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DCS-120 MP System Records Multiphoton FLIM and PLIM

The DCS-120 MP multiphoton FLIM system is an extended version of the bh DCS-120 confocal scanning FLIM System. It uses multiphoton excitation by a femtosecond titanium-sapphire laser, fast galvanometer scanning, non-descanned detection, hybrid detector technology, and single-photon recording by bh’s multi-dimensional TCSPC process. An AOM is included to control the laser power and to modulate the laser for PLIM acquisition. The system records FLIM data in two fully parallel recording channels, runs Z stacks, accumulates fast FLIM time series, and records simultaneously FLIM and PLIM.

Keywords: FLIM, PLIM, Multiphoton

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DCS-120 MP System Records Multiphoton FLIM and PLIM

The DCS‑120 MP is an extended version of the bh DCS‑120 confocal scanning FLIM System. It uses multiphoton excitation by a femtosecond titanium-sapphire laser, fast galvanometer scanning, non-descanned detection, hybrid detector technology, and single-photon recording by bh’s multi-dimensional TCSPC process. An AOM is included to control the laser power and to modulate the laser for PLIM acquisition. The system records FLIM data in two fully parallel recording channels, runs Z stacks, accumulates fast FLIM time series, and records simultaneously FLIM and PLIM. All components, including the laser and the AOM, are controlled by bh’s SPCM 64 bit data acquisition software. By using bh’s 64 bit Megapixel FLIM technology, images of the full field of view of the microscope can be recorded at diffraction-limited resolution. Image formats as large as 2048 x 2048 pixels with 256 time channels per pixel are available.

Convallaria sample with 1024 x 1024 pixels, 256 time channel per pixel. DCS‑120 scan head, Nikon Eclipse inverted microscope, Spectra Physics Mai Tai laser. Microscope lens 20x NA = 0.5. Excitation wavelength 800 nm

Due to its fast scan rates and its high sensitivity, the DCS-120 MP is compatible with live cell and life tissue imaging. Typical applications are measurements of local molecular environment parameters, protein interaction experiments by FRET, imaging of metabolic parameters derived from the fluorescence decay functions of endogenous fluorophores, and correlated metabolic and oxygen saturation imaging.

Phosphorescence Lifetime Image, recorded by bh’s PLIM technique based on laser modulation and dual-time-base recording. SPCImage FLIM / PLIM data analysis.

Main Panel of SPCM Software. Images in two spectral channels, control panels for scanner, laser and AOM, detectors, and predefined setup panel for easy selection of imaging mode.

Key Specifications

Excitation                                                           Femtosecond Titanium:Sapphire Laser

Wavelength                                                        Typ. 750 to 980 nm, depends on laser

Excitation pulse frequency                             75 to 80 MHz, depends on laser

Coupling into scan head                                  Free beam

Power control                                                    Acousto-optical modulator (AOM). AOM is optional for FLIM but required for 2p PLIM. AOM can be retrofitted.

Laser modulation for PLIM                             Acousto-optical modulator (AOM).

AOM response time                                          200 ns in PLIM mode

Laser and AOM control                                   via SPCM TCSPC/FLIM data acquisition software

Additional excitation sources                         ps diode lasers, supercontinuum laser with AOTF. Optional, can be retrofitted to the system.

Microscopes                                                      All inverted microscopes of Zeiss, Nikon, and Olympus

Detection beam path                                        Non-descanned (direct) detection for 2p excitation

                                                                             Optional transmission path for SHG recording

                                                                             2 confocal detection channels for 1p excitation

                                                                             Piezo actuators for confocal alignment

Detectors                                                            Two HPM‑100-40 GaAsP hybrid detectors. No afterpulsing background, no secondary pulses in IRF

                                                                             Option: HPM-100-50 GaAs hybrid detectors

                                                                             Option: MW-FLIM GaAsP 16-wavelength detector

Detector protection                                          Electronic overload shutdown

Detection wavelength selection                     Beamsplitter / filter cube in front of detectors

Scanner                                                               bh DCS-120 scan head

Alignment                                                          via internal piezo actuators

Scanner control                                                 integrated in SPCM TCSPC/FLIM data acquisition software

Scan format, pixels                                            2048 x 2048             1024 x 1024                512 x 512 256 x 256

Scan format, time channels (max)                          256                            1024                           4096              4096

Scan rate, frames per sec., at zoom 4                     0.37                            0.65                            1.47                2.95

Scan rate, lines per sec., at zoom 4                         500                             750                             750                 750

Additional scanner ports                                 Additional port for visible-wavelength laser

                                                                             Two outputs for additional confocal detectors

TCSPC System                                                   Two parallel SPC-150, SPC-150N or SPC-160 channels

                                                                             Upgrade to three or four parallel channels possible

FLIM modes                                                       X-Y scan, fast intensity preview mode, fast lifetime preview mode, Z Stack by record-and-save procedure, Z Stack by Mosaic FLIM function, time series FLIM by record-and-save procedure, time series FLIM by Mosaic FLIM function, fast accumulated Mosaic FLIM time series, fast accumulated line scan time series (FLITS), PLIM, simultaneous FLIM and PLIM

FCS mode                                                           Online FCS, by correlating photon macro times, spot selected by beam park function of scanner

Selection of operation mode                           Via predefined-setup panel

FLIM data analysis                                           By bh SPCImage data analysis software. 1-2-3 exponential fit, incomplete-decay model, 1st. moment analysis. No IRF recording necessary. Images of lifetime components, amplitudes of components, intensity and amplitude-weighted lifetime, relative intensity contribution, FRET efficiency. 1D histograms in region of interest, 2D histograms of decay parameters, phasor plot.

 

For details, please see Handbook of DCS-120 Confocal Scanning FLIM System, 6th ed. or later or bh TCSPC Handbook, 6th ed. or later, both available for free download at www.becker-hickl.com. Printed copies available from bh.

 

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