DCS-120 MP Multiphoton FLIM System

Description

The DCS-120 MP system uses multiphoton excitation by femtosecond laser pulses, fast scanning by galvanometer mirrors, confocal detection, and FLIM by bh’s multidimensional TCSPC technique. It records fluorescence lifetime images at unprecedented temporal resolution, unprecedented reproducibility, high spatial resolution, high sensitivity, and near-ideal photon efficiency. Fluorescence lifetimes can be detected down to 10 ps; the decay data can be resolved into 4096 time channels of down to 203 fs width. The pixel format can be increased to 2048 x 2048.

To make use of the high penetration depth of multiphoton excitation the DCS-120 MP used non-descanned detection.  The MP system is available with inverted microscopes of Nikon, Zeiss, and Olympus. Due to its fast beam scanning and its high sensitivity the DCS-120 system is compatible with live-cell imaging. DCS-120 functions include simultaneous recording of FLIM or steady-state fluorescence images simultaneously in two fully parallel wavelength channels, laser wavelength multiplexing, time-series FLIM, time-series recording, Z-Stack FLIM, phosphorescence lifetime imaging (PLIM), fluorescence lifetime-transient scanning (FLITS) and FCS recording. Applications focus on lifetime variations by interactions of fluorophores with their molecular environment. Typical applications are ion concentration measurement, FRET experiments, metabolic imaging, imaging of fast physiological effects, and plant physiology.

DCS-120 MP Multiphoton FLIM systems are available with titanium-sapphire lasers and with femtosecond fiber lasers. The two versions are shown in the figure below, left and right. Ti:Sa-laser systems have the advantage of tuneability and high laser power. The high power allows the use of an AOM (acousto-optical modulator) for software-controlled intensity regulation, fast beam blanking, and pixel-synchonous ON / OFF modulation for simultaneous FLIM / PLIM. Fiber-laser systems, on the other hand, are compact, easy to use, and virtually maintanance-free. On the negative side, the wavelength is fixed to typically 780 nm. However, the lack of tuneability is less of a problem than commonly believed: The two-photon-absorption bands of most fluorophores are broad enough to excite them at this wavelength. Moreover, 780 nm is an excellent wavelength to excite NADH. Fiber laser systems therefore make almost ideal multiphoton metabolic FLIM systems.

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The realm of the bh FLIM systems is in molecular imaging. Typical applications are the imaging of ion concentrations, pH, or local viscosity, protein interaction experiments by FRET, and metabolic imaging by fluorescence decay of NADH an FAD in combination with oxygen measurement. In these applications, the bh FLIM systems benefit from their high sensitivity, high time resolution, high timing stability, and their capability to resolve multi-exponential-decay profiles into their components. Other advantages are the capability to record FLIM of fast physiological effects down to the millisecond range, and to record at several excitation and emission wavelengths simultaneously.

Principles

Multiphoton Excitation

Multiphoton excitation uses two or more photons of the excitation light to excite one fluorescence photon, see figure below, left. To get the process working at noticeable efficiency extremely high power density, both in space and in time, is required. Both are achieved by using femtosecond pulses and focussing through a high-NA microscope objective lens, see below, right. With a pulse duration of 100 fs, a repetition rate of 80 MHz, and focusing through a lens of NA = 1.3, no more than 5 mW are required to obtain bright fluorescence of most fluorescent dyes used in microscopy. In most instances 'multiphoton' excitation is performed by using just two photons. To obtain fluorescence in the visible region the laser wavelength must then be in the near-infrared (NIR) region. Ti:Sa lasers, OPOs, and femtosecond fibre lasers can be used to generate the required excitation wavelength.

Multiphoton excitation has a number of advantages over one-photon excitation.
First, the absorption of the sample at the excitation wavelength is low. Also the scattering coefficient is lower than at visible or ultraviolet wavelengths. Two-photon (or, in the general case, multiphoton) excitation therefore goes deeper into tissue than one-photon excitation, see figure below, left and right. Multiphoton excitation can therefore be used to excite fluorescence in deep layers of biological tissue.

Second, the excitation is confined to the focal volume, where the power density is high. In a scanning system, this automatically implies that the excitation is confined to the focal plane. That means no confocal pinhole is needed to reject light from outside the focal plane. Multiphoton excitation intrinsically provides out-of-focus suppression and, consequently, optical-sectioning capability.
The fact that no fluorescence is excited outside the focal plane leads to the third advantage of multiphoton excitation, and this is detection of light scattered inside the sample. Visible light from a deep focal plane is massively scattered on its way out of the sample, see figure below, left. This light cannot be collimated, and, consequently, not focused into a confocal pinhole. However, there is no need to feed the light back through the scanner and through a pinhole. Instead, it can be diverted immediately behind the microscope lens and sent to a detector. By projecting an image of the microscope lens (not of the image plane in the sample) a large part of the scattered photons is transferred to the detector. The principle is called non-descanned detection, see figure below, right.

In combination with the bh TCSPC FLIM systems a fourth advantage is the extremely high time resolution. The excitation pulses have femtosecond pulse width. The instrument-response function (IRF) of the FLIM system is therefore not broadened by the excitation pulses. With fast hybrid detectors, IRF widths down to 20 ps (FWHM) are reached. The capability to directly record ultra-fast fluorescence decay phenomena opens a entirely new world of FLIM applications. Please see application notes about ultra-fast decay in mushroom spores and in pollen grains, and its measurement in the sub-10-ps range.

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