DCS-120 Confocal FLIM System

Description

The DCS-120 confocal FLIM system uses excitation by ps diode lasers, 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 25 ps; the decay data can be resolved into 4096 time channels of down to 405 fs width. The pixel format can be increased to 2048 x 2048.

The DCS-120 system is available with inverted microscopes of Nikon, Zeiss, and Olympus. It can also be used to convert an existing conventional microscope into a fully functional confocal or multiphoton laser scanning microscope with TCSPC detection. Due to its fast beam scanning and its high sensitivity the DCS-120 system is compatible with live-cell imaging.

DCS-120 functions include recording of FLIM or steady-state fluorescence images simultaneously in two fully parallel wavelength channels, laser wavelength multiplexing, multi-wavelength FLIM, time-series FLIM, ultra-fast time-series recording by temporal-mosaic FLIM, spatial mosaic FLIM, Z-Stack FLIM, phosphorescence lifetime imaging (PLIM), fluorescence lifetime-transient scanning (FLITS) and FCS recording. Applications focus on metaboloc imaging, i.e. the use of lifetime changes 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.

Data analysis is performed by bh's new SPCImage NG FLIM analysis software. SPCImage NG is a combination of time-domain and phasor analysis. By running the calculations on a GPU (Graphics Processor Unit) the results are available within seconds. Other features are the availability of image segmentation via the phasor plot or via 2D time-domain histograms, and automatic modelling of the system IRF. Repeated recalibration by recording the actual IRF is thus unnecessary.

Specifications

For Complete Specifications Please See:

DCS-120 Confocal and Multiphoton FLIM Systems, User Handbook

Downloads

Documents

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

Confocal Scanning

In a conventional fluorescence microscope fluorescence is excited and detected in a double cone throughout the entire depth of the sample, see figure below. The sharp image seen in the focal plane is therefore surrounded by out-of-focus haze from above and below the focal plane. For structural imaging of single cells this may still be acceptable. For FLIM, however, even a small amount of out-of focus blur is unacceptable because it adds unwanted decay components to the fluorescence decay in the focal plane. For FLIM of thick tissue the situation becomes entirely hopeless. The out-of-focus blur becomes entirely overwhelming, and results in an almost complete loss in image contrast and a total mixup of decay components.

The solution to the out-of-focus problem is confocal detection. The principle is illustrated in the figure below, left. A laser beam is focused into the sample by the microscope objective lens. Although this beam excites fluorescence through the entire depth of the sample the fluorescence is brightest in the focus of the laser beam. The light from the focus of the laser beam is collected back through the microscope lens, separated from the laser beam by a dichroic mirror, and focused into a pinhole in the upper focal plane of the microscope lens. Only light from the focal plane can pass the pinhole - light from other planes is not focused into the pinhole and, consequently, cannot pass it with any appreciable efficiency.

Scanning
The principle of confocal detection solves the problem of out-of-focus light but, taken by itself, does not provide an image of the sample. To obtain an image of the an object in the focal plane confocal detection has to be combined with scanning. Scanning can be achieved by different techniques, such as moving the sample, moving the objective lens, or deflecting the laser beam and the detection beam by fast moving mirrors. The third option is the only one which is applicable to imaging of biological samples. It is fast, it does not exert mechanical forces to the sample, and it can be used for any object that can be placed under the microscope. Confocal detection in combination with beam scanning is illustrated in the figure below, left and right. The laser beam is deflected by a pair of fast-moving galvanometer mirrors. The scan lens projects the beam down towards the microscope lens. As the mirrors are moving, the beam angle in the plane of the microscope lens changes, and so does the position of the laser focus in the sample plane. The fluorescence goes back through the same beam path. Being reflected at the galvanometer mirrors, it forms a stationary beam of light coincident with the laser beam, see figure below, left. The fluorescence is separated from the laser by a dichroic mirror, and projected into a pinhole in a plane conjugate with the focal plane in the sample. Please see figure below, right.

As explained above, confocal detection in combination with scanning solves the problem of out-of-focus light in a microscope. However, scanning does more than that. It also avoids that light scattered in the sample causes lateral crosstalk between the pixels. Surprisingly, this is rarely mentioned in the microscopy literature. Consider an imaging system that simultaneously illuminates the entire sample and detects an image from the focal plane by a camera. In all pixels of this image the camera will detect scattered light from all other pixels in the illuminated area, see below, left. A scanning system - scanning the sample by a focused laser beam and detecting light only from the excited spot - records only light from the current pixel (right). Even if a part of this light is scattered in the sample the imaging system will assign the scattered photons to the correct pixel.

The supression of laterally scattered light means that already scanning alone - without the help of confocal detection - massively improves the image quality in optically thick samples. An example is shown in the figure below. From left to right, it shows images of a pig skin sample recorded by an ordinary camera, by scanning and detection without a pinhole, and by confocal detection though a pinhole. The camera image (left) shows nothing from inside the sample, the scan image (middle) shows the internal structure, and the confocal image (right) shows the internal structure without out-of-focus haze.

Combination with TCSPC FLIM
The bh FLIM systems use a multi-dimensional TCSPC technique. That means, single photons of the light passing the pinhole are detected, the times of the photons within the excitation pulse period and the position of the laser beam in the moment of the photon detection are determined, and a photon distribution over these parameters is build up. The result is an array of pixels, each containing photon numbers in a large number of consecutive time channels. Please see figure below.

The TCSPC-FLIM recording process works at any scan rate, achieves a near-ideal photon efficiency, delivers a beautifully resolved decay function in every pixel, and reaches an extremely high time resolution. The signal-to-noise ratio depends only on the photon rate available from the sample and the total acquisition time. By adding additional dimensions to the photon distribution, multi-wavelength FLIM images, multi-excitation-wavelength images, combined fluorescence / phosphorescence image, and images of fast physiological effects in the sample can be accumulated. As all bh FLIM systems, the DCS-120 TCSPC system has two parallel TCSPC channels detecting in different wavelength intervals or under different angle of polarisation.
Please see The bh TCSPC Handbook and Handbook of the DCS-120 Confocal and Multiphoton FLIM Systems for details.

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