We present a time-correlated single photon counting (TCPSC) technique that allows time-resolved multi-wavelength imaging in conjunction with a laser scanning microscope and a pulsed excitation source. The technique is based on a four-dimensional histogramming process that records the photon density over the time of the fluorescence decay, the x-y coordinates of the scanning area, and the wavelength. The histogramming process avoids any time gating or wavelength scanning and, therefore, yields a near-perfect counting efficiency. The time resolution is limited only by the transit time spread of the detector. The technique can be used with almost any confocal or two-photon laser scanning microscope and works at any scanning rate. We demonstrate the application to samples stained with several dyes and to CFP-YFP FRET.

Fluorescence lifetime imaging by time‐correlated single‐photon counting

Photodynamic therapy (PDT) is a clinical modality used to treat cancer and infectious diseases. The main agent is the photosensitizer (PS), which is excited by light and converted to a triplet excited state. This latter species leads to the formation of singlet oxygen and radicals that oxidize biomolecules. The main motivation for this review is to suggest alternatives for achieving high-efficiency PDT protocols, by taking advantage of knowledge on the chemical and biological processes taking place during and after photosensitization. We defend that in order to obtain specific mechanisms of cell death and maximize PDT efficiency, PSes should oxidize specific molecular targets. We consider the role of subcellular localization, how PS photochemistry and photophysics can change according to its nanoenvironment, and how can all these trigger specific cell death mechanisms. We propose that in order to develop PSes that will cause a breakthrough enhancement in the efficiency of PDT, researchers should first consider tissue and intracellular localization, instead of trying to maximize singlet oxygen quantum yields in in vitro tests. In addition to this, we also indicate many open questions and challenges remaining in this field, hoping to encourage future research.

https://www.mdpi.com/1422-0067/16/9/20523/pdf

Photodynamic Efficiency: From Molecular Photochemistry to Cell Death

Surgical smoke is omnipresent in the day-to-day life of the surgeon and other medical personnel who work in the operating room. In addition, patients are also exposed, especially and uniquely so in laparoscopic cases where smoke is created and trapped in a closed and absorptive space. Surgical smoke has typically been produced by electrocautery but is now ever more present in a new form with the burgeoning use of the laser and the harmonic scalpel. Several cases of transmission of human papillomavirus (HPV) from patient to treating professional via laser smoke have alerted us to the reality that surgical smoke in certain situations is far form benign. However, surgeons rarely take measures to protect themselves, their co-coworkers and patients from surgical smoke. Should we and, if so, how do we differentiate between different types of smoke and should we move toward increasing our efforts to protect ourselves, our co-workers, and patients from it? This article attempts to sort through the available data and draw some reasonable conclusions regarding surgical smoke. In general, surgical smoke is a biohazard and cannot be ignored. At a minimum, surgical smoke is a toxin similar to cigarette smoke. However, other dangers exist. This is especially true in specific circumstances such as when tissue infected with dangerous viruses is aerosolized by lasers. In addition, smoke generated by the harmonic scalpel, being a relatively cold vapor similar to laser smoke, should be further investigated for its potential ill effects and until then, looked upon with reasonable caution. Although not a high-priority in most surgical cases, surgeons should support efforts to minimize OR personnel, patients, and their own exposure to surgical smoke.

Surgical smoke: a review of the literature. Is this just a lot of hot air?

Background Hepatitis B virus (HBV) transmission is known to occur through direct contact with infected blood. There has been some suspicion that the virus can also be detected in aerosol form. However, this has never been directly shown. The purpose of this study was to sample and analyse surgical smoke from laparoscopic surgeries on patients with hepatitis B to determine whether HBV is present. Methods A total of 11 patients who underwent laparoscopic or robotic abdominal surgeries between October 2014 and February 2015 at Korea University Anam Hospital were included in this study. A high efficiency collector was used to obtain surgical smoke in the form of hydrosol. The smoke was analysed by using nested PCR. Results Robotic or laparoscopic colorectal resections were performed in 5 cases, laparoscopic gastrectomies in 3 cases and laparoscopic hepatic wedge resections in another 3 cases. Preoperatively, all of the patients had positive hepatitis B surface antigen (HBsAg). 2 patients had detectable HBsAb, and 2 were positive for hepatitis B e antigen. 3 patients were taking antihepatitis B viral medications at the time of the study. The viral load measured in the patients’ blood was undetectable to 1.7×10 8  IU/mL. HBV was detected in surgical smoke in 10 of the 11 cases. Conclusions HBV is detectable in surgical smoke. This study provides preliminary data in the investigation of airborne HBV infection.

https://oem.bmj.com/content/oemed/73/12/857.full.pdf

Detecting hepatitis B virus in surgical smoke emitted during laparoscopic surgery.

This review traces the development and study of the second-generation photosensitizer 5,10,15,20-tetra(m-hydroxyphenyl)chlorin through to its acceptance and clinical use in modern photodynamic (cancer) therapy. The literature has been covered up to early 2011.

https://onlinelibrary.wiley.com/doi/pdf/10.1111/j.1751-1097.2011.00986.x

Temoporfin (Foscan®, 5,10,15,20-tetra(m-hydroxyphenyl)chlorin)--a second-generation photosensitizer

Photodynamic therapy (PDT) and on-line fluorescence spectroscopy were carried out on human tumors after 5-aminolevulinic acid (ALA) administration using 633-nm light of a dye laser as therapeutic radiation and as fluorescence excitation radiation. This has the advantages of (1) enabling use of one laser for PDT and fluorescence diagnosis only, (2) enabling the possibility of on-line fluorescence measurements, and (3) exciting protoporphyrin molecules in deep tissue layers. Monte Carlo calculations were carried out to determine excitation and fluorescence photon distribution in case of red and violet excitation radiation. The results show the possibility of depth-resolved measurements on the fluorophore distribution by variation of excitation wavelength. The high penetration depth of 633-nm radiation results in a higher ratio of the 700-nm protoporphyrin fluorescence of the xenotransplanted tumor It to the skin Is compared with 407-nm excitation. No values greater than 1 for the ratio It/Is were found in the case of intravenous ALA injection even for red excitation. Therefore, a large amount of ALA will be metabolized in the skin and can cause photosensitivity of the patient when applied systematically. In contrast, protoporphyrin fluorescence limited to the pretreated skin area was detected in case of topically applied ALA to patients with mycosis funcoides and erythroplasy of Queyrat. The influence of remitted excitation light and of the spontaneous radiation from the laser as well as the possible excitation of food-based degradation products of chlorophyll has to be considered in high-sensitivity fluorescence measurements.

Photodynamic tumor therapy and on-line fluorescence spectroscopy after ALA administration using 633-nm light as therapeutic and fluorescence excitation radiation

The use of lasers in medical applications has grown enormously in the last few years. Recent chemical analysis of the laser pyrolysis products revealed that aerosols generated by pyrolytic decomposition of tissue could be health hazards. Therefore we analysed the genotoxic and mutagenic effects of laser pyrolysis products from different types of porcine tissue. The tissues were irradiated with a surgical CO2 laser and the generated aerosols were sampled as particulate fractions as well as low and highly volatile fractions. Then human leukocytes were incubated with the pyrolysis products and subjected to the comet assay. The results of the comet assay indicated the pyrolysis products being inducers of DNA damage. The ability to induce genotoxic effects turned out to be strongly dependent on the type of tissue that had been irradiated during laser treatment. To check whether the pyrolysis products also have mutagenic properties the Salmonella mutagenicity assay was performed. The particulate aerosol fractions of skin, muscle tissue and liver tissue clearly proved to be mutagenic in TA98 in the presence of S9 mix. There was no mutagenic effect detectable without metabolic activation. In conclusion, our experiments showed that the laser pyrolysis products originating from porcine tissues induced very potent genotoxic as well as mutagenic effects and therefore they could be potential health hazards for humans.

Laser pyrolysis products: sampling procedures, cytotoxic and genotoxic effects

Various tissue samples have been irradiated with lasers generally used for surgical laser applications Laser generated fumes were collected on charcoal tubes and chemical analysis of the pyrolytic products was performed by means of gaschromatographic and mass spectrometric (GC/MS) methods First experimental results show clearly distinguishable features in the component spectra from various combinations of tissues and lasers or laser parameters, respectively A method of standardization and calibration is presented and preliminary estimations of emission concentrations of particular substances are given© (1994) COPYRIGHT SPIE--The International Society for Optical Engineering Downloading of the abstract is permitted for personal use only

Influence of laser parameters on by-products during laser treatment of biological tissues

This article describes a setup for subcellular time-resolved fluorescence spectroscopy and fluorescence lifetime measurements using a confocal laser scanning microscope in combination with a short pulsed diode laser for fluorescence excitation and specimen illumination. The diode laser emits pulses at 398 nm wavelength with 70 ps full width at half maximum (FWHM) duration. The diode laser can be run at a pulse repetition rate of 40 MHz down to single shot mode. For time resolved spectroscopy a spectrometer setup consisting of an Czerny Turner spectrometer and a MCP-gated and -intensified CCD camera was used. Subcellular fluorescence lifetime measurements were achieved using a time-correlated single photon counting (TCSPC) module instead of the spectrometer setup. The capability of the short pulsed diode laser for fluorescence imaging, fluorescence lifetime measurements and time-resolved spectroscopy in combination with laser scanning microscopy is demonstrated by fluorescence analysis of several photosensitizers on a single cell level.© (2001) COPYRIGHT SPIE--The International Society for Optical Engineering. Downloading of the abstract is permitted for personal use only.

Short-pulsed diode lasers as an excitation source for time-resolved fluorescence applications and confocal laser scanning microscopy in PDT

Laser Interaction with Hard and Soft Tissue II

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