Combining multiple optical trapping with microflow manipulation for the rapid bioanalytics on microparticles in a chip.

An array of four independent laser traps is combined with a polydimethylsiloxane microfluidic chip to form a very compact system allowing parallel processing of biological objects. Strong three dimensional trapping allows holding objects such as functionalized beads in flows at speeds near 1 mm/s, enabling rapid processing. By pressure control of the inlet flows, the trapped objects can be put in contact with different solutions for analysis purpose. This setup, including a fluorescence excitation-detection scheme, offers the potential to perform complex biochemical manipulations on an ensemble of microparticles.

MRI thermodosimetry in laser-induced interstitial thermotherapy.

BACKGROUND AND OBJECTIVES: The aim of this study was to establish a correlation between a thermal measurement and a magnetic resonance imaging (MRI) signal during laser-induced interstitial thermotherapy (LITT) in liver. STUDY DESIGN/MATERIALS AND METHODS: Pig liver was irradiated for 15 minutes with a diode laser at two different powers, 0.5 W (450 J) and 1.5 W (1,350 J). Tissue temperature was monitored every 20 seconds using thermocouples. Thermosensitive MRI sequences (T(1)-weighted Turbo-Flash) were acquired with the same irradiation parameters. Correlation between MRI signals (SI) and temperature measures was defined at two different distances from the fiber (5 and 10 mm). RESULTS: At 0.5 W, temperatures rose progressively up to a maximum increase of 9.5 degrees C at 5 mm and 4 degrees C at 10 mm after 15 minutes. The corresponding MRI signal decreased progressively to -27.6 SI at 5 mm and -18.5 SI at 10 mm. At 1.5 W, temperatures rose dramatically at 5 mm, reaching a plateau. The temperature elevation measured at the end of the irradiation was of 30 degrees C whereas at 10 mm it was only 14.5 degrees C. The MRI signal varied accordingly, remaining inversely proportional to temperature (-76 SI at 5 mm and -35.5 SI at 10 mm). CONCLUSIONS: An inversely proportional relationship was observed between MRI signal in sequential Turbo-Flash and temperature. MRI should allow to analyze heat diffusion in the liver, and thus to monitor real-time LITT treatments.

Laser-induced immobilization and plasma membrane permeabilization in human spermatozoa.

We evaluated the potential use of a non-contact, 1.48 microm wavelength diode laser for immobilization of human spermatozoa and permeabilization of the sperm membrane in different culture media. When we applied a single laser shot near to the middle region of the sperm tail, spermatozoa could be immobilized either temporarily or permanently, depending on the energy used. Above an energy of 2 mJ in polyvinylpyrrolidone and 2-3 mJ in culture medium, a reliable permanent immobilization was achieved by permeabilization of the sperm tail membrane. We then explored the use of a double laser shot technique. Spermatozoa were temporarily immobilized by a first laser shot applied near to the sperm tail followed by permeabilization with a second laser shot aimed directly at the sperm tail. This sequential approach yielded permanent immobilization at much lower energy values compared with the single shot technique. Following the injection of laser-treated spermatozoa, mouse oocytes underwent normal activation and pronuclear formation. We conclude that a non-contact 1.48 microm diode laser system can be used for immobilization of spermatozoa and for permeabilization of the sperm tail membrane. This laser procedure may offer an alternative to currently used sperm pretreatment prior to intracytoplasmic sperm injection.

Temperature-related reversible birefringence changes in rat tail tendon.

By use of a highly sensitive method for measuring slight variations in birefringence it is shown here that a strong reversible correlation exists between rat tail tendon birefringence and temperature. This phenomenon is totally different from the loss of birefringence that results from a denaturation process. Below the threshold temperature leading to denaturation, an increase in temperature is systematically accompanied by a reversible increase in birefringence (0.25% degrees C(-1)). This phenomenon is observed at very fast heating rates (250,000 degrees C s(-1)), such as those induced by pulsed infrared lasers, and confirmed by experiments conducted with slow homogeneous heating of the sample medium (0.1 degrees C s(-1)). The good correlation between birefringence and temperature observed during the fast heating suggests that there are only small modifications of the tissue structure at the fibril level.

Biliary structures lead to tumour recurrences after laser-induced interstitial thermotherapy.

BACKGROUND AND OBJECTIVE: Thermal diffusion during laser-induced interstitial thermotherapy (LITT) has not yet been fully investigated in heterogeneous tissue architecture such as liver. LITT was performed on rabbit liver tumours to analyse the role of biliary structures in thermal diffusion. STUDY DESIGN/MATERIALS AND METHODS: Twenty-four VX2 tumours were grafted onto 12 rabbit livers. The animals were randomly separated into two groups when tumour size reached 8 mm. Thermotherapy was performed by delivering the 830-nm output of a diode laser to the centre of the tumour with a 300-,microm fibre. Irradiation conditions were 1.5 W over 900 sec. On day 7 or 14, the tumours were removed and stained with haematoxylin-eosin and picrosirius red F3BA (PR). Thermal damage was evaluated by PR and electron microscopic examinations. RESULTS: Among the treated tumours, recurrences were found both at the periphery (one on day 7, seven on day 14) and within the treated area (two on day 7, two on day 14). All recurrences were located in the vicinity of the biliary structures, which are frequently spared from thermal injury. CONCLUSION: Biliary ducts lead to a heat sink, thereby facilitating tumour recurrences.

Laser-assisted cryopreservation of single human spermatozoa in cell-free zona pellucida.

Improved procedure for efficient cryopreservation of single human spermatozoa in cell-free human zona pellucida is reported. We used a diode laser system for efficient and precise creation of a single hole into the zona pellucida of a degenerated or immature human oocyte. This allowed the extraction of the cytoplasm using a micropipette with a diameter of 10-15 microns. Through the same opening, human spermatozoa were inserted into the empty zona. We used motile and laser immobilized spermatozoa. Immobilized sperm were obtained by a single laser irradiation delivered in the vicinity of the sperm tail prior to insertion. This new immobilization procedure was shown to have no deleterious effect on membrane integrity and sperm viability. Following sperm transfer into the zona, the laser-drilled hole was closed with an oil droplet which was expelled from the micropipette during withdrawal to avoid loss of spermatozoa. This facilitated detection of the otherwise translucent zona during the cryopreservation procedure. After thawing, all cryopreserved zonae (20/20) could be successfully retrieved. Spermatozoa were recovered from the zona pellucida through the hole used for insertion. The rate of sperm recovery for initially motile spermatozoa was 80% vs. 92% for laser immobilized spermatozoa. Sperm viability was 81% and 84%, respectively, detected by a Hoechst stain. This technique makes cryopreservation of single human spermatozoa easy and feasible and appears beneficial for couples with severe male infertility and for those facing repeated surgical sperm extraction.

Laser-assisted microdissection of the zona pellucida facilitates polar body biopsy.

OBJECTIVE: To investigate whether polar body biopsy can be performed after laser microdissection of the zona pellucida (ZP). DESIGN: Mouse zygotes were allocated randomly to three groups. The zygotes were subjected to laser microdissection of the ZP and polar body biopsy (group 1), laser microdissection alone (group 2), or no treatment (group 3). SETTING: University-based IVF program. PATIENT(S): Animal study. INTERVENTION(S): A hole was drilled in the ZP of mouse zygotes using a 1.48-micron noncontact diode laser. A microneedle was inserted and the polar body was aspirated. MAIN OUTCOME MEASURE(S): The efficacy of polar body biopsy after laser microdissection of the ZP was evaluated. RESULT(S): The laser diode beam allowed for precise drilling of a 14- to 18-micron hole in the ZP. Polar bodies could be aspirated without damaging the zygote and did not disintegrate during the biopsy. Zygotes developed to blastocysts and underwent the same hatching as control zygotes. Lower hatching rates were observed in untreated zygotes. CONCLUSION(S): Laser microdissection of the ZP with a noncontact laser system facilitates subsequent polar body biopsy. The use of blunt-ended micropipettes greatly reduces the risk of damage to the zygote or the polar body. This procedure makes polar body biopsy more accurate and effective for preimplantation genetic diagnosis.

[Vaporization of urethral stenosis using the KTP 532 laser]. / Vaporisation des sténoses urétrales au laser KTP 532.

The authors treated 16 patients presenting with a total of 20 anterior urethral strictures using the KTP 16 Laser. The aetiology was iatrogenic in 50% of cases, infectious in 20% of cases, traumatic in 20% of cases and unknown in 10% of cases. The stricture was situated in the bulbous urethra (80%), membranous urethra (10%) or penil urethra (10%). Laser vaporization of the urethral stricture was performed over the entire circumference of the urethra when necessary, followed by bladder drainage by urethral catheter for 24 hours. All patients were prospectively reviewed at 3 weeks, 3 months and 6 months (clinical symptoms, uroflowmetry, cystourethrography). A complete symptom and urodynamic success was obtained in 13 patients (81%) at 3 and 6 months. The stricture recurred in 4 patients, but only three of them (19%) required treatment (reoperation of repeat dilatations). The mean maximum flow rate increased from 6 mL/s to 20 mL/s at 3 months and was maintained at 19 mL/s at 6 months. No intraoperative or postoperative complications were observed. In conclusion, our results confirm that KTP 532 laser urethral strictures is a reliable and effective method in the medium term. These good results also suggest an advantage in terms of the recurrence rate in comparison with internal urethrotomy. However, our series needs to be evaluated with a longer follow-up and prospective, randomized trials comparing the two methods need to be conducted.

Morphologic changes in collagen fibers after 830 nm diode laser welding.

BACKGROUND AND OBJECTIVE: The mechanism of laser tissue welding is elusive, but collagen transitions are somehow involved. Collagen fiber modifications observed after 830 nm diode laser welding are presented in this study. STUDY DESIGN/MATERIALS AND METHODS: A 830 nm diode laser assisted longitudinal aortorrhaphy was performed on 37 Wistar rats, with shots of 0.5 W in power, 8 sec in duration and 250 W/cm2 in irradiance. Energy utilized ranged from 400-550 J/ mm2 for 1 cm-length of anastomosis. After laser welding, histological modifications in collagen fibers were observed through optic, scanning electron, and electron microscopic examination. RESULTS: After laser welding, collagen fibers lost a proportion of birefringence. Under electron microscope, the different changes in collagen fibers were visualized being either fused, "roped," swollen, or dissolved, surrounded by normal ones situated in the same zone. CONCLUSION: These data suggest that diode laser heating denatured part of the collagenic fibers, and that these morphologic changes play an important role in laser welding.

Morphological analysis of microarterial media repair after 830 nm diode laser assisted end-to-end anastomosis: Comparison with conventional manual suture.

A series of 240 diode laser assisted end-to-end microvascular anastomoses (LAMA) and conventional manual anastomoses (CMA) were performed in the left and right common carotid of Wistar rats, respectively. In comparison with the two anastomotic methods, optic and scanning electron microscopic examinations were achieved from Day 0 to Day 210, in order to clarify the mechanism of media repair after diode laser welding, especially the long-term results. In the LAMA group, the cut vessel edges were welded without obvious thermal necrosis after laser treatment. On Day 10, media repair was underlined by circular bulges corresponding to the folds of cut vessel endings brought together. Inflammatory cells were regularly scattered in the adventitia in the vicinity of the anastomotic site, and were gaining ground intensively in the media by Day 20. At this time, the parallel organization of elastic laminae disappeared while the collagen network developed. On Day 120, irregular elastic fibres aggregated in the anastomotic site. On Day 210, reconstituted elastic lamina was present. In the CMA group, on Day 20, fibrotic repair appeared between cut vessel edges, and the injury incorporated by sutures was important. The elastic laminae were not reconstituted by Day 210 in any case. This microscopic study proves that the long-term repair of diode LAMA facilitates media repair and prevents fibrotic scarring of the media.

Enhancement of tissue lesion depth by dual wavelength irradiation with the Nd-YAG/KTP laser: Perspectives for laser prostatectomy.

The Nd-YAG/KTP laser coagulates and vaporizes prostate tissue. The objective of this study was to investigate the combined effects of both wavelengths and to determine the irradiation parameters allowing the largest lesion volume. Chicken breast tissue was irradiated ex vivo. Consecutive 1064 and 532 nm Nd-YAG/KTP laser irradiations were performed for different combinations (30 W/10 W, 20 W/20 W, 10 W/30 W) with variable total fluence (1200 J, 2400 J, 3600 J) and compared to isofluent single wavelengths at 40 W irradiation. The depths, diameters and volumes of the total lesion as well as the vaporization effects of the 532 nm wavelength on normal and on priorly coagulated tissue were analysed. Maximum total lesion depths (p< 0.001) were found under combined Nd-YAG/KTP (20 W/20 W) irradiation conditions. Ablation efficacy of the 532 nm wavelength was reduced after prior 1064 nm irradiation, but crater depths were increased. Dual wavelength irradiation with the Nd-YAG/KTP laser induces a specific denaturation process. This may represent a new approach to increase the depth of coagulation necrosis, and thus the treated volume, thereby improving long-term results.

Improved fertilization and implantation rates after non-touch zona pellucida microdrilling of mouse oocytes with a 1.48 microm diode laser beam.

The safety of microdrilling the zona pellucida of mouse oocytes with a 1.48 microm diode laser has been investigated by determining the ability of mouse oocytes to fertilize in vitro and develop in vivo. Mice born after transfer of control and zona pellucida-microdrilled embryos into foster mothers were submitted to anatomical and immunohistochemical investigations, and their aptitude to breed was assessed in two subsequent generations. Decoronization of the oocytes with hyaluronidase induced a reduction of the fertilization and implantation rates, which was attributed to a zona hardening phenomenon. After laser zona pellucida microdrilling, these rates were restored to those obtained with embryos derived from untreated oocyte-cumulus complexes. Pups derived from zona pellucida microdrilled embryos were comparable with those obtained from control embryos, confirming the lack of deleterious effects of the laser treatment. In conclusion, the 1.48 microm diode laser allows safe microdrilling of the zona pellucida of mouse oocytes after decoronization with hyaluronidase. Based on the health of the F2 generation and the lack of neuroanatomical and neurochemical differences, we concluded that this technology may be investigated in the human, particularly when the zona pellucida represents the main impediment for fertilization or embryo hatching.

[Biophysical bases of the effects of holmium laser on articular cartilage and their impact on clinical application technics]. / Biophysikalische Grundlagenuntersuchungen zur Wirkung der Holmium-Laserstrahlung am Knorpelgewebe und deren Konsequenzen für die klinische Applikationstechnik.

The in vitro study presented helps to clarify the biophysical mechanisms and tissue interactions of the holmium laser at the point of impact on the surface of cartilage-bone specimens investigated in different experimental settings. A striking event is the creation of a vapor bubble that opens up access for the laser beam through the fluid medium. This bubble shows a reproducible dynamic behavior function of the laser irradiance and the angle of incidence of the delivery fiber. These determine directly the amplitude of the pressure waves induced when the bubble collapses. Apart from this acoustic effect, which is correlated with epicentric histological features that can hardly be considered biologically relevant, a thermal effect is recognized that is finally responsible for the ablation and tissue damage. It induces typical histological alterations that can be observed along the laser beam axis, with a penetration function mainly of the irradiance but also of the angle of incidence. Nevertheless, at a pulse energy of 1 J and an irradiation angle of 30 degrees, the recorded overall depth of the immediate histological changes was down to 500-600 micrometers. Thus, in realistic working conditions, the damage observed after cartilage sealing with the holmium laser remains within an absolutely acceptable range. This is in agreement with the better results compared to mechanical cartilage debridement that have been reported in previous prospective clinical studies.

Effect of pulse duration on bubble formation and laser-induced pressure waves during holmium laser ablation.

BACKGROUND AND OBJECTIVE: One concern during laser ablation of tissue is the mechanical injury that may be induced in tissue in the vicinity of the ablation site. This injury is primarily due to rapid bubble expansion and collapse or due to laser-induced pressure waves. In this study, the effect of laser pulse duration on the thermodynamics of bubble formation and accompanying acoustic pressure wave generation has been investigated. STUDY DESIGN/MATERIALS AND METHODS: Q-switched holmium:YAG laser pulses (pulse duration 500 ns, pulse energy 14 mJ) and free-running holmium:YAG laser pulses (pulse duration 100-1,100 microseconds, pulse energy 200 mJ) were delivered in water and tissue phantoms via a 200- and 400-microns fiber, respectively. The tissue phantoms consisted of polyacrylamide gels with varying mechanical strengths. Bubble formation was recorded with a fast flash photography setup, while acoustic transients were measured with a needle hydrophone. RESULTS: It was observed that, as the pulse length was increased the bubble shape changed from almost spherical for Q-switched pulses to a more elongated cylinder shape for longer pulse durations. The bubble expansion velocity was larger for shorter pulse durations. Only the Q-switched pulse induced a measurable thermo-elastic expansion wave. All pulses that induced bubble formation generated pressure waves upon collapse of the bubble in gels as well as in water. However, the magnitude of the pressure wave depended strongly on the size and geometry of the induced bubble. CONCLUSION: The magnitude of the collapse pressure wave decreased as laser pulse duration increased. Hence it may be possible to reduce collateral mechanical tissue damage by stretching the holmium laser pulse.

Non-contact microdrilling of mouse zona pellucida with an objective-delivered 1.48-microns diode laser.

BACKGROUND AND OBJECTIVE: A non-touch laser-induced microdrilling procedure is studied on mouse zona pellucida (ZP). STUDY DESIGN/MATERIALS AND METHODS: A 1.48-microns diode laser beam is focused in a 8-microns spot through a 45x objective of an inverted microscope. Mouse zygotes, suspended in a culture medium, are microdrilled by exposing their ZP to a short laser irradiation and allowed to develop in vitro. RESULTS: Various sharp-edged holes can be generated in the ZP with a single laser irradiation. Sizes can be varied by changing irradiation time (3-100 ms) or laser power (22-55 mW). Drilled zygotes present no signs of thermal damage under light and scanning electron microscopy and develop as expected in vitro, except for a distinct eight-shaped hatching behavior. CONCLUSION: The microdrilling procedure can generate standardized holes in mouse ZP, without any visible side effects. The hole formation can be explained by a local photothermolysis of the protein matrix.

Interstitial diode laser hyperthermia in the treatment of subcutaneous tumor.

BACKGROUND AND OBJECTIVE: Interstitial Laser Hyperthermia (ILH) has been investigated since the early 80s in the treatment of deep seated tumors. The purpose of this study was to evaluate the efficiency of diode ILH (830 nm) in a subcutaneous tumor model. STUDY DESIGN/MATERIALS AND METHOD: The antitumoral effect of diode ILH was assessed in a randomized study performed on 80 Swiss nu/nu mice. The tumor model was a subcutaneously implanted HT29 colonic carcinoma. The animals were assigned to four groups of 20 mice: Groups 1 and 3 were treated by ILH, groups 2 and 4 were not treated (control groups). Tumors were removed on day 3 in groups 1 and 2, and on day 30 in groups 3 and 4. The treatment was performed on tumors of 8 mm in diameter and a volume of 140 mm3. A laser irradiation of 360 J (power: 0.2 W; irradiation time: 1800 s) was delivered through a 300 microns optical fiber implanted in the tumor. The laser parameters insured temperatures of 46 degrees C in the central part of the tumor and 42 degrees C at the periphery. Tumor features were evaluated on day 3 and day 30. RESULTS: Untreated tumors grew rapidly up to a mean volume of 241 mm3 on day 3 (group 2) and 2,000 mm3 on day 30 (group 4). Treated tumors regressed to a mean volume of 32 mm3 on day 3 (group 1). On day 30, 40% of the tumors had totally disappeared and 60% showed partial response with small and peripheral residual tumor of 172 mm3 on an average, as to say 11.2 times smaller than in group 4. CONCLUSION: ILH with a low power 830 nm diode laser is an efficient treatment of subcutaneous tumor model. Partial responses are attributed to an insufficient heating at the tumor periphery. More precise control of the peripheral tumor temperature will improve the ILH results.

Microdissection of mouse and human zona pellucida using a 1.48-microns diode laser beam: efficacy and safety of the procedure.

OBJECTIVE: To investigate the efficacy and safety of a small and affordable 1.48-microns continuous wave diode laser for zona pellucida (ZP) microdissection. DESIGN: Mouse and human oocytes and zygotes were submitted to ZP drilling. The hole characteristics and possible laser-induced structural alterations of the neighboring cytoplasm were investigated with scanning and transmission electron microscopy. The safety of the procedure was checked on control and drilled zygotes by determining their ability to develop in vitro and in vivo. SETTING: Collaborative study between three Swiss academic centers. INTERVENTIONS: The collimated diode laser beam was delivered through a 45x objective of an inverted microscope and focused through the culture dish and culture medium in 1- to 3-microns spots. MAIN OUTCOME MEASURE: Safety assessment of the laser drilling procedure. RESULTS: The 1.48-microns radiation achieves a rapid, precise, and easily controlled lysis of the ZP without any micromanipulative handling of the eggs. Different shapes of holes can be produced by varying the laser beam intersection site on the ZP, laser power, and irradiation time. The energy needed to drill holes of a given diameter is greater for zygotes than for oocytes. Safety of the drilling procedure is confirmed by the lack of damage at the ultrastructural and biologic levels. CONCLUSIONS: The low-cost 1.48-microns diode laser allows an easy, objective-driven, nontouch microdissection of the ZP. The procedure is safe, as drilled embryos give rise to normal and fertile offspring.

Importance of the implosion of ESWL-induced cavitation bubbles.

The damage induced by an extracorporeal shock wave lithotripter is observed with a fiber optic stress sensing technique. When a stone is placed in the focus, besides the expected stress induced by the incoming shock wave emitted by the ESWL apparatus, a second delayed stress is observed some hundreds of microseconds later. The second stress is induced by a shock wave generated at the collapse of a cavitation bubble. Partial reflection of the incoming shock wave at the stone boundary is at the origin of the large cavitation bubble formation. Sensing fiber fracture results always from the second shock wave due to the collapse of the cavitation bubble. Thus the largest stress is generated at the collapse. When no target is placed in the focus of the lithotripter, no large cavitation bubble is formed and no delayed shock wave is observed. Our results demonstrate unambiguously the decisive role of cavitation in ESWL procedures.