Roman Amberger

Dual color localization microscopy of cellular nanostructures

The dual color localization microscopy (2CLM) presented here is based on the principles of spectral precision distance microscopy (SPDM) with conventional autofluorescent proteins under special physical conditions. This technique allows us to measure the spatial distribution of single fluorescently labeled molecules in entire cells with an effective optical resolution comparable to macromolecular dimensions. Here, we describe the application of the 2CLM approach to the simultaneous nanoimaging of cellular structures using two fluorochrome types distinguished by different fluorescence emission wavelengths. The capabilities of 2CLM for studying the spatial organization of the genome in the mammalian cell nucleus are demonstrated for the relative distributions of two chromosomal proteins labeled with autofluorescent GFP and mRFP1 domains. The 2CLM images revealed quantitative information on their spatial relationships down to length‐scales of 30 nm.

Using conventional fluorescent markers for far-field fluorescence localization nanoscopy allows resolution in the 10-nm range

We present a novel technique of far‐field localization nanoscopy combining spectral precision distance microscopy with widely used fluorochromes like the Green Fluorescent Protein (GFP) derivatives eGFP, EmGFP, Yellow Fluorescent Protein (YFP) and eYFP, synthetic dyes like Alexa 488 and Alexa 568, as well as fluoresceine derivates. Spectral precision distance microscopy allows the surpassing of conventional resolution limits in fluorescence far‐field microscopy by precise object localization after the optical isolation of single signals in time. Based on the principles of this technique, our novel nanoscopic method was realized for laser optical precision localization and image reconstruction with highly enhanced optical resolution in intact cells. This allows for spatial assignment of individual fluorescent molecules with nanometre precision. The technique is based on excitation intensity dependent reversible photobleaching of the molecules used combined with fast time sequential imaging under appropriate focusing conditions. A meaningful advantage of the technique is the simple applicability as a universal tool for imaging and investigations to the major part of already available preparations according to standard protocols. Using the above mentioned fluorophores, the positions of single molecules within cellular structures were determined by visible light with an estimated localization precision down to 3 nm; hence distances in the range of 10–30 nm were resolved between individual fluorescent molecules allowing to apply different quantitative structure analysis tools.

SPDM: single molecule superresolution of cellular nanostructures

Novel methods of visible light microscopy have overcome the limits of resolution hitherto thought to be insurmountable. The localization microscopy technique presented here based on the principles of Spectral Precision Distance Microscopy (SPDM) with conventional fluorophores under special physical conditions allows to measure the spatial distribution of single fluorescence labeled molecules in entire cells with macromolecular precision which is comparable to a macromolecular effective optical resolution. Based on detection of single molecules, in a novel combination of SPDM and Spatially Modulated Illumination (SMI) microscopy, a lateral (2D) effective optical resolution of cellular nanostructures around 10 - 20 nm (about 1/50th of the exciting wavelength) and a three dimensional (3D) effective optical resolution in the range of 40 - 50 nm are achieved.

Structured illumination microscopy of autofluorescent aggregations in human tissue.

Sections from human eye tissue were analyzed with Structured Illumination Microscopy (SIM) using a specially designed microscope setup. In this microscope the structured illumination was generated with a Twyman-Green Interferometer. This SIM technique allowed us to acquire light-optical images of autofluorophore distributions in the tissue with previously unmatched optical resolution. In this work the unique setup of the microscope made possible the application of SIM with three different excitation wavelengths (488, 568 and 647 nm), thus enabling us to gather spectral information about the autofluorescence signal.

[Exposure to light during vitreoretinal surgery. II: Characteristics of endoilluminators].

PURPOSE Light can cause phototoxic retinal damage. The aim of this study was to evaluate the risk of retinal hazard by endoilluminators during vitreoretinal surgery. METHODS The spectra, radiance, and irradiance of six light sources with different associated fibre optics (20 G, 23 G, standard collimated, wide-angle diffuse) were measured and compared with thresholds published by international standardisation committees. RESULTS The spectra of the endoilluminators differed significantly in the short wavelength band. The maximum radiance ranged from 15 mW to 190 mW and the calculated irradiance from 36 mW/cm2 to 1,130 mW/cm2 (distance 5 mm) and from 9 mW/cm2 to 376 mW/cm2 (distance 10 mm). Compared with published thresholds for surgery, time limits ranging from 0.7 min to 264 min (distance 5 mm) and 2.7 min to 1,052 min (distance 10 mm) seem to be safe. CONCLUSIONS Light systems used for vitreoretinal surgery differ considerably in spectra, radiance, and irradiance; these differences have an impact on the maximum tolerable exposure times during surgery.

Light-emitting diode technology in vitreoretinal surgery.

Background: Systems for vitreoretinal illumination during surgery usually consist of an external light source and a light fiber. We introduce a new illumination system for vitreoretinal surgery based on the light-emitting diode technology, with an embedded light source in the handle of the light fiber, making a separate light source unnecessary. Methods: A prototype of a new illumination system for vitreoretinal surgery (ocuLED; Geuder, Heidelberg, Germany) was tested. This system consists of a handle with a built-in light-emitting diode, supported by an external power source. The OcuLED was analyzed in regards to wavelength, maximum radiant power, and maximum irradiance and was compared with three commercially available vitreoretinal illumination systems. Furthermore, the first intraoperative application and handling were evaluated. Results: The ocuLED system works with a cool white or a neutral white light-emitting diode and is powered externally. The wavelength spectrum shows a maximum at 565 nm and a second peak at 455 nm. Compared with other light sources, the proportion of potentially harmful blue light is low. Maximum radiant power and irradiance are in line with xenon and mercury vapor light sources. The intrasurgical light is bright and offers good visibility. The handle of ocuLED is slightly wider than commonly used light fiber handles, which do not affect its use during surgery. Conclusion: Technical progress in light-emitting diode technology allows minimizing the equipment for vitreoretinal illumination. The OcuLED provides bright illumination without an external light source. Wavelength spectrum, maximum radiant power, and irradiance are safe from the risk of phototoxic damage. Intrasurgical handling is identical to conventional light fibers.

Light exposition in vitreoretinal surgery. I. Basics

ZusammenfassungDas Auge ist aufgrund seiner Funktion einer großen Strahlenbelastung im optischen Spektrum ausgesetzt. Der größte Teil der UV- und Infrarotstrahlung wird in der Hornhaut und Linse absorbiert, sodass die Netzhaut fast nur durch Strahlung im sichtbaren Wellenlängenbereich gefährdet wird. Sichtbares Licht kann über fotomechanische, fotothermische oder fotochemische Mechanismen zu einer Schädigung der Netzhaut führen. Der wichtigste Schädigungsmechanismus in der Netzhaut ist dabei unter Alltagsbedingungen oder bei der Anwendung ophthalmologischer Lichtquellen die fotochemische Lichttoxizität, die durch lichtinduzierte chemische Reaktionen zustande kommt. Das Ausmaß der Schädigung wird entscheidend durch verschiedene Faktoren wie die Wellenlänge des Lichts, die Expositionszeit und die Bestrahlungsstärke beeinflusst. Insbesondere der kurzwellige Anteil des sichtbaren Lichts (blaues Licht) ist für die fotochemische Schädigung der Netzhaut von Bedeutung.AbstractDue to its function of light perception, the eye is exposed to high levels of radiation of the optical spectrum. Most of the ultraviolet and infrared radiation is absorbed in the cornea and lens, and mostly only radiation of the visible spectrum can reach the retina. Visible light can cause retinal damage by photomechanical, photothermal, and photochemical mechanisms. The most important mechanism of light damage to the retina under daily conditions or when using ophthalmologic light sources is the photochemical light toxicity caused by light-induced chemical reactions. The extent of damage depends on several factors, such as wavelength, exposure time, and irradiance. Particularly the shorter portion of the visible light spectrum (blue light) is responsible for photochemical damage to the retina.Due to its function of light perception, the eye is exposed to high levels of radiation of the optical spectrum. Most of the ultraviolet and infrared radiation is absorbed in the cornea and lens, and mostly only radiation of the visible spectrum can reach the retina. Visible light can cause retinal damage by photomechanical, photothermal, and photochemical mechanisms. The most important mechanism of light damage to the retina under daily conditions or when using ophthalmologic light sources is the photochemical light toxicity caused by light-induced chemical reactions. The extent of damage depends on several factors, such as wavelength, exposure time, and irradiance. Particularly the shorter portion of the visible light spectrum (blue light) is responsible for photochemical damage to the retina.