhuber and K. König, “Modern laser scanning microscopy in biology, biotechnology and medicine,” Ann. Anat.185(1), 1–20 (2003).
[CrossRef] [PubMed]

Appl. Opt. (1)

Appl. Spectrosc. (2)

Biophys. J. (1)

P. J. Caspers, G. W. Lucassen, and G. J. Puppels, “Combined in vivo confocal Raman spectroscopy and confocal microscopy of human skin,” Biophys. J.85(1), 572–580 (2003).
[CrossRef] [PubMed]

Biospectroscopy (1)

P. J. Caspers, G. W. Lucassen, R. Wolthuis, H. A. Bruining, and G. J. Puppels, “In vitro and in vivo Raman spectroscopy of human skin,” Biospectroscopy4(S5), S31–S39 (1998).
[CrossRef] [PubMed]

ChemPhysChem (1)

R. Petry, M. Schmitt, and J. Popp, “Raman spectroscopy--a prospective tool in the life sciences,” ChemPhysChem4(1), 14–30 (2003).
[CrossRef] [PubMed]

Geology (1)

K. D. McKeegan, A. B. Kudryavtsev, and J. W. Schopf, “Raman and ion microscopic imagery of graphitic inclusions in apatite from older than 3830 Ma Akilia supracrustal rocks, west Greenland,” Geology35(7), 591–594 (2007).
[CrossRef]

J. Archaeol. Sci. (1)

M. A. Pabst, I. Letofsky-Papst, M. Moser, K. Spindler, E. Bock, P. Wilhelm, M. D. Leopold Dorfer, J. B. Geigl, M. Auer, M. R. Speicher, and F. Hofer, “Different staining substances were used in decorative and therapeutic tattoos in a 1000-year-old Peruvian mummy,” J. Archaeol. Sci.37(12), 3256–3262 (2010).
[CrossRef]

J. Invest. Dermatol. (4)

M. Rajadhyaksha, S. González, J. M. Zavislan, R. R. Anderson, and R. H. Webb, “In vivo confocal scanning laser microscopy of human skin II: advances in instrumentation and comparison with histology,” J. Invest. Dermatol.113(3), 293–303 (1999).
[CrossRef] [PubMed]

M. Rajadhyaksha, G. Menaker, T. Flotte, P. J. Dwyer, and S. González, “Confocal examination of nonmelanoma cancers in thick skin excisions to potentially guide mohs micrographic surgery without frozen histopathology,” J. Invest. Dermatol.117(5), 1137–1143 (2001).
[CrossRef] [PubMed]

P. J. Caspers, G. W. Lucassen, E. A. Carter, H. A. Bruining, and G. J. Puppels, “In vivo confocal Raman microspectroscopy of the skin: noninvasive determination of molecular concentration profiles,” J. Invest. Dermatol.116(3), 434–442 (2001).
[CrossRef] [PubMed]

A. Nijssen, T. C. Bakker Schut, F. Heule, P. J. Caspers, D. P. Hayes, M. H. A. Neumann, and G. J. Puppels, “Discriminating basal cell carcinoma from its surrounding tissue by Raman spectroscopy,” J. Invest. Dermatol.119(1), 64–69 (2002).
[CrossRef] [PubMed]

J. Microsc. (1)

D. B. Hovis and A. H. Heuer, “The use of laser scanning confocal microscopy (LSCM) in materials science,” J. Microsc.240(3), 173–180 (2010).
[CrossRef] [PubMed]

J. Phys. Chem. (1)

A. Palm, “Raman spectrum of polystyrene,” J. Phys. Chem.55(8), 1320–1324 (1951).
[CrossRef]

Lasers Surg. Med. (1)

C. A. Lieber, S. K. Majumder, D. L. Ellis, D. D. Billheimer, and A. Mahadevan-Jansen, “In vivo nonmelanoma skin cancer diagnosis using Raman microspectroscopy,” Lasers Surg. Med.40(7), 461–467 (2008).
[CrossRef] [PubMed]

Nature (1)

R. S. Scott, P. S. Ungar, T. S. Bergstrom, C. A. Brown, F. E. Grine, M. F. Teaford, and A. Walker, “Dental microwear texture analysis shows within-species diet variability in fossil hominins,” Nature436(7051), 693–695 (2005).
[CrossRef] [PubMed]

Opt. Express (1)

Phys. Chem. Earth, Part A Solid Earth Geod. (1)

J. T. Fredrich, “3D imaging of porous media using laser scanning confocal microscopy with application to microscale transport processes,” Phys. Chem. Earth, Part A Solid Earth Geod.24(7), 551–561 (1999).
[CrossRef]

Sci. Justice (1)

M. Claybourn and M. Ansell, “Using Raman spectroscopy to solve crime: inks, questioned documents and fraud,” Sci. Justice40(4), 261–271 (2000).
[CrossRef] [PubMed]

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Figures (8)

Fig. 1
Fig. 1

The CM-CRS system. (a) Schematic diagram of the microscope. (b) Photograph of the hand-piece. The overall size of the hand-piece is 7” (L) x 4.5” (W) x 2.25” (H). (c) Photograph of the CM-CRS system. The controls for the MEMS scanner, picomotor, camera, avalanche-photodiode (APD), and electronic gain box for CM, and laser system are within the hardware enclosure, while the laser controller and Raman spectrometer are on the lower shelf of the cart.

Fig. 2
Fig. 2

Effect of CRS detection fiber size on SNR and axial resolution. Measurements of SNR and axial resolution were made using a polished Si substrate, and evaluating the intensity of the 520 cm−1 peak. The plot of SNR vs. detection fiber size (a) shows a sharp improvement when going from an 8 μm detection fiber to a 50 μm fiber. The plot of axial resolution vs. detection fiber size (b) shows the degradation in axial resolution with increasing fiber size. Both (a) and (b) demonstrate the performance improvements offered by the BK7 hemisphere. All measurements were made using the 50x objective lens.

Fig. 3
Fig. 3

Representative axial response functions. Measured LSCM axial response function (a) and the measured CRS axial response (black dots) and Gaussian fit (blue line) (b) for the 100x objective with aperture front piece. CRS was measured using the 50 µm core fiber.

Fig. 4
Fig. 4

Representative edge response functions for the 50x objective with the hemisphere front-piece. Measured data (black line) and fit for the CM channel (a). Measured data and fit for the CRS channel (b). Data is fit to the error function.

Fig. 5
Fig. 5

Co-registration of Raman sampling location and confocal imaging was done using 5 µm polystyrene microspheres in gelatin solution. Crosshairs mark the Raman sampling location. On (a) and off (b) the microsphere shows distinction in Raman spectra (c), where the spectra corresponding to (a) is shown as the solid black line, and the spectra corresponding to (b) is shown as the dashed red line. The spectrum acquired on the bead clearly shows the characteristic spectral features of polystyrene, including the prominent peaks at 628, 1011, 1034, and 1613 cm−1 [14]. Image dimensions in (a) and (b) is 520 x 396 µm.

Fig. 6
Fig. 6

Images and spectra acquired from the fingertip using the 100x objective and hemisphere (a,c) and 50x objective and hemisphere (b,d). Cell membranes and nuclei are easily distinguished in (a), but nuclear detail is less evident when using the lower magnification objective (b). Image size is 260 x 198 µm and 520 x 396 µm for (a) and (b) respectively. Corresponding Raman spectra demonstrate the fact that the 50x objective allows for greater detected Raman signal intensity.

Fig. 7
Fig. 7

In vivo CM image and CRS spectra of a sebaceous gland and the adjacent skin taken with 50x objective. (a) CM image shows the presence of the large circular sebaceous gland (SG) in the center of the image, and indicates the positions from which spectra were acquired (+)(Red-Dermis, Purple-Sebaceous). (b) Mean spectra acquired from within the sebaceous gland (purple line) and from the adjacent dermis (red line). Spectral features characteristic of the fatty acids within sebum are seen at 884,, 1062, 1157 cm−1, and 1297 cm−1 (arrows), while typical tissue protein features (*) such as those at 936 and 1339 cm−1 are more prevalent in the adjacent skin.

Fig. 8
Fig. 8

CM and CRS from normal stratum corneum (SC) and stratum granulosum (SG) on the cheek of a Caucasian male. SC image (a) shows the bright polygonal corneocytes distinguished by the dark regions separating them. The SG image (b), acquired just 20 μm below the (a), shows distinctly different round epidermal cells with the hyporeflective cell soma and bright membranes. The corresponding CRS spectra (c), show differences in the spectral lineshape around 1300 cm−1 and 1600 cm−1 (arrows), and in the relative intensities of the CHx peak at 1440 cm−1 and the amide I band at 1650 cm−1 (*). Spectral features characteristic of the SC, including the sharp lipid peak at 1297 cm−1, while the SG is characterized by increased spectral intensity at the amide I shoulder at 1600 cm−1, and the increased relative intensity of the amide I peak.

Tables (2)

Tables Icon

Table 1 Axial resolutions of the confocal channel and Raman channel (50µm core fiber)

Tables Icon

Table 2 Transverse resolution of the confocal and Raman channel (50µm core fiber)

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