Johan F. Beek

Double-integrating-sphere system for measuring the optical properties of tissue

A system is described and evaluated for the simultaneous measurement of the intrinsic optical properties of tissue: the scattering coefficient, the absorption coefficient, and the anisotropy factor. This system synthesizes the theory of two integrating spheres and an intervening scattering sample with the inverse adding-doubling algorithm, which employs the adding-doubling solution of the radiative transfer equation to determine the optical properties from the measurement of the light flux within each sphere and of the unscattered transmission. The optical properties may be determined simultaneously, which allows for measurements to be made while the sample undergoes heating, chemical change, or some otherexternal stimulus. An experimental validation of the system with tissue phantoms resulted in the determination of the optical properties with a < 5% deviation when the optical density was between 1 and 10 and the albedo was between 0.4 and 0.95.

In vitro double-integrating-sphere optical properties of tissues between 630 and 1064 nm

The optical properties (absorption and scattering coefficients and the scattering anisotropy factor) were measured in vitro for cartilage, liver, lung, muscle, myocardium, skin, and tumour (colon adenocarcinoma CC 531) at 630, 632.8, 790, 850 and 1064 nm. Rabbits, rats, piglets, goats, and dogs were used to obtain the tissues. A double-integrating-sphere setup with an intervening sample was used to determine the reflectance, and the diffuse and collimated transmittances of the sample. The inverse adding-doubling algorithm was used to determine the optical properties from the measurements. The overall results were comparable to those available in the literature, although only limited data are available at 790-850 nm. The results were reproducible for a specific sample at a specific wavelength. However, when comparing the results of different samples of the same tissue or different lasers with approximately the same wavelength (e.g. argon dye laser at 630 nm and HeNe laser at 632.8 nm) variations are large. We believe these variations in optical properties should be explained by biological variations of the tissues. In conclusion, we report on an extensive set of in vitro absorption and scattering properties of tissues measured with the same equipment and software, and by the same group. Although the accuracy of the method requires further improvement, it is highly likely that the other existing data in the literature have a similar level of accuracy.

Mitochondrial PO2 measured by delayed fluorescence of endogenous protoporphyrin IX

Molecular oxygen is the primary oxidant in biological systems. The ultimate destination of oxygen in vivo is the mitochondria where it is used in oxidative phosphorylation. The ability of this process to produce an amount of high-energy phosphates adequate to sustain life highly depends on the available amount of oxygen. Despite a vast array of techniques to measure oxygen, major (patho)physiological questions remain unanswered because of the unavailability of quantitative techniques to measure mitochondrial oxygen in situ. Here we demonstrate that mitochondrial PO2 can be directly measured in living cells by harnessing the delayed fluorescence of endogenous protoporphyrin IX (PpIX), thereby providing a technique with the potential for a wide variety of applications. We applied this technique to different cell lines (V-79 Chinese hamster lung fibroblasts, HeLa cells and IMR 32-K1 neuroblastoma cells) and present the first direct measurements of the oxygen gradient between the mitochondria and the extracellular volume.

In Vivo Mitochondrial Oxygen Tension Measured by a Delayed Fluorescence Lifetime Technique

Mitochondrial oxygen tension (mitoPO(2)) is a key parameter for cellular function, which is considered to be affected under various pathophysiological circumstances. Although many techniques for assessing in vivo oxygenation are available, no technique for measuring mitoPO(2) in vivo exists. Here we report in vivo measurement of mitoPO(2) and the recovery of mitoPO(2) histograms in rat liver by a novel optical technique under normal and pathological circumstances. The technique is based on oxygen-dependent quenching of the delayed fluorescence lifetime of protoporphyrin IX. Application of 5-aminolevulinic acid enhanced mitochondrial protoporphyrin IX levels and induced oxygen-dependent delayed fluorescence in various tissues, without affecting mitochondrial respiration. Using fluorescence microscopy, we demonstrate in isolated hepatocytes that the signal is of mitochondrial origin. The delayed fluorescence lifetime was calibrated in isolated hepatocytes and isolated perfused livers. Ultimately, the technique was applied to measure mitoPO(2) in rat liver in vivo. The results demonstrate mitoPO(2) values of approximately 30-40 mmHg. mitoPO(2) was highly sensitive to small changes in inspired oxygen concentration around atmospheric oxygen level. Ischemia-reperfusion interventions showed altered mitoPO(2) distribution, which flattened overall compared to baseline conditions. The reported technology is scalable from microscopic to macroscopic applications, and its reliance on an endogenous compound greatly enhances its potential field of applications.

Modeling the effect of wavelength on the pulsed dye laser treatment of port wine stains

To determine the influence of wavelength on the depth of vascular injury in port wine stains following pulsed dye laser treatment, we calculated fluence rates at wavelengths varying from 415 to 590 nm in a two-layer Monte Carlo model representing the epidermis and the dermis. Calculations were made for four different volumetric fractions of blood in the dermis: 0%, 1%, 5%, and 10%. The depth of the selective vascular injury was determined to be the depth at which the rate of temperature rise at some point within the vessel just equals that at the epidermal-dermal junction. This was maximal between 577 and 590 nm with the maximum shifted toward 590 nm for a greater dermal blood content and for larger vessels. The effect of greater epidermal pigmentation was not only to reduce the depth of vascular injury but to shift slightly the wavelength of the maximum vascular injury to a shorter wavelength.

Changes in the optical properties (at 632.8 nm) of slowly heated myocardium

The three transport equation optical properties, the absorption coefficient, the scattering coefficient, and the average cosine of the scattering angle, or anisotropy factor have been measured (at 632.8 nm) for canine myocardium after it is heated in a water bath at room temperature and at 37-75 degrees C for 1000 s. The measurement system was a double integrating sphere with collimated light and utilized the adding-doubling solution to the equation of radiative transfer. The absorption coefficient (room temperature control, 2.0 +/- 0.4 cm(-1)) began to increase and the anisotropy factor (room temperature control, 0.93 +/- 0.02) to decrease at above 45 degrees C. At 75 degrees C the changes were significant at the p < 0.0005 level (absorption, 4.5 +/- 1.3 cm(-1); anisotropy, 0.78 +/- 0.05). There was no significant change in the scattering coefficient (room temperature controls, 161 +/- 33 cm(-1)).

Mitochondrial oxygen tension within the heart

By using a newly developed optical technique which enables non-invasive measurement of mitochondrial oxygenation (mitoPO(2)) in the intact heart, we addressed three long-standing oxygenation questions in cardiac physiology: 1) what is mitoPO(2) within the in vivo heart?, 2) is mitoPO(2) heterogeneously distributed?, and 3) how does mitoPO(2) of the isolated Langendorff-perfused heart compare with that in the in vivo working heart? Following calibration and validation studies of the optical technique in isolated cardiomyocytes, mitochondria and intact hearts, we show that in the in vivo condition mean mitoPO(2) was 35+/-5 mm Hg. The mitoPO(2) was highly heterogeneous, with the largest fraction (26%) of mitochondria having a mitoPO(2) between 10 and 20 mm Hg, and 10% between 0 and 10 mm Hg. Hypoxic ventilation (10% oxygen) increased the fraction of mitochondria in the 0-10 mm Hg range to 45%, whereas hyperoxic ventilation (100% oxygen) had no major effect on mitoPO(2). For Langendorff-perfused rat hearts, mean mitoPO(2) was 29+/-5 mm Hg with the largest fraction of mitochondria (30%) having a mitoPO(2) between 0 and 10 mm Hg. Only in the maximally vasodilated condition, did the isolated heart compare with the in vivo heart (11% of mitochondria between 0 and 10 mm Hg). These data indicate 1) that the mean oxygen tension at the level of the mitochondria within the heart in vivo is higher than generally considered, 2) that mitoPO(2) is considerably heterogeneous, and 3) that mitoPO(2) of the classic buffer-perfused Langendorff heart is shifted to lower values as compared to the in vivo heart.

Non-ablative 1,550 nm fractional laser therapy versus triple topical therapy for the treatment of melasma: A randomized controlled split-face study†‡

Melasma is a uichronic, often relapsing skin disorder, with poor long‐term results from all current therapies.

Nonablative 1550-nm fractional laser therapy versus triple topical therapy for the treatment of melasma: A randomized controlled pilot study

BACKGROUND Various treatments are currently available for melasma. However, results are often disappointing. OBJECTIVE We sought to assess the efficacy and safety of nonablative 1550-nm fractional laser therapy and compare results with those obtained with triple topical therapy (the gold standard). METHODS Twenty female patients with moderate to severe melasma and Fitzpatrick skin types II to V were treated either with nonablative fractional laser therapy or triple topical therapy (hydroquinone 5%, tretinoin 0.05%, and triamcinolone acetonide 0.1% cream) once daily for 8 weeks in a randomized controlled observer-blinded study. Laser treatment was performed every 2 weeks for a total of 4 times. Physician Global Assessment was assessed at 3 weeks, 3 months, and 6 months after the last treatment. RESULTS Physician Global Assessment improved (P < .001) in both groups at 3 weeks. There was no difference in Physician Global Assessment between the two groups. Mean treatment satisfaction and recommendation were significantly higher in the laser group at 3 weeks (P < .05). However, melasma recurred in 5 patients in both groups after 6 months. Side effects in the laser group were erythema, burning sensation, facial edema, and pain; in the triple group side effects were erythema, burning, and scaling. LIMITATIONS Limitations were: small number of patients; only one set of laser parameters; and a possible difference in motivation between groups. CONCLUSIONS Nonablative fractional laser therapy is safe and comparable in efficacy and recurrence rate with triple topical therapy. It may be a useful alternative treatment option for melasma when topical bleaching is ineffective or not tolerated. Different laser settings and long-term maintenance treatment should be tested in future studies.

Cryoablation induces greater inflammatory and coagulative responses than radiofrequency ablation or laser induced thermotherapy in a rat liver model

BACKGROUND Cryoablation (CA), radiofrequency ablation (RFA), and laser induced thermotherapy (LITT) are alternative therapies for patients with unresectable liver tumors. We investigated whether there are different inflammatory and coagulative responses between these techniques. METHODS Livers of 48 rats were subjected to either CA, RFA, LITT, or sham operation (n = 12 in each group). Blood was withdrawn before, and 1, 3, 6, and 24 h after ablation. Liver enzymes as well as inflammatory and coagulation parameters were determined. Whole liver sections from the coagulated liver lobe were stained for quantification of necrosis and morphologic examination. RESULTS Histologic examination showed similar volume of complete destruction of liver parenchyma after CA, RFA, or LITT. Transaminase levels as well as the inflammatory response upon CA, as reflected by white blood cell count and cytokine levels, were significantly higher than following RFA or LITT. The systemic intravascular procoagulative state in rats that underwent CA, as reflected by platelets, and levels of sensitive markers for activation of coagulation and fibrinolyis, was also significantly higher. CONCLUSION CA of liver in rats induces greater inflammatory and coagulative responses than RFA or LITT. The combined activation of inflammation and coagulation may importantly contribute to the higher morbidity after CA.