Gereon Hüttmann

Origin of retinal pigment epithelium cell damage by pulsed laser irradiance in the nanosecond to microsecond time regimen

Selective photodamage of the retinal pigment epithelium (RPE) is a new technique to treat a variety of retinal diseases without causing adverse effects to surrounding tissues such as the neural retina including the photoreceptors and the choroid. In this study, the mechanism of cell damage after laser irradiation was investigated.

Optimizing Descemet Membrane Endothelial Keratoplasty Using Intraoperative Optical Coherence Tomography

IMPORTANCE Descemet membrane endothelial keratoplasty (DMEK) is a challenging procedure for the surgeon, particularly because of deficient visibility of the delicate tissue due to the natural en face view through the operating microscope. A cross-sectional view would greatly enhance intraoperative overview and enable the surgeon to better control the procedure. OBJECTIVE To retrospectively analyze the use of intraoperative optical coherence tomography (iOCT) for improving the safety of DMEK. DESIGN Intraoperative OCT during DMEK was performed in 26 eyes of 26 patients. We retrospectively analyzed imaging and video data. SETTING Department of Ophthalmology, University of Cologne. PARTICIPANTS Seven men and 19 women aged 39 to 93 years with corneal endothelial dysfunction undergoing DMEK. EXPOSURE Descemet membrane endothelial keratoplasty. MAIN OUTCOMES AND MEASURES Visibility of surgical steps, overall duration of DMEK, overall time for complete intraoperative air filling of the anterior chamber, and correlation between donor age and Descemet rolling behavior. RESULTS Intraoperative OCT enables visualization of all steps of the DMEK procedure. Overall mean (SD) duration of the DMEK procedure was 25.7 (6.9) minutes when using iOCT. Overall mean (SD) complete intraoperative anterior chamber air-filling time was 236 (108) seconds in contrast to 60 to 90 minutes for standard air-filling time. Descemet membrane rolling behavior showed significant inverse correlation between donor age (range, 39-93 years) and the extent of rolling (R2 = 0.5 [P = .006]). CONCLUSIONS AND RELEVANCE Intraoperative OCT enhances the visibility of graft orientation and unfolding, thereby improving safety of the DMEK procedure. Overall, iOCT is a helpful device that may support surgeons in all steps of DMEK procedures.

Laparoscopic Fluorescence Detection of Ovarian Carcinoma Metastases Using 5-Aminolevulinic Acid-Induced Protoporphyrin IX

The aim of the current clinical study was to evaluate the in vivo fluorescence detection of ovarian carcinoma metastases in a second‐look laparoscopic procedure after intraperitoneally applied 5‐aminolevulinic acid (ALA).

Chromophore-assisted light inactivation of pKi-67 leads to inhibition of ribosomal RNA synthesis

Abstract.  Objectives: Expression of the nuclear Ki‐67 protein (pKi‐67) is strongly associated with cell proliferation. For this reason, antibodies against this protein are widely used as prognostic tools for the assessment of cell proliferation in biopsies from cancer patients. Despite this broad application in histopathology, functional evidence for the physiological role of pKi‐67 is still missing. Recently, we proposed a function of pKi‐67 in the early steps of ribosomal RNA (rRNA) synthesis. Here, we have examined the involvement of pKi‐67 in this process by photochemical inhibition using chromophore‐assisted light inactivation (CALI). Materials and methods: Anti‐pKi‐67 antibodies were labelled with the fluorochrome fluorescein 5(6)‐isothiocyanate and were irradiated after binding to their target protein. Results: Performing CALI in vitro on cell lysates led to specific cross‐linking of pKi‐67. Moreover, the upstream binding factor (UBF) necessary for rRNA transcription was also partly subjected to cross‐link formation, indicating a close spatial proximity of UBF and pKi‐67. CALI in living cells, using micro‐injected antibody, caused a striking relocalization of UBF from foci within the nucleoli to spots located at the nucleolar rim or within the nucleoplasm. pKi‐67‐CALI resulted in dramatic inhibition of RNA polymerase I‐dependent nucleolar rRNA synthesis, whereas RNA polymerase II‐dependent nucleoplasmic RNA synthesis remained almost unaltered. Conclusions: Our data presented here argue for a crucial role of pKi‐67 in RNA polymerase I‐dependent nucleolar rRNA synthesis.

On the possibility of high-precision photothermal microeffects and the measurement of fast thermal denaturation of proteins

The precision of laser-induced effects is often limited by thermal and thermomechanical collateral damage. Adjusting the pulsewidth of the laser to the size of the absorbing structure can at least avoid thermal side effects and facilitates a selective treatment of vessels or pigmented cells. Further extending the precision of thermal effects below cellular dimensions by using nanometer sized particles could open up new fields of applications for lasers in medicine and biology. Calculations show that under irradiation with nano- or picosecond laser pulses gold particles of submicrometer size can easily be heated by several hundred K. High temperatures have to be used for subcellular thermal effects, because heat confinement to such small structures requires the thermal damage to occur in extremely short times. Estimating the denaturation temperature by extrapolating the Arrhenius equation from a time range of minutes and seconds into a time range of nano- and picoseconds leads to temperatures between 370 K-470 K. There is evidence that in aqueous media, due to the surface tension, these temperatures can be generated at the surface of nanometer sized particles without vaporization of the surrounding water. In order to show whether or not an extrapolation of the damage rates over six to nine orders of magnitude gives correct data, a temperature-jump experiment was designed and tested which allows to measure denaturation rates of proteins in the millisecond time range. Denaturation of chymotrypsin was observed within 300 ps at temperatures below 380 K. The rate constants for the unfolding of chymotrypsin followed the Arrhenius equation up to rates of 3000 s/sup -1/.

Elevation of plasma membrane permeability by laser irradiation of selectively bound nanoparticles.

Irradiation of nanoabsorbers with pico- and nanosecond laser pulses could result in thermal effects with a spatial confinement of less than 50 nm. Therefore absorbing nanoparticles could be used to create controlled cellular effects. We describe a combination of laser irradiation with nanoparticles, which changes the plasma membrane permeability. We demonstrate that the system enables molecules to penetrate impermeable cell membranes. Laser light at 532 nm is used to irradiate conjugates of colloidal gold, which are delivered by antibodies to the plasma membrane of the Hodgkins disease cell line L428 and/or the human large-cell anaplastic lymphoma cell line Karpas 299. After irradiation, membrane permeability is evaluated by fluorescence microscopy and flow cytometry using propidium iodide (PI) and fluorescein isothiocyanate (FITC) dextran. The fraction of transiently permeabilized and then resealed cells is affected by the laser parameter, the gold concentration, and the membrane protein of the different cell lines to which the nanoparticles are bound. Furthermore, a dependence on particle size is found for these interactions in the different cell lines. The results suggest that after optimization, this method could be used for gene transfection and gene therapy.

Chemical instability of 5-aminolevulinic acid used in the fluorescence diagnosis of bladder tumours

Aqueous solutions of 5-aminolevulinic acid (ALA) prepared for intravesical instillation in the framework of a clinical study on the fluorescence diagnosis of urothelial bladder cancer were found to be unstable. This chemical instability of ALA was studied in aqueous solution of 37 degrees C as a function of concentration, pH and reaction time. Our investigations showed that the reaction of ALA is an irreversible process, which yields at least two reaction products in the pH range studied (pH lower than 8): the 2,5-(beta-carboxyethyl)dihydropyrazine and the 2,5-(beta-carboxyethyl)pyrazine. As a result of these studies, the conditions for the preparation of ALA solutions to be used for intravesical instillation were optimized: solution of ALA in phosphate-buffered saline at a concentration of 0.18 M (3 g of ALA in 100 ml) neutralized to pH 5, were prepared and stored on ice until use. Solutions prepared under these conditions were stable and were used for fluorescence diagnosis of bladder tumours with successful results. The effect of the pH and the composition of the urine on the extent of the reaction of ALA and on the nature of its reaction products formed during instillation was investigated by comparing the urine of patients before and immediately after instillation of ALA.

Imaging of brain and brain tumor specimens by time-resolved multiphoton excitation microscopy ex vivo.

Multiphoton excitation fluorescent microscopy is a laser-based technology that allows subcellular resolution of native tissues in situ. We have recently applied this technology to the structural and photochemical imaging of cultured glioma cells and experimental gliomas ex vivo. We demonstrated that high microanatomical definition of the tumor, invasion zone, and normal adjacent brain can be obtained down to single-cell resolution in unprocessed tissue blocks. In this study, we used multiphoton excitation and four-dimensional microscopy to generate fluorescence lifetime maps of the murine brain anatomy, experimental glioma tissue, and biopsy specimens of human glial tumors. In murine brain, cellular and noncellular elements of the normal anatomy were identified. Distinct excitation profiles and lifetimes of endogenous fluorophores were identified for specific brain regions. Intracranial grafts of human glioma cell lines in mouse brain were used to study the excitation profiles and fluorescence lifetimes of tumor cells and adjacent host brain. These studies demonstrated that normal brain and tumor could be distinguished on the basis of fluorescence intensity and fluorescence lifetime profiles. Human brain specimens and brain tumor biopsies were also analyzed by multiphoton microscopy, which demonstrated distinct excitation and lifetime profiles in glioma specimens and tumor-adjacent brain. This study demonstrates that multiphoton excitation of autofluorescence can distinguish tumor tissue and normal brain based on the intensity and lifetime of fluorescence. Further technical developments in this technology may provide a means for in situ tissue analysis, which might be used to detect residual tumor at the resection edge.

Intravital Two-Photon Microscopy of Immune Cell Dynamics in Corneal Lymphatic Vessels

Background The role of lymphatic vessels in tissue and organ transplantation as well as in tumor growth and metastasis has drawn great attention in recent years. Methodology/Principal Findings We now developed a novel method using non-invasive two-photon microscopy to simultaneously visualize and track specifically stained lymphatic vessels and autofluorescent adjacent tissues such as collagen fibrils, blood vessels and immune cells in the mouse model of corneal neovascularization in vivo. The mouse cornea serves as an ideal tissue for this technique due to its easy accessibility and its inducible and modifiable state of pathological hem- and lymphvascularization. Neovascularization was induced by suture placement in corneas of Balb/C mice. Two weeks after treatment, lymphatic vessels were stained intravital by intrastromal injection of a fluorescently labeled LYVE-1 antibody and the corneas were evaluated in vivo by two-photon microscopy (TPM). Intravital TPM was performed at 710 nm and 826 nm excitation wavelengths to detect immunofluorescence and tissue autofluorescence using a custom made animal holder. Corneas were then harvested, fixed and analyzed by histology. Time lapse imaging demonstrated the first in vivo evidence of immune cell migration into lymphatic vessels and luminal transport of individual cells. Cells immigrated within 1–5.5 min into the vessel lumen. Mean velocities of intrastromal corneal immune cells were around 9 µm/min and therefore comparable to those of T-cells and macrophages in other mucosal surfaces. Conclusions To our knowledge we here demonstrate for the first time the intravital real-time transmigration of immune cells into lymphatic vessels. Overall this study demonstrates the valuable use of intravital autofluorescence two-photon microscopy in the model of suture-induced corneal vascularizations to study interactions of immune and subsequently tumor cells with lymphatic vessels under close as possible physiological conditions.

Imaging corneal crosslinking by autofluorescence 2-photon microscopy, second harmonic generation, and fluorescence lifetime measurements

PURPOSE: To evaluate the use of 2‐photon microscopy (TPM), which excites tissue autofluorescence, in detecting and calculating the grade of collagen corneal crosslinks, which are not visible through the slitlamp and in vivo confocal microscopy. SETTING: Departments of Ophthalmology, University of Lübeck, Lübeck, and University of Rostock, Rostock, Germany. DESIGN: Experimental study. METHODS: Corneas of rabbits were treated with different crosslinking (CXL) protocols. Two weeks after treatment, the corneas were evaluated in vivo by confocal microscopy. Eyes were enucleated and TPM was performed at 710 nm and 826 nm excitation wavelengths to detect tissue autofluorescence, second harmonic generation, and fluorescence lifetime measurements (FLIM). Eyes were then fixed and analyzed by histology. RESULTS: Crosslinking following the standard protocol generated a strong autofluorescence signal in the stroma that was detected by TPM. This signal was weakly present in the control specimens, and a sharp transition zone between the peripheral zone and the CXL zone was seen. On FLIM, an increase in corneal crosslinks was measured when the standard protocol was used. CONCLUSIONS: Two‐photon microscopy, a noninvasive method, was able to detect the effects of therapeutic CXL and measure the grade of CXL. In addition to postoperative treatment control, the technique has possibilities for use in online dosimetry during 2‐photon triggered CXL. Financial Disclosure: No author has a financial or proprietary interest in any material or method mentioned.