3D microscopy images cells with nanoscale resolution

The highest resolution 3D images of the inside of single cells have been generated by scientists at the Max Planck Institute in Germany. The group says that its scanning fluorescent microscope obtains a spatial resolution far below the wavelength of light, allowing it to image the interior of cells with unprecedented detail (Nature Methods DOI:10.1038).

"We have produced the smallest focal spots that have been attained so far in a scanning fluorescent microscope, and thus attained the best 3D resolution inside a transparent object such as a cell," Stefan Hell, from the Department of NanoBiophotonics in Göttingen, told optics.org. "Contrary to previous systems, the focal spot, which measures 40–45 nm in diameter, is both spherical and of subdiffraction dimensions enabling isotropic 3D resolution on the nanoscale."

The resolution of a standard confocal system is limited to over 200 nm in the focal plane and over 500 nm along the optic axis. "We have achieved a resolution of 40–45 nm in all directions and this can be improved even further since our scheme allows further confinement of fluorescent spot (i.e. the effective PSF of the microscope)," commented Hell.

3D microscopy offers the only method of non-invasively visualizing the interior of cells. According to Hell, the level of 3D resolution is approaching that of an electron microscope but with the advantage of being able to use fluorescent tags to identify specific proteins.

The group's setup is a lens-based fluorescence microscope that merges the fundamentals from two existing microscopy techniques. "We have developed an effective microscopy scheme called isoSTED, which combines elements of stimulated emission depletion (STED) and 4Pi microscopy to compress the fluorescent spot to subdiffraction dimensions," explained Hell.

In STED microscopy, very short laser pulses excite a fluorescent tag attached to the sample under observation. This excitation pulse is immediately followed by a depletion pulse, tuned to an emission line of the fluorescent tag. The depletion pulse causes stimulated emission, which moves electrons from the excited state from which fluorescence occurs to a lower energy state. This decreases the effective spot size of the excited region resulting in higher resolution imaging.

The idea behind 4Pi microscopy is to illuminate the sample with a pair of synchronized laser pulses in such a way that the pulses interfere constructively. This is equivalent to increasing the total aperture of the system.

Source: optics.org

STED microscopy sees details on the nanoscale

Stimulated emission depletion (STED) microscopy has demonstrated that, contrary to a longstanding notion, diffraction-unlimited spatial resolution is viable with conventional lenses and visible light. Currently providing 15–70 nm resolution, it is entering the life sciences at a fast pace, while still undergoing technical improvements. Scientists from Max Planck Institute summarized its principles and recent outcomes. The whole summary should be found from optics.org.

A simple stage-scanning STED setup. Inset: overlay of the excitation focus (green) and the STED efficiency (red) for three different STED laser powers. Credit: Max Planck Institute for Biophysical Chemistry.

Lasers Break an Optical Barrier -- Stimulated Emission Depletion (STED)

This year's 10th annual German Innovation Award has been awarded to Stefan W. Hell, the director of the Max Planck Institute for Biophysical Chemistry, for his project "Light Microscopy with Unprecedented Resolution".

More than 100 years ago, Ernst Abbe laid down the law that the wave nature of light limits the smallest resolvable spot size to one-third of a wavelength in diameter, or approximately 200 nm. He predicted that any features smaller than this must always be blurred by diffraction and hence be indistinguishable by focusing light microscopes. Professor Hell resolved a spot that was 30 percent smaller than Abbe's minimum using a focusing fluorescence microscope and a pair of laser beams operating at different wavelengths. One beam -- a frequency-doubled UV-pulse -- caused excited molecules to be distributed by the fluorophore, while the other, slightly red-shifted fundamental beam quenched them through stimulated emission.

The researchers generated the two pulses with a Mira 900 Ti:sapphire laser, pumped by an Innova 400 argon-ion laser, both from Coherent Inc. of Santa Clara, Calif. The Ti:sapphire operated at a wavelength of 766 nm and a 120-fs pulse length. The outgoing beam was divided into an excitation beam and a fundamental. While the pulses of the first beam were frequency-doubled with a nonlinear optical crystal made by Fuzhou, China-based Casix Inc., the pulses of the fundamental were stretched to 40 ps by a Coherent grating compressor/decompressor. The technique requires pulsed lasers because, to eliminate fluorescence efficiently, the fundamental pulse must do its job in a much shorter time -- a few picoseconds --than the fluorescence lifetime of the dye -- a few nanoseconds.

related link: STED microscopy

(from Photonics.com)