STED Microscopy

Fluorescence Microscopy allows highly specific imaging of cellular compartments in a minimally invasive fashion while obtaining the highest contrast. However, conventional microscopes are limited in their resolution due to the diffraction barrier. In the past decades, several different approaches have shown the ability to circumvent the diffraction limit by either switching on and off single fluorophores, utilizing structured illumination, optical fluctuation or alternatively depleting excited chromophores from the excitation volume.

STED Microscopy

STED microscopy is widely used to study luminescent samples with a high spatial resolution far below the diffraction barrier in the fields of biology, medicine as well as materials science. Therefore, in a confocal laser scanning microscope, the sample is excited with a diffraction-limited, pulsed laser, followed by a doughnut-shaped second laser pulse which is red-shifted with regard to the emission spectrum of the chromophore.

This document provides a quick overview of the STED technique introduced by Stefan W. Hell in 1994. NKT Photonics’ SuperK and KATANA HP lasers are a perfect combination to realize flexible pulsed excitation as well as synchronized depletion within the visible and near-infrared range.

STED illumination

Diffraction-limited excitation from SuperK laser
Doughnut-shaped depletion from Katana-HP laser
Super-resolved fluorescence signal after depletion

This leads to a depletion of the outer ring of the confocal excitation volume.  The remaining fluorescence after the depletion pulse is therefore only emitted from a shrunk region in the center of the excitation volume.

Temporal behavior of the fluorescence signal, above: from the central minimum of the doughnut, below: within the depleted region in the doughnut.

The SuperK Supercontinuum lasers deliver a continuous spectrum over the visible (Vis) and near-infrared (nIR) range, with excellent single-mode beam profile (M2 < 1.1) and picosecond (ps) pulse duration. In combination with our filter technology, it can be transformed into a tunable laser source, allowing optimized excitation of every chromophore absorbing in the Vis and nIR regions.

Multiline filter transmission of Supercontinuum light from a SuperK laser through SuperK SELECT.

The KATANA HP lasers are available at various wavelengths within the Vis-nIR and offer high pulse energy at ps pulse duration making them ideal for STED depletion.

The spectral power density of the SuperK Extreme EXW-12 (blue),
available wavelength for KATANA HP lasers (red).

In addition to using the proper combination of wavelengths and laser power levels, in STED microscopy it is also crucial to precisely adjust the excitation and depletion laser pulses, to synchronize for efficient depletion of the excited chromophores at the beginning of each fluorescence cycle.

NKT Photonics’ mode-locked Supercontinuum lasers are equipped with a NIM trigger output and built-in adjustable trigger delay as a standard feature. The KATANA HP lasers can run as a slave on external trigger input, which allows for simple software-controlled adjustment of the excitation and depletion pulses.

Timing of excitation and depletion pulses can be easily adjusted to
optimize depletion for the best resolution.

In the experiment, the resolution is mainly limited by the photophysics of the chromophore used. That is why new fluorescent labels are developed rapidly. Consequently, the flexibility of a Supercontinuum laser to freely choose the excitation wavelength helps to prepare for upcoming labels in the VIS-nIR.

The combination of the SuperK platform with the KATANA HP laser family becomes a turnkey solution for the most flexible and modular implementation of STED microscopy on the market successfully proven through multiple installations worldwide.

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Suitable hardware configuration for STED microscopy:

Supercontinuum excitation:

Pulsed depletion:

Optical Coherence Tomography (OCT) enables cross-sectional or 3D imaging of the subject under investigation. This is a huge advantage compared to alternative microscopy techniques where you can only investigate the surface or shallow layers of the subject.

Cross-sectional or 3D imaging is relevant for various applications, ranging from the analysis of tissue in medical applications to visualization of sub-micron structures in manufacturing.

The OCT principle for imaging was first demonstrated in 1991 by Professor Huang et al.. A very thorough description of principles and applications has been provided by Professor Drexler from the Medical University Vienna and Professor Fujimoto from MIT in “Optical Coherence Tomography: Technology and Applications”.

Over the last 20 years, OCT has become an essential imaging tool in ophthalmology with particular emphasis on detailed analysis of the retina and the surrounding tissue.

OCT applications are, however, not limited to ophthalmology. An increasing amount of research is made in OCT outside the ophthalmology field.

The SuperK supercontinuum lasers offer several key parameters relevant for UHR-OCT:

  • Extreme optical bandwidths
  • Excellent spatial coherence
  • High optical power density

The number of OCT applications using SuperK sources is increasing rapidly.

OCT in a nutshell

OCT is based on interferometry. Light from one arm is reflected or scattered off the subject under investigation and interferes with light from a reference arm.

The light from both arms originates from the same light source. The two light beams will interfere if the difference in the path lengths is within the coherence length of the optical signal.

This coherence gating lets the detection system to discriminate between reflections from closely spaced reflectors enabling high-resolution imaging.

The sensitivity of OCT is so high it can detect even the weak signals from sub-surface reflections. In this way, cross-sectional imaging can be realized – much like ultrasound – but with a much higher resolution. Image depths of several mm into the tissue can be achieved.

Practical realizations of OCT

There are different practical realizations of OCT:

  • Time-domain OCT (TD-OCT): The reference mirror is moving hence enabling coherence gating at different depths in the sample arm. This was the very first realization of OTC and it is still relevant e.g. for Full-Field OCT where the interference pattern for a full 2-dimensional array is detected simultaneously by a 2D detector array (e.g. CCD or CMOS).
  • Spectral-domain OCT (SD-OCT): Also known as Fourier-domain OCT (FD-OCT) where the reference mirror is fixed and the interference pattern is detected spectrally and converted to spatial information by the Fourier transformation.
  • Spectrometer based OCT (Sp-OCT): A broadband source (such as a SuperK source) is used to generate the interference spectrum which is detected with a high-speed spectrometer, typically with several thousand pixels and sub-nm optical resolution.
  • Swept-source OCT (SS-OCT): A tunable source is rapidly scanning the relevant spectral range. The spectral response of the interferometer is detected by a single or a balanced detector.

Each of these techniques has different advantages and disadvantages, which make them more or less relevant for certain applications.

SuperK sources can be used in all of the above realizations. For SS-OCT, a SuperK can be used with a rapidly scanning bandpass filter to effectively sweep the center wavelength. Most SuperK sources, however, have been applied to SD-OCT based on spectrometer detection (Sp-OCT).

Low noise ensures high-contrast images

The SuperK FIANIUM supercontinuum laser series has the lowest noise on the market. Optimized for low-noise performance, it gives high-contrast low-noise images in OCT systems. Its performance matches that of bulky Ti:Sapphire lasers.

Below are shown two OCT images of a human eye. The image on the right is recorded using a SuperK EXTREME OCT source, while the image on the left is obtained using a Ti:Sapphire laser. Both images are recorded by Angelika Unterhuber from Prof. Dr. Wolfgang Drexlers group at Medical University Vienna.

How others have used supercontinuum white light lasers for OCT

Papers describing OCT using a SuperK supercontinuum source: