Rolf T. Borlinghaus, Dr.

Fluorescence Lifetime Imaging (FLIM) in combination with Förster Resonance Energy Transfer (FRET) has proven to be very beneficial for investigations in biomedical research for a wide range of structural elements and dynamic changes in cells. FRET allows to monitor molecular interactions, as the FRET signal depends strongly on the distance of the two FRET partners. This allows to investigate interaction of molecules, like ligand-receptor pairs, protein-protein interactions or interactions of effectors with DNA.

„Way too complicated!“ - the notorious feedback when it comes to fluorescence lifetime measurements. This will change now! New technologies and new concepts for data evaluation, all implemented in the new Leica SP8 FALCON, render fluorescence lifetime imaging (FLIM) as fuss-free as ordinary confocal imaging. And by the way: with Leica FALCON you can record frames 10 times faster compared to the classical standard. And three (or more) dimensional image stacks or time series are generated in a snap. Four channels simultaneously? No problem! And of course there are tunable excitation wavelength both visible with white light lasers (WLL) and infrared (the latter for multiphoton microscopy). That should be reason enough to delve into fluorescence lifetime imaging. The picture shows a lifetime image of a mouse embryo. Recorded in 722 stitched tiles and fitted for four separate characteristic times. Recording time ca 1 hour – compared to ca 1 day with the classical approach.

The colorful picture shows colon tumor cells, fluorescently labelled and lineage traced from a multicolor tracer. The gray color codes for the second harmonic generation (SHG) signal from Collagen 1. Lineage traced tumor cells are shown in magenta, blue, green, yellow and red. All channels were recorded with two-photon excitation, using the SP8 DIVE by Leica Microsystems. Sample and image were kindly provided by J. van Rheenen, H. Snippert, Utrecht (the Nederlands,) and I. Steinmetz, Leica Microsystems Mannheim.

Leica Microsystems’ 4Tune detector, the key component of the SP8 DIVE Deep In Vivo Explorer, provides spectrally tunable image recording with non-descanning detection. An innovative solution for multiparameter multiphoton microscopy. The colorful image on the right shows multiphoton microscopy of an unstained mouse skin section acquired using the 4Tune detector. The green color codes for autofluorescence of muscle tissue. Red shows second harmonic generation of fibers upon illumination with 900 nm. The blue pattern is generated by third harmonic generation at lipid boundaries from illumination at 1230 nm.

Multiphoton Microscopy is one of the current hot topics in life science research. The new Leica TCS SP8 DIVE from Leica Microsystems presents a series of beneficial new innovations, including a freely tunable non-descanning detector and an ingenious beam manipulating device VBE. The variable beam expander offers free tuning of both beam diameter and axial IR-correction for up to four IR beams simultaneously.

Current fluorescence microscopy employs incident illumination which requires separation of illumination and emission light. The classical device performing this separation is a color-dependent beam splitting mirror which has fixed spectral parameters and transmits the emission usually between 90% and 98% within the designated bands. Transmission is wavelength dependent and also differs by technology, requirements and design. An alternative is the acousto optical beam splitter which has freely tunable reflection notches and transmits the emission on average at 95% between these notches.

When operating a confocal microscope, or when discussing features and parameters of such a device, we inescapably mention the pinhole and its diameter. This short introductory document is meant to explain the significance of the pinhole for those, who did not want to spend too much time to dig into theory and details of confocal microscopy but wanted to have an idea about the effect of the pinhole.

Since the invention of the microscope, there has been continual discussion about the possibility of showing more detailed features of specimens as compared to just magnifying them. In this article we describe the HyVolution concept and how the combination of confocal multiparameter fluorescence imaging at the confocal super-resolution regime with psf-based real deconvolution allows high-speed multicolor imaging with a resolution down to 140 nm.

This article outlines the most important sensors used in confocal microscopy. By confocal microscopy, we mean "True Confocal Scanning", i.e. the technique that illuminates and measures one single point only. The aim is not to impart in-depth specialist knowledge, but to give the user a small but clear overview of the differences between the various technologies and to advise on which sensor may be most suitable for which applications.

Optical imaging instrumentation can magnify tiny objects, zoom in on distant stars and reveal details that are invisible to the naked eye. But it notoriously suffers from an annoying problem: the limited depth of field. Our eye-lens (an optical imaging instrument) has the same trouble, but our brain smartly removes all not-in-focus information before the signal reaches conscious cognition.

Since super-resolution has become one of the most favored methods in biomedical research, the term has become increasingly popular. Still, there is much of confusion about what is super-resolution and what is resolution at all. Here, the classical view of microscopic resolution is discussed and some techniques that resolve better than classical are briefly introduced. The picture on the right shows the intensity distribution of an image of two points whose distance is just the Rayleigh criterion (false color coding).

Multi-channel multiphoton microscopy with dedicated optics for CLARITY. Why clearing? Curiosity is human nature. And nothing attracts as much curiosity as the inside of living organisms. While in ancient times those who cut human bodies open to do research were put to death, and modern anatomy started only after Pope Clement VII allowed dissection, we can now watch brains working in living animals – and have a good chance of soon being able to interfere with the observed activities for healing (or control) purposes.

High time-resolution confocal microscopy (HTRCLSM) requires fast scanning devices. Whereas non-resonant galvo scanners allow full position control, but only at slow speed, resonant scanners allow ~25,000 lines per second, but offer much less positioning freedom. To still allow zoom and pan functions, several approaches have been tried, with varying success. The Leica confocal microscopes od the TCS series use a very smart solution that enables stepless zooming with short switching times.

Optical imaging devices have a finite depth of field and diffraction limited resolution. The depth of field problem was tackled first with confocal microscopes, diffraction unlimited resolution is available since a few years with super-resolution microscopes. Super-resolution microscopes with a solved depth of field problem are now available.

Acousto-optical elements have successfully replaced planar filters in many positions. The white confocal, regarded as the fully spectrally tunable confocal microscope, was not possible without this technique. Acousto-optical elements are highly transparent, quickly tunable and allow many colors to be managed simultaneously. As they show a strong dependence in polarization and have comparably small dimensions, their active part is used to modify and guide the laser illumination light, thereby leaving the principal beam (0th order) unaffected. Excitation color selection and attenuation (excitation filtering), as well as separation of illumination and detection light (beam splitting) are the main fields of application.

To specifically collect emission from multiple probes, the light is first separated spatially and then passes through a device that defines a spectral band. Classically, this is a common glass-based bandpass filter. More recent approaches employ arrays for fixed-band detection or moving mirror sliders for fully tunable band characteristics.

Fluorescence microscopy assumed a pivotal role in cell biology once it was possible to stain cell components selectively by fluorescing dyes. One of the first explorers of targeted stainings, Paul Ehrlich, had the idea that something that stains specifically should also kill specifically – which was associated with the term “magic bullet”, the essential idea of chemotherapy. His group discovered Salvarsan, a tailored drug against syphilis – though not specific enough not to cause substantial side effects. Screening many fluorescent dyes led to a long list of stainings which are used in histology, including dyes like DAPI or hematoxylin and eosin.