Abstract

Due to the limited number of suitable intravital microscopy techniques, relatively little is known about the opto-thermal (endo)vascular responses to selective photothermolysis, used as a default treatment modality for superficial vascular anomalies such as port wine stains, telangiectasias, and hemangiomas. In this preliminary study we present a novel microscopy technique for studying (endo)vascular laser-tissue interactions in vivo, in which conventional orthogonal polarized spectral (OPS) imaging is combined with darkfield (DF) illumination. DFOPS imaging of rat mesenteric vasculature irradiated at increasing powers revealed the following (tissular) responses: formation of translucent aggregates, retrograde flow, gradual and immediate hemostasis, reinstatement of flow, vessel disappearance, and perivascular collagen damage. DFOPS imaging therefore constitutes a useful tool for examining (endo)vascular events following selective photothermolysis.

© 2005 Optical Society of America

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References

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  1. D. W. Slaaf, G. J. Tangelder, R. S. Reneman, K. Jager, and A. Bollinger, �??A versatile incident illuminator for intravital microscopy,�?? Int. J. Microcirc. Clin. Exp. 6, 391-397 (1987).
    [PubMed]
  2. K. R. Mathura, G. J. Bouma, and C. Ince, �??Abnormal microcirculation in brain tumours during surgery,�?? Lancet 358, 1698-1699 (2001).
    [CrossRef] [PubMed]
  3. F. A. Pennings, G. J. Bouma, and C. Ince, �??Direct observation of the human cerebral microcirculation during aneurysm surgery reveals increased arteriolar contractility,�?? Stroke 35, 1284-1288 (2004).
    [CrossRef] [PubMed]
  4. P. E. Spronk, C. Ince, M. J. Gardien, K. R. Mathura, H. M. Oudemans-van Straaten, and D. F. Zandstra, �??Nitroglycerin in septic shock after intravascular volume resuscitation,�?? Lancet 360, 1395-1396 (2002).
    [CrossRef] [PubMed]
  5. K. R. Mathura, K. C. Vollebregt, K. Boer, J. C. De Graaff, D. T. Ubbink, and C. Ince, �??Comparison of OPS imaging and conventional capillary microscopy to study the human microcirculation,�?? J. Appl. Physiol. 91, 74-78 (2001).
    [PubMed]
  6. W. Groner, J. W. Winkelman, A. G. Harris, C. Ince, G. J. Bouma, K. Messmer, and R. G. Nadeau, �??Orthogonal polarization spectral imaging: a new method for study of the microcirculation,�?? Nat. Med. 5, 1209-1212 (1999).
    [CrossRef] [PubMed]
  7. S. Langer, P. Biberthaler, A. G. Harris, H. U. Steinau, and K. Messmer, �??In vivo monitoring of microvessels in skin flaps: introduction of a novel technique,�?? Microsurgery 21, 317-324 (2001).
    [CrossRef]
  8. A. G. Harris, I. Sinitsina, and K. Messner, �??Validation of OPS imaging for microvascular measurements during isovolumic hemodilution and low hematocrits,�?? Am. J. Physiol. Heart Circ. Physiol. 282, H1502-H1509 (2002).
    [PubMed]
  9. S. Pahernik, A. G. Harris, M. Schmitt-Sody, S. Krasnici, A. E. Goetz, M. Dellian, and K. Messmer, �??Orthogonal polarisation spectral imaging as a new tool for the assessment of antivascular tumour treatment in vivo: a validation study,�?? Br. J. Cancer 86, 1622-1627 (2002).
    [CrossRef] [PubMed]
  10. K. C. Vollebregt, K. Boer, K. R. Mathura, J. C. de Graaff, D. T. Ubbink, and C. Ince, �??Impaired vascular function in women with pre-eclampsia observed with orthogonal polarisation spectral imaging,�?? BJOG. 108, 1148-1153 (2001).
    [CrossRef]
  11. H. Sherman, S. Klausner, and W. A. Cook, �??Incident dark-field illumination: a new method for microcirculatory study,�?? Angiology 22, 295-303 (1971).
    [CrossRef] [PubMed]
  12. R. R. Anderson and J. A. Parrish, �??Selective photothermolysis: precise microsurgery by selective absorption of pulsed radiation,�?? Science 220, 524-527 (1983).
    [CrossRef] [PubMed]
  13. M. M. Hamilton, �??Laser treatment of pigmented and vascular lesions in the office,�?? Facial Plast. Surg. 20, 63-69 (2004).
    [CrossRef] [PubMed]
  14. S. Falati, P. Gross, G. Merrill-Skoloff, B. C. Furie, and B. Furie, �??Real-time in vivo imaging of platelets, tissue factor and fibrin during arterial thrombus formation in the mouse,�?? Nat. Med. 8, 1175-1180 (2002).
    [CrossRef] [PubMed]
  15. E. D. Rosen, S. Raymond, A. Zollman, F. Noria, M. Sandoval-Cooper, A. Shulman, J. L. Merz, and F. J. Castellino, �??Laser-induced noninvasive vascular injury models in mice generate platelet- and coagulation-dependent thrombi,�?? Am. J. Pathol. 158, 1613-1622 (2001).
    [CrossRef] [PubMed]
  16. W. I. Rosenblum, G. H. Nelson, B. Wormley, P. Werner, J. Wang, and C. C. Shih, �??Role of platelet-endothelial cell adhesion molecule (PECAM) in platelet adhesion/aggregation over injured but not denuded endothelium in vivo and ex vivo,�?? Stroke 27, 709-711 (1996).
    [CrossRef] [PubMed]
  17. S. Mordon, S. Begu, B. Buys, C. Tourne-Peteilh, and J. M. Devoiselle, �??Study of platelet behavior in vivo after endothelial stimulation with laser irradiation using fluorescence intravital videomicroscopy and PEGylated liposome staining,�?? Microvasc. Res. 64, 316-325 (2002).
    [CrossRef] [PubMed]
  18. W. Verkruysse, J. F. Beek, E. van Bavel, M. J. C. van Gemert, and J. Spaan, �??Laser pulse impact on rat mesenteric blood vessels in relation to laser treatment of port wine stain,�?? Lasers Surg. Med. 28, 461-468 (2001).
    [CrossRef] [PubMed]
  19. O. T. Tan, D. Whitaker, J. M. Garden, and G. Murphy, �??Pulsed dye laser (577 nm) treatment of portwine stains: ultrastructural evidence of neovascularization and mast cell degranulation in healed lesions,�?? J. Invest. Dermatol. 90, 395-398 (1988).
    [CrossRef] [PubMed]
  20. O. T. Tan, J. G. Morelli, D. Whitaker, J. Boll, and G. Murphy, �??Ultrastructural changes in red blood cells following pulsed irradiation in vitro,�?? J. Invest. Dermatol. 92, 100-104 (1989).
    [CrossRef] [PubMed]
  21. J. F. Black and J. K. Barton, �??Chemical and structural changes in blood undergoing laser photocoagulation,�?? Photochem. Photobiol. 80, 89-97 (2004).
    [CrossRef] [PubMed]
  22. K. Suthamjariya, W. A. Farinelli, W. Koh, and R. R. Anderson, �??Mechanisms of microvascular response to laser pulses,�?? J. Invest. Dermatol. 122, 518-525 (2004).
    [CrossRef] [PubMed]
  23. J. Lindert, J. Werner, M. Redlin, H. Kuppe, H. Habazettl, and A. R. Pries, �??OPS imaging of human microcirculation: a short technical report,�?? J. Vasc. Res. 39, 368-372 (2002).
    [CrossRef] [PubMed]
  24. J. K. Barton, D. Popok, and J. F. Black, �??Thermal analysis of blood undergoing laser photocoagulation,�?? IEEE J. Sel. Topics Quant. Electron. 7, 936-943 (2001).
    [CrossRef]
  25. J. K. Barton, G. Frangineas, H. Pummer, and J. F. Black, �??Cooperative phenomena in two-pulse, two-color laser photocoagulation of cutaneous blood vessels,�?? Photochem. Photobiol. 73, 642-650 (2001).
    [CrossRef] [PubMed]
  26. K. Takeda, A. Wada, K. Yamamoto, Y. Moriyama, and K. Aoki, �??Conformational change of bovine serum albumin by heat treatment,�?? J. Protein Chem. 8, 653-659 (1989).
    [CrossRef] [PubMed]
  27. A. R. Tall, D. M. Small, G. Shipley, and R. S. Lees, �??Apoprotein stability and lipid-lipid protein interactions in human plasma high density lipoproteins,�?? Proc. Nat. Acad. Sci. 72, 4940-4942 (1975).
    [CrossRef] [PubMed]
  28. M. Heger, J. F. Beek, N. I. Moldovan, C. M. A. M. van der Horst, and M. J. C. van Gemert, �??Towards optimization of selective photothermolysis: prothrombotic pharmaceutical agents as potential adjuvants in laser treatment of port wine stains. A theoretical study,�?? Thromb. Haemost. 92, 242-257 (2005).
  29. S. P. Jackson, W. S. Nesbitt, and S. Kulkarni, �??Signaling events underlying thrombus formation,�?? J. Thromb. Haemost. 1, 1602-1612 (2003).
    [CrossRef] [PubMed]
  30. O. Genevois, M. Paques, M. Simonutti, R. Sercombe, J. Seylaz, A. Gaudric, J. P. Brouland, J. Sahel, and E. Vicaut, �??Microvascular remodeling after occlusion-recanalization of a branch retinal vein in rats,�?? Invest. Ophthalmol. Vis. Sci. 45, 594-600 (2004).
    [CrossRef] [PubMed]
  31. S. Thomsen, �??Pathological analysis of photothermal and photomechanical effects of laser-tissue interactions,�?? Photochem. Photobiol. 53, 825-835 (2001).
  32. R. Dong, X. Yan, X. Pang, and S. Liu, �??Temperature-dependent Raman spectra of collagen and DNA,�?? Spectrochim. Acta A 60, 557-561 (2004).
    [CrossRef]
  33. T. V. Belopolskaya, G. I. Tsereteli, N. A. Grunina, and O. L. Vaveliouk, �??DSC study of the postdenatured structures in biopolymer-water systems,�?? J. Therm. Anal. Calorim. 62, 75-88 (2000).
    [CrossRef]
  34. S. S. Chen, N. T. Wright, and J. D. Humphrey, �??Heat-induced changes in the mechanics of a collagenous tissue: isothermal free shrinkage,�?? J. Biomech. Eng. 119, 372-378 (1997).
    [CrossRef]

. Invest. Dermatol.

O. T. Tan, J. G. Morelli, D. Whitaker, J. Boll, and G. Murphy, �??Ultrastructural changes in red blood cells following pulsed irradiation in vitro,�?? J. Invest. Dermatol. 92, 100-104 (1989).
[CrossRef] [PubMed]

Am. J. Pathol.

E. D. Rosen, S. Raymond, A. Zollman, F. Noria, M. Sandoval-Cooper, A. Shulman, J. L. Merz, and F. J. Castellino, �??Laser-induced noninvasive vascular injury models in mice generate platelet- and coagulation-dependent thrombi,�?? Am. J. Pathol. 158, 1613-1622 (2001).
[CrossRef] [PubMed]

Am. J. Physiol. Heart Circ. Physiol.

A. G. Harris, I. Sinitsina, and K. Messner, �??Validation of OPS imaging for microvascular measurements during isovolumic hemodilution and low hematocrits,�?? Am. J. Physiol. Heart Circ. Physiol. 282, H1502-H1509 (2002).
[PubMed]

Angiology

H. Sherman, S. Klausner, and W. A. Cook, �??Incident dark-field illumination: a new method for microcirculatory study,�?? Angiology 22, 295-303 (1971).
[CrossRef] [PubMed]

BJOG.

K. C. Vollebregt, K. Boer, K. R. Mathura, J. C. de Graaff, D. T. Ubbink, and C. Ince, �??Impaired vascular function in women with pre-eclampsia observed with orthogonal polarisation spectral imaging,�?? BJOG. 108, 1148-1153 (2001).
[CrossRef]

Br. J. Cancer

S. Pahernik, A. G. Harris, M. Schmitt-Sody, S. Krasnici, A. E. Goetz, M. Dellian, and K. Messmer, �??Orthogonal polarisation spectral imaging as a new tool for the assessment of antivascular tumour treatment in vivo: a validation study,�?? Br. J. Cancer 86, 1622-1627 (2002).
[CrossRef] [PubMed]

Facial Plast. Surg.

M. M. Hamilton, �??Laser treatment of pigmented and vascular lesions in the office,�?? Facial Plast. Surg. 20, 63-69 (2004).
[CrossRef] [PubMed]

IEEE J. Sel. Topics Quant. Electron.

J. K. Barton, D. Popok, and J. F. Black, �??Thermal analysis of blood undergoing laser photocoagulation,�?? IEEE J. Sel. Topics Quant. Electron. 7, 936-943 (2001).
[CrossRef]

Int. J. Microcirc. Clin. Exp.

D. W. Slaaf, G. J. Tangelder, R. S. Reneman, K. Jager, and A. Bollinger, �??A versatile incident illuminator for intravital microscopy,�?? Int. J. Microcirc. Clin. Exp. 6, 391-397 (1987).
[PubMed]

Invest. Ophthalmol. Vis. Sci.

O. Genevois, M. Paques, M. Simonutti, R. Sercombe, J. Seylaz, A. Gaudric, J. P. Brouland, J. Sahel, and E. Vicaut, �??Microvascular remodeling after occlusion-recanalization of a branch retinal vein in rats,�?? Invest. Ophthalmol. Vis. Sci. 45, 594-600 (2004).
[CrossRef] [PubMed]

J. Appl. Physiol.

K. R. Mathura, K. C. Vollebregt, K. Boer, J. C. De Graaff, D. T. Ubbink, and C. Ince, �??Comparison of OPS imaging and conventional capillary microscopy to study the human microcirculation,�?? J. Appl. Physiol. 91, 74-78 (2001).
[PubMed]

J. Biomech. Eng.

S. S. Chen, N. T. Wright, and J. D. Humphrey, �??Heat-induced changes in the mechanics of a collagenous tissue: isothermal free shrinkage,�?? J. Biomech. Eng. 119, 372-378 (1997).
[CrossRef]

J. Invest. Dermatol.

K. Suthamjariya, W. A. Farinelli, W. Koh, and R. R. Anderson, �??Mechanisms of microvascular response to laser pulses,�?? J. Invest. Dermatol. 122, 518-525 (2004).
[CrossRef] [PubMed]

O. T. Tan, D. Whitaker, J. M. Garden, and G. Murphy, �??Pulsed dye laser (577 nm) treatment of portwine stains: ultrastructural evidence of neovascularization and mast cell degranulation in healed lesions,�?? J. Invest. Dermatol. 90, 395-398 (1988).
[CrossRef] [PubMed]

J. Protein Chem.

K. Takeda, A. Wada, K. Yamamoto, Y. Moriyama, and K. Aoki, �??Conformational change of bovine serum albumin by heat treatment,�?? J. Protein Chem. 8, 653-659 (1989).
[CrossRef] [PubMed]

J. Therm. Anal. Calorim.

T. V. Belopolskaya, G. I. Tsereteli, N. A. Grunina, and O. L. Vaveliouk, �??DSC study of the postdenatured structures in biopolymer-water systems,�?? J. Therm. Anal. Calorim. 62, 75-88 (2000).
[CrossRef]

J. Thromb. Haemost.

S. P. Jackson, W. S. Nesbitt, and S. Kulkarni, �??Signaling events underlying thrombus formation,�?? J. Thromb. Haemost. 1, 1602-1612 (2003).
[CrossRef] [PubMed]

J. Vasc. Res.

J. Lindert, J. Werner, M. Redlin, H. Kuppe, H. Habazettl, and A. R. Pries, �??OPS imaging of human microcirculation: a short technical report,�?? J. Vasc. Res. 39, 368-372 (2002).
[CrossRef] [PubMed]

Lancet

K. R. Mathura, G. J. Bouma, and C. Ince, �??Abnormal microcirculation in brain tumours during surgery,�?? Lancet 358, 1698-1699 (2001).
[CrossRef] [PubMed]

P. E. Spronk, C. Ince, M. J. Gardien, K. R. Mathura, H. M. Oudemans-van Straaten, and D. F. Zandstra, �??Nitroglycerin in septic shock after intravascular volume resuscitation,�?? Lancet 360, 1395-1396 (2002).
[CrossRef] [PubMed]

Lasers Surg. Med.

W. Verkruysse, J. F. Beek, E. van Bavel, M. J. C. van Gemert, and J. Spaan, �??Laser pulse impact on rat mesenteric blood vessels in relation to laser treatment of port wine stain,�?? Lasers Surg. Med. 28, 461-468 (2001).
[CrossRef] [PubMed]

Microsurgery

S. Langer, P. Biberthaler, A. G. Harris, H. U. Steinau, and K. Messmer, �??In vivo monitoring of microvessels in skin flaps: introduction of a novel technique,�?? Microsurgery 21, 317-324 (2001).
[CrossRef]

Microvasc. Res.

S. Mordon, S. Begu, B. Buys, C. Tourne-Peteilh, and J. M. Devoiselle, �??Study of platelet behavior in vivo after endothelial stimulation with laser irradiation using fluorescence intravital videomicroscopy and PEGylated liposome staining,�?? Microvasc. Res. 64, 316-325 (2002).
[CrossRef] [PubMed]

Nat. Med.

S. Falati, P. Gross, G. Merrill-Skoloff, B. C. Furie, and B. Furie, �??Real-time in vivo imaging of platelets, tissue factor and fibrin during arterial thrombus formation in the mouse,�?? Nat. Med. 8, 1175-1180 (2002).
[CrossRef] [PubMed]

W. Groner, J. W. Winkelman, A. G. Harris, C. Ince, G. J. Bouma, K. Messmer, and R. G. Nadeau, �??Orthogonal polarization spectral imaging: a new method for study of the microcirculation,�?? Nat. Med. 5, 1209-1212 (1999).
[CrossRef] [PubMed]

Photochem. Photobiol.

J. F. Black and J. K. Barton, �??Chemical and structural changes in blood undergoing laser photocoagulation,�?? Photochem. Photobiol. 80, 89-97 (2004).
[CrossRef] [PubMed]

J. K. Barton, G. Frangineas, H. Pummer, and J. F. Black, �??Cooperative phenomena in two-pulse, two-color laser photocoagulation of cutaneous blood vessels,�?? Photochem. Photobiol. 73, 642-650 (2001).
[CrossRef] [PubMed]

S. Thomsen, �??Pathological analysis of photothermal and photomechanical effects of laser-tissue interactions,�?? Photochem. Photobiol. 53, 825-835 (2001).

Proc. Nat. Acad. Sci.

A. R. Tall, D. M. Small, G. Shipley, and R. S. Lees, �??Apoprotein stability and lipid-lipid protein interactions in human plasma high density lipoproteins,�?? Proc. Nat. Acad. Sci. 72, 4940-4942 (1975).
[CrossRef] [PubMed]

Science

R. R. Anderson and J. A. Parrish, �??Selective photothermolysis: precise microsurgery by selective absorption of pulsed radiation,�?? Science 220, 524-527 (1983).
[CrossRef] [PubMed]

Spectrochim. Acta A

R. Dong, X. Yan, X. Pang, and S. Liu, �??Temperature-dependent Raman spectra of collagen and DNA,�?? Spectrochim. Acta A 60, 557-561 (2004).
[CrossRef]

Stroke

W. I. Rosenblum, G. H. Nelson, B. Wormley, P. Werner, J. Wang, and C. C. Shih, �??Role of platelet-endothelial cell adhesion molecule (PECAM) in platelet adhesion/aggregation over injured but not denuded endothelium in vivo and ex vivo,�?? Stroke 27, 709-711 (1996).
[CrossRef] [PubMed]

F. A. Pennings, G. J. Bouma, and C. Ince, �??Direct observation of the human cerebral microcirculation during aneurysm surgery reveals increased arteriolar contractility,�?? Stroke 35, 1284-1288 (2004).
[CrossRef] [PubMed]

Thromb. Haemost.

M. Heger, J. F. Beek, N. I. Moldovan, C. M. A. M. van der Horst, and M. J. C. van Gemert, �??Towards optimization of selective photothermolysis: prothrombotic pharmaceutical agents as potential adjuvants in laser treatment of port wine stains. A theoretical study,�?? Thromb. Haemost. 92, 242-257 (2005).

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Figures (7)

Fig. 1.
Fig. 1.

Diagram of the experimental setup. The Cytoscan probe (insert top left) is positioned onto an exteriorized rat large intestine. Source light incident on tissue is passed through a polarizer and a green spectral filter (548 nm). Remitted light is guided through an orthogonally oriented polarizer (analyzer) to a CCD camera by a beam splitter, and the images are video-recorded. A frequency-doubled Nd:YAG laser (532 nm) is used to inflict photothermal damage during DFOPS imaging.

Fig. 2.
Fig. 2.

OPS imaging of superficially located microcirculation in the large intestine of a rat. (a) (2.2 MB) Movie: Hierarchical organization of mesenteric vasculature (14.6 MB version). Arrows indicate direction of flow, arrowheads indicate vessel segment with clearly visible leukocyte rolling. (b) (1.4 MB) Movie: Leukocyte rolling and red blood cell-devoid strips of plasma (14.5 MB version). Arrows indicate translucent strips of plasma, arrowheads point to sites of clearly visible leukocyte rolling. Scale bar=100 µm. The original movies in DV.avi format can be downloaded from http://www.opsimaging.net.

Fig. 3.
Fig. 3.

(a) (1.5 MB) Movie: Formation of translucent matter in laser-irradiated microcirculation (14.0 MB version). The laser was set to a power of 1000 mW, 500 ms pulse duration, and a spot size of 0.35±0.05 mm2. Laser irradiation (white screen in movie) induced the formation of translucent matter (encircled) in the vasculature imaged in Fig 2(a). All yellow crosses designate hemostatic vessels after lasing, white arrows indicate direction of flow before irradiation, and green arrows signify retrograde flow after irradiation. (b) (2.5 MB) Movie: Vasculature in (a), two minutes after laser irradiation (9.3 MB version). The open arrowhead marks the encircled area in (a), the green arrow indicates main flow path of blood, all yellow crosses signify hemostatic vessels, black arrowheads designate translucent, endoluminal obstructions, red arrowhead designates detaching translucent matter, and the red circles demarcate translucent vessel segments. (c) Encircled area in (a) enlarged. Arrowheads point to translucent material attached to the vessel wall. (d) (2.4 MB) Movie: Fig. 3(a), inverted contrast (14.4 MB version). (e) (2.3 MB) Movie: Fig. 3(b), inverted contrast (14.6 MB version). Scale bar=100 µm. The original movies in DV.avi format can be downloaded from http://www.opsimaging.net.

Fig. 4.
Fig. 4.

(1.3 MB) Movie: Vessel disappearance following laser irradiation (14.2 MB version). The laser was set to a power of 1500 mW and 500 ms pulse duration with a spot size of 0.35±0.05 mm2. (a) shows a video frame right before laser irradiation (white screen in movie), and (b) displays a video frame immediately after the laser pulse. Small arrowheads in (a) indicate affected vessels in (b), large arrowheads in (b) designate unaffected vessel, and circle marks intact loop vessel. Scale bar=100 µm. The original movie in DV.avi format can be downloaded from http://www.opsimaging.net.

Fig. 5.
Fig. 5.

(1.5 MB) Movie: Acute hemorrhage in partially affected vessel 2.5 minutes after laser irradiation at 1500 mW, 500 ms pulse duration, and a 0.35±0.05 mm2 spot size (10.0 MB version). Hemorrhage (encircled) occurs from the large vessel depicted in Fig. 4 without exogenous stimuli. Scale bar=100 µm. The original movie in DV.avi format can be downloaded from http://www.opsimaging.net.

Fig. 6.
Fig. 6.

(a) (1.3 MB) Movie: Disappearance of partially affected vessels following a second laser pulse (9.7 MB version). Vessels from Figs. 4 and 5 were irradiated again 3:45 minutes after the first laser pulse. The laser was set to maximum power (2000 mW) at 500 ms pulse duration and a spot size of 0.35±0.05 mm2. (a) shows a video frame right before laser irradiation (white screen in movie), and (b) displays a video frame immediately after the laser pulse. The demarcated higher order damage region (Fig. 6(b), dotted line) corresponds to the center of the beam profile. Vessel marked by small arrowheads in (a) represents the similarly marked vessel in Fig. 4(a). In (b), black arrowhead indicates hemorrhage, red arrowhead designates translucent material, and green arrows signify retrograde flow. (c) (2.4 MB) Movie: Fig. 6(a), inverted contrast (14.6 MB version). (c) shows an inverted contrast video frame right before laser irradiation (black screen in movie), and (d) displays an inverted contrast video frame immediately after the laser pulse. Scale bar=100 µm. The original movies in DV.avi format can be downloaded from http://www.opsimaging.net.

Fig. 7.
Fig. 7.

(a) Light microscopic image of a laser-induced coagulum in vitro (532 nm, 500 ms, 1000 mW). The red column represents the blood-filled cuvette, the coagulum appears black. Original magnification x4. (b) Coagulum in (a) imaged with DFOPS. Original magnification x10. (c) Light microscopic image of a laser-induced coagulum in vitro (532 nm, 500 ms, 1500 mW). The red column represents the blood-filled cuvette, the coagulum appears black. Original magnification x4. (d) Coagulum in (c) imaged with DFOPS. Original magnification x10.

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