Effects of Different Wavelengths of Low Level Laser Irradiation on Murine Immunological Activity and Intracellular Ca<Superscript>2+</Superscript> in Human Lymphocytes and Cultured Cortical Neurogliocytes

. The purpose of this study was to determine the wavelength-response effects of low level laser irradiation (LLLI) on immunocompetence of mice in vivo and intracellular free calcium ([Ca²⁺]_i) in human lymphocytes and cultured cortical neurogliocytes (CCN) in vitro. Mice were first immune compromised by cyclophosphamide (CTX) injection, and the immunological activities including the production of interleukin 2 (IL-2), the murine mixed lymphocyte reaction (MLR), the mitogenic response of murine thymocytes (MRT), the proliferation of murine bone marrow cells (BMC) and the natural killer (NK) cells activity, were investigated after intravascular low level laser irradiation (ILLLI) (1 mW, 1.1×10⁴ J/cm²) at wavelengths of 532 nm, 632.8 nm, 650 nm and 1520 nm, respectively. In addition, using Ca²⁺ sensitive indicator Fura-2 AM with the Spex AR-CM-MIC cation measurement system ([Ca²⁺]_i) in single human lymphocytes and CCN were measured after LLLI (7.5 J/cm²) at wavelengths of 532 nm, 632.8 nm, 650 nm, 810 nm and 1300 nm, respectively. Results showed that the ILLLI at wavelengths of 532 nm, 632.8 nm and 650 nm, produced a significant increase in the proliferation of BMC and the NK activity. The production of IL-2 was greatly promoted after irradiation at 632.8 nm and 650 nm. After irradiation at 532 nm and 650 nm, the murine MLR was evidently enhanced, and MRT was dramatically increased only after irradiation at 632.8 nm. In contrast, no significant effects were found on the above mentioned indexes by irradiation at 1520 nm in comparison to the control. In addition, [Ca²⁺]_i in single human lymphocytes and CCN were increased after LLLI at wavelengths of 532 nm, 632.8 nm and 650 nm, respectively, whereas they were not significantly affected by the wavelengths of 810 nm and 1300 nm. Our results indicated that LLLI could induce significant and different effects on the immunological activities of the mice and cause an increase in [Ca²⁺]_i in single human lymphocytes and CCN. Furthermore, these effects are dependent on the wavelengths, for example, more positive effects produced by the wavelengths of 532 nm, 632.8 nm and 652 nm than those produced by the wavelengths of 810 nm, 1300 nm and 1520 nm. Paper received 18 October 1999; accepted after revision 24 February 2000.     

Scanning Force Microscopy of the Fine Structure of Cartilage Irradiated with a CO2 Laser

. Alterations of the cartilage matrix structure under non-destructive laser irradiation have been investigated by scanning force microscopy. Porcine nasal septum cartilage was irradiated with a CO₂ laser with a power density of 50 W/cm² under two different time regimes: for 3 s and for 30 s. Short-time irradiation had little effect on the structure of the cartilage matrix. In comparison with non-irradiated cartilage, small channels of 100–400 nm in cross-section appeared. This observation gives evidence that the underlying mechanism of laser-induced stress relaxation of cartilage is based on short-time depolymerisation and subsequent re-formation of proteoglycan units. The 30 s laser treatment results in melting and denaturation of the matrix. For the first time, small crystals, 100–800 nm, were found on cut sections of the laser treated cartilage. The crystals mainly consist of resolvable sodium carbonate. Thus, they cannot be responsible for the formation of a stable cartilage configuration after laser treatment. Paper received 24 February 1998; accepted after revision 22 June 1999.     

Changes in Local Hepatic Blood Perfusion During Interstitial Laser-Induced Thermotherapy of Normal Rat Liver Measured by Interstitial Laser Doppler Flowmetry

Interstitial laser Doppler flowmetry was used to measure the effect of interstitial laser-induced thermotherapy on local blood perfusion in normal rat liver in the peripheral treatment region elevated to hyperthermic temperatures. The Nd:YAG laser emitting at 1064 nm was utilised as heat generation source. The plane-cut tip of an optical fibre was placed in the middle of the exteriorised left liver lobe. Blood perfusion and temperature were measured in the liver parenchyma 4 mm from the laser fibre. The temperature at the location of the liver temperature sensor was maintained at 41 or 44°C during 30 min by regulating the power of the heating laser. The laser Doppler signal was recorded during and after heat treatment, for a total time of 60 min. At 41°C, a significant increase in perfusion up to 1.3 times the initial value was observed 2–16 min after start of treatment. At 44°C, perfusion decreased continuously during and after treatment, and was significantly different from control 40 min after start of treatment. The results may be valuable in assessing the thermal response of tissues surrounding the target in interstitial laser-induced thermotherapy of liver tumours during conditions of normal blood flow. Paper received 28 September 1998; accepted after revision 24 November 1998.     

Effects of composite fissure sealants on IR laser fluorescence measurements

The influence of composite fissure sealants on caries detection with IR laser fluorescence measurements should be assessed. Thirty-five extracted human teeth with 105 initial carious lesions were included. Six groups containing 15 lesions each were sealed with either a clear or a white version of three sealants. Group 7 was sealed with an experimental nanofilled material. Occlusal surfaces were irradiated by a diode laser (<1 mW, 655 nm). Fluorescence was measured before and after acid etching, directly and 1 week after application of the sealants. Values significantly increased after etching (p < 0.05). Compared to initial measurements, values decreased after sealing with the white materials (p < 0.05). There was no difference between values before and after sealing with the clear and the experimental materials (p > 0.05). All values were reproducible. The study indicates that it might be possible to monitor caries activity under clear or nanofilled fissure sealants by means of laser fluorescence.     

Fluorescence Imaging of Cutaneous Malignant Melanomas and Naevi

Fluorescence images of skin lesions of 140 patients were recorded in vivo, including 53 melanomas and 42 naevi. All lesions (140) were validated by histology. Fluorescence was excited at λ_ex=365 nm and observed within the optical band pass 450 nm<λ_obs<500 nm using a cooled CCD camera. For discrimination of melanomas and naevi, recorded fluorescence intensities were averaged over the lesions (I _lesion) and over selected areas of healthy skin (I _skin). Normalised cumulative frequencies of naevi and melanomas differ slightly, when plotted versus the ratio I _skin/I _lesion, with the cumulative frequency of melanomas shifted towards higher ratios. However, because of strong overlap, diagnosis of melanomas and naevi is not improved significantly contrary to reports in the literature. More specifically, 26% of melanomas (43% of naevi) are falsely classified as naevi (melanomas) when using the ratio I _skin/I _lesion exclusively. Furthermore, spatial distribution of fluorescence intensities (450 nm<λ_obs<500 nm) does not allow discrimination between melanomas and naevi. Paper received 19 September 1997; accepted after revision 7 April 1998.     

Picosecond Laser Ablation of Dentine in Endodontics

. The interaction of picosecond laser radiation with human dental tissue was investigated in this study, in order to determine the ablation rates and the surface characteristics of the dentine by using scanning electron microscopy (SEM). Dentine ablation was performed by using tooth sections of different thicknesses (0.5–2.0 mm). Dental tissue samples were irradiated in air with the fundamental wavelength and first harmonic of a regenerative amplifier Nd:YAG laser system, at 1064 nm and 532 nm, respectively, with a pulse duration of 100 ps and a pulse repetition rate of 10 Hz. The results showed very clean craters surrounded by minimum melting of the surface of dentine when the 1064 nm pulses were used. In contrast, when the first harmonic 532 nm pulses were used, the SEM examinations revealed cracks and melting of dentine with irregular surface modification. Consequently, it seems that cleaning and shaping of the root canal walls during endodontic therapy with the picosecond Nd:YAG laser application may be possible in the future. The, as yet unexplored, field of the picosecond laser interaction with hard dental tissue is expected to be a potential alternative for powerful laser processing of biomedical structures. Paper received 24 February 1998; accepted following revision 20 November 1998.     

Effect and mechanism of 5-aminolevulinic acid-mediated photodynamic therapy in esophageal cancer

5-aminolevulinic acid-mediated photodynamic therapy (ALA-PDT) provides a novel and promising treatment for esophageal cancer. However, its specific mechanism has not been fully elucidated and its efficacy is remarkably varied. This study investigated the effect of ALA-PDT on esophageal squamous carcinoma cell line Eca-109 in vitro and vivo to explore optimal parameters, and evaluated the significance of cell apoptosis, cell cycle, ALA-protoporphyrin IX (ALA-PpIX) subcellular localization, and expression of Bcl-2 and Bax mRNA in cells to understand the mechanism of ALA-PDT for esophageal cancer. How ALA concentration, incubation time, and laser irradiation dose influenced the cell proliferation was determined by MTT assay. ALA-PpIX subcellular localization was analyzed by confocal microscopy. The mRNA changes were detected by quantitative real-time polymerase chain reaction (QRT-PCR). Tumor models transplanted with Eca-109 cells in nude mice were established (n = 10) and killed (n = 4) at 24 h post-PDT for malondialdehyde (MDA) detection and histological study. The remaining mice were measured the tumor size for 3 weeks after treatment. Our data show that ALA-PDT significantly inhibits cell proliferation (p < 0.05), the PDT efficacy depends on the saturation of ALA concentration, incubation time, and laser irradiation dose, and the best effect in tumor destruction is at 7–14 days post-PDT. ALA-PpIX is localized in mitochondria and cytoplasm. ALA-PDT induces cell apoptosis and arrests cell cycle at G0/G1 phase. Bcl-2 is significantly down-regulated while Bax is up-regulated (p < 0.05). The results of this study provide references in choosing clinical optimal PDT parameters and help in better understanding the PDT mechanism for esophageal cancer.     

IR Lasers and Application Systems for Myringotomy

. The study examines an Er:YAG laser (2940 nm) and different application systems of the CO₂ laser (10 600 nm) with regard to their suitability for a one-shot laser myringotomy of an adequate perforation size (∼2 mm). The laser–tissue interaction of the Er:YAG laser and the CO₂ laser in fresh tympanic membranes of horses (thickness: 80–100 μm) as well as in formalin-fixed human tympanic membranes (thickness: 100 μm) is studied correlating perforation diameters to the applied power/energy density and the effects demonstrated by light and scanning electron microscopy are analysed. Using the Er:YAG laser with a focused laser beam (spot diameter: 400 μm) or with a maximally defocused laser beam (spot diameter: 1600 μm) perforations of an adequte size (2 mm) can only be achieved with multiple laser pulses. Histological studies disclose only minimal thermic side effects in the adjacent tissue in both specimens. If the CO₂ laser radiation is transmitted via a silver halide polycrystalline fibre (diameter: 900 μm) a maximal perforation diameter of 1300 μm is achieved with significant thermic side effects such as coagulation. Using an Acuspot™ 710 micromanipulator (focused beam diameter: 180 μm) combined with a SilkTouch™ scanner a maximal perforation diameters of 1700 μm can be achieved in horse tympanic membrane with one laser pulse. A prototype of a hand-held CO₂ laser otoscope in combination with the SilkTouch™ scanner is suitable for performing laser myringotomies with a diameter of 2 mm with a single laser pulse in fresh horse tympanic membrane. Paper received July 1999; accepted after revision December 1999.     

Photodynamic Inactivation of the Single Crayfish Nerve Cell: Dynamics of Electrophysiological Responses and Comparison of Photosensitisers

. The study of single neuron response to photodynamic effect provides a means for the study of the dynamics of cytotoxic events leading to cell death and allows comparison of the phototoxicity of different photosensitisers. Isolated crayfish stretch receptor neurons were photosensitised for 30 min, then irradiated with a He-Ne laser (632.8 nm; 0.3 W/cm²) until irreversible firing cessation. The dynamics of neuron firing frequency were continuously recorded throughout. The following photosensitisers were studied: methylene blue, janus green B, protoporphyrin IX, chlorins e ₆ and p ₆, haematoporphyrin derivative (Photoheme) and sulphonated aluminium phthalocyanine (Photosens). Nerve cells were found to be insensitive to either He-Ne laser irradiation or photosensitisation alone, but very vulnerable to the photodynamic effect: neurons changed firing rate and died at nanomolar concentrations of photosensitisers. The dynamics of neuron responses was found to depend on photosensitiser type and concentration. The current approach provides a means of evaluation of initial threshold cell membrane alteration and cytotoxic events leading to cell death. The dependence of firing acceleration rate and neuron lifetime on photosensitiser concentration additionally allowed comparison of efficiencies of different photosensitisers. Photosens, Photoheme and chlorin p ₆ were found to be the most potent photosensitisers: neurons responded to their photodynamic effects at concentrations as low as 1–5 nM. Paper received 23 February 1998; accepted following revision 7 December 1998.     

Muscular Trauma Treated with a Ga-Al-As Diode Laser: In Vivo Experimental Study

The aim of the study was to verify in an experimental model the effects of laser therapy performed with Ga-Al-As diode lasers (780 nm, 2500 mW) on traumatised muscles. Forty adult New Zealand male rabbits were divided into four groups (A, B, C and D) of ten animals each. Each group of animals was further divided into two subgroups of five animals each. The animals were submitted to muscular trauma for 7 min by clamping the posterior muscles of the left thigh under general anaesthesia. Four days later, the rabbits in the B₁, B₂, C₁, C₂, D₁ and D₂ subgroups started daily laser therapy. The parameters utilised were: 150 J/cm² energy density, 3 W, 50 Hz in group B; 250 J/cm², 3 W, 100 Hz in group C; and 800 J/cm², 3 W, 0 Hz (continuous output) in group D. The animals in subgroups A₁ and A₂ were used as untreated controls and allowed to heal spontaneously. In order to prepare samples for histological, histochemical and histomorphometrical studies, dissection of the posterior muscle of the thigh was performed under general anaesthesia and before sacrifice, after five days of laser therapy in the subgroups B₁, C₁ and D₁ and after ten days of laser therapy in subgroups B₂, C₂ and D₂. The samples of untreated subgroups A₁ and A₂ were subjected to the same procedure and at the same times as the corresponding laser-treated groups. The following parameters were analysed on muscular samples: qualitative histological aspect (lactate dehydrogenase (LDH), cytochrome oxidase, acid phosphatase and alkaline phosphatase concentration with histoenzymatic methods) and quantitative histomorphometric evaluation of muscular damage and tissue repair. Blood samples were drawn from each subgroup before the trauma and again before sacrifice to measure the creatine phosphokinase (CK) and LDH levels. The results obtained in the tables are shown. Analysis of the results showed a better qualitative and quantitative healing process in traumatised muscles treated with Ga-Al-As diode laser therapy than in spontaneously healed ones. The results obtained with laser therapy were confirmed as haematic, histoenzymatic and histomorphometric values. According to these results, there is a positive relationship between the biostimulation properties of the laser and the healing of traumatised muscular tissue. Received for publication 15 August 1997; accepted following revision 17 March 1998     

A Preliminary Study of the In Vivo Behaviour of an Emulsion Formulation of Indocyanine Green

Indocyanine green (ICG) is a fluorescent dye largely used as functional indicator, fluorescent imaging contrast agent and recently as enhancer during diode laser photocoagulation. In this study, indocyanine green was incorporated in an emulsion to increase its residence time in blood. In vitro results to show that indocyanine green absorption peak is shifted towards longer wavelength (from 778 nm to 794 nm) close to the peak emission wavelength of the near infrared diode laser (805 nm) used during laser-induced photocoagulation. Plasmatic clearance is slower (in the 15–60 min time interval) compared to the plasmatic clearance of ICG administered as an aqueous solution. The absorbing capacity of ICG at 805 nm shows a two- to threefold increase when indocyanine green is incorporated in this emulsion. Received for publication 23 July 1997; accepted following revision 6 March 1998     

Performance of laser fluorescence at tooth surface and histological section

This study aimed to evaluate a laser fluorescence device (the DIAGNOdent) and a visual classification system (ICDAS-II) for occlusal caries diagnosis. It also aimed to determine whether fluorescence measurements taken at the tooth surface correlate with the fluorescence measurements taken within the body of the lesion. The occlusal surfaces of 100 extracted permanent teeth were examined using ICDAS-II and DIAGNOdent (LF-tooth). Serial sections were made and lesion depth was assessed histologically. DIAGNOdent readings were also taken from the sections (LF-section). There were significant positive strong correlations between ICDAS-II and histology (r_S = 0.71) and LF-section and histology (r_S = 0.70), and only moderate correlations between LF-tooth and histology (r_S = 0.51) and LF-tooth and LF-section (r_S = 0.60). Diagnostic accuracy for ICDAS-II was generally better than for LF-tooth. While the DIAGNOdent device provides an objective reading for detection and monitoring of carious lesions, using the cut-off ranges previously suggested leads to inferior performance.     

Long Exposure Growth of In-Vivo Interstitial Laser Photocoagulation Lesions

. An investigation of the temperature response and growth of thermal lesions resulting from in vivo, interstitial laser photocoagulation at long exposures was conducted to assess extended lesion growth characteristics and test the applicability of first order unimolecular rate kinetics (Arrhenius theory) to thermal lesion growth. Irradiations were performed in vivo in rabbit muscle using a continuous 805 nm diode laser source operating at 1.0 W coupled to an optical fibre with a precharred tip (i.e. point heat source). Temperature responses were measured using a linear array of five microthermocouples. Each temperature–time profile was fitted to a solution of the Weinbaum–Jiji bioheat transfer equation (W–J BHTE). Lesions were resected 48 h post-irradiation and the necrosis boundaries were determined histologically. Numerical integration of the Arrhenius damage integral using temperature–time data at the lesion boundary produced corresponding pairs of activation energy and pre-exponential factor (E _a, α) consistent with reported values for various other end-points and tissue types. Lesion radii were 6.0±0.6, 8.7±0.4 and 9.7±0.5 mm for 10, 20 and 30 min irradiations respectively. Thermal lesion growth predicted from Arrhenius theory was consistent with experimental results and is non-asymptotic by 30 min. Thermal parameters generally assumed to be constant when solving the W–J BHTE were found to vary with radial distance from the source, presumably due to a temperature dependence. Paper received 23 July 1998; accepted after revision 25 June 1999.     

Endogenous Porphyrin Production in Bacteria by δ-Aminolaevulinic Acid and Subsequent Bacterial Photoeradication

. In the present study we examined the effects of the accumulation of endogenous porphyrins on the microorganisms Staphylococcus aureus and Escherichia coli. δ-Aminolaevulinic acid (δ-ALA) 50 μg/ml can induce production of large amounts of porphyrins when incubated with the bacteria in the dark. This porphyrin production within the cells was examined directly by a fluorescence activated cell sorter (FACS) instrument and a significant increase in red fluorescence was observed. Incubation of δ-ALA with these microorganisms did not slow down their growth. δ-ALA or δ-ALA methyl ester induced accumulation of uroporphyrin in S. aureus cells and excretion of coproporphyrin into the growth medium. In E. coli, these inducers resulted in the accumulation of uroporphyrin and protoporphyrin within the cells and excretion of coproporphyrin and protoporphyrin from the cells. Photoinactivation of the porphyrin-loaded bacteria can be achieved by various light sources, depending on the dose. The most effective photokilling of S. aureus and of E. coli by its endogenic porphyrins was achieved by blue light (400–450 nm) at approximately 50 J/cm². With red light (600–700 nm), a 10-fold higher light dose was required to get a similar result for S. aureus killing. With a laser beam (632.8 nm), 50 J/cm² was necessary for 3 orders of decrease in the viability of S. aureus. With white light, 75 J/cm² was needed for 2–3 orders of decrease of S. aureus viability. With the last two light sources eradication of E. coli required at least 10 times higher doses of light. It seems that S. aureus is more susceptible than E. coli to photosensitisation when it is loaded with endogenous porphyrins. Ultrastructural alterations were observed in the bacteria incubated with δ-ALA in the dark and photosensitised by blue light (400–450 nm). Filamentous chromosomes were observed in E. coli, whereas honeycomb mesosomes were observed in S. aureus, with a destruction of the area around these mesosomes in the chromosomal area. The method described here is an additional approach to the photoinactivation of bacteria by porphyrins, with photoinactivation being accomplished by endogenous porphyrins. Paper received 6 October 1998; accepted after revision 12 April 1999.     

Effect of Helium-Neon (He-Ne) Laser Irradiation on Dog Neoplasm Cells in Culture

The effects of laser light on the cellular proliferation have been extensively characterised. Low-power laser sources, such as the helium–neon (He-Ne) laser irradiation with a wavelength of 632.8 nm, have been found to produce photobiological and photodamaging effects with evidence of interference with cell proliferation functions. The present study has investigated the in vitro effect of He-Ne laser irradiation on the proliferative action of dog tumour cells in culture. Dose–response studies showed that repeated He-Ne irradiation (irradiance 12.8 mW/cm²) once a day for 4 consecutive days in a dose range between 0.13 and 2.08 J/cm² significantly increased with increasing energy density up to a laser dose of 0.26 J/cm², whereas at >1.04 J/cm², the cell proliferation decreased with increasing energy densities. It is concluded that the application of He-Ne laser irradiation at energy densities ranging from 0.13 J/cm² to 2.08 J/cm² produced different effects on cell proliferation in dog tumour cells in culture. Paper received for publication 27 June 1997; accepted following revision 6 February 1998.     

Bone healing of the sheep tibia shaft after carbon dioxide laser osteotomy: histological results

The aim of the study was to compare the histological results after complete osteotomies of the sheep tibia using either the prototype carbon dioxide (CO₂) laser osteotome ‘OsteoLAS’ (n = 12) or an oscillating saw (n = 12). The laser parameters were as follows: wavelength 10.6 μm; energy of laser pulses 75–85 mJ; pulse duration 80 μs; pulse repetition rate 200 Hz; spot diameter 460 μm (1/e² level); radiant exposure 45–51 J/cm²; peak irradiance 0.56–0.64 MW/cm². Both groups were divided into two subgroups (n = 6), and the animals were killed after 4 weeks or 12 weeks, respectively. Light and fluorescence microscopy with semiquantitative analysis and histomorphometry were performed to compare bone healing. Charring-free laser osteotomies were possible up to a depth of 20 mm with the short-pulsed CO₂ laser. The laser, however, required a significantly longer time to perform, and a wedge-shaped gap was present on the cis-cortex. After 4 weeks the osteotomy gaps were almost unchanged in both groups and filled with connective tissue. After 12 weeks the gaps were filled with newly formed bone in both groups. Primary gap healing was predominant in the laser group and longitudinal cortical remodelling in the control group. On a cellular level, no fundamental differences were observed for early and late stages of bone healing. Further research has to be focussed on improving the CO₂ laser ostetome in order to reduce the long duration of the laser osteotomy and the necessity of creating a wedge-shaped cut in thick bones.     

Improvements in colour fundus imaging using scanning laser ophthalmoscopy

. The objective of this study was to examine the properties of two laser lines 514 nm and 532 nm when used to image the retina with a scanning laser ophthalmoscope (SLO), and to compare the images taken with a simultaneous multiple wavelength SLO, to those taken with a fundus camera. From this we concluded that the 514 nm line is the preferred line for visualising the nerve fibre layer whereas the 532 nm line is preferred for visualising retinal vessels. Based on these results the 532 nm laser light source was selected as the green line for imaging with the simultaneous colour SLO. Cases are presented where the colour SLO images contain more information than traditional digitised fundus photographs. Received for publication 7 October 1999; Accepted after revision 7 July 2000.     

Experience with non-ablative fractional photothermolysis with a dual-mode laser device (1,440/1,320 nm): no considerable clinical effect on hypertrophic/acne scars and facial wrinkles

In the literature, non-ablative fractionated photothermolysis (nFP) is accredited with improvement of wrinkles and scars combined with a reduced downtime. The purpose of this work was to evaluate the impact of a combination laser (1,320/1,440 nm) for nFP on hypertrophic scars, acne scars, and facial wrinkles. Thirty-six patients suffering from hypertrophic scars (n = 7), acne scars (n = 9), and wrinkles (n = 20) were treated using a combination Nd:YAG laser [λ_em = 1,320 and 1,440 nm, pulse duration: 3-ms single pulse, fluence: 8.0–9.0 J/cm² (1,320 nm); 2.0–2.5 J/cm² (1,440 nm)]. The appearance of the treated condition was evaluated in a retrospective study by two blinded investigators based on follow-up photographs and by patient self-assessment. The frequency of side-effects was also assessed. Both patients and blinded observers rated the treatment results for hypertrophic scars and acne scars as slight improvement, and for wrinkles as equal as compared to baseline. No serious side-effects were reported. The light device used did not lead to a considerable clinical improvement of hypertrophic scars, acne scars, or wrinkles in this study.     

Thermally Induced Irreversible Conformational Changes in Collagen Probed by Optical Second Harmonic Generation and Laser-induced Fluorescence

Irreversible thermal conformational changes induced to collagen have been studied by optical methods. More specifically, second harmonic generation (SHG) from incident nanosecond Ng:YAG 1064 nm radiation and laser-induced fluorescence by 337 nm, pulsed nanosecond nitrogen laser excitation, at 405, 410 and 415 nm emission wavelengths were registered at eight temperatures (40°, 50°, 55°, 60°, 65°, 70°, 75° and 80°C) and normalised with respect to the corresponding values at the ambient temperature of 30°C. The heating protocol used in this work, was selected to monitor only permanent changes reflecting in the optical properties of the samples under investigation. In this context, the SHG, directly related to the collagen fibril population in triple helix conformation, indicated on irreversible phase transition around 64°C. On the other hand fluorescence related to the destruction of cross-linked chromophores in collagen, some of which are related to the triple helix tertiary structure, also indicated a permanent phase transition around 63°C. These results are in agreement with previous results from studies with differential scanning calorimetry. However SHG and fluorescence, being non-invasive optical methods are expected to have a significant impact in the fields of laser ablative surgery and laser tissue welding. Paper received 2 February 2001; accepted after revision 7 May 2001.