iMIC TIRF FD-FLIM and TGL
The microscope is custom build around the iMIC stand (Till Photonics/FEI) with H101 motorized stage (Prior).
The iMIC stand is equipped with:
Objective carousel (up to 4 Olympus objectives). Available objectives: UPLFLN 4x (WD 17, NA 0.13), LUCPLFLN 20x (WD 6.6-7.8, NA 0.45) and 60x (WD 1.5-2.2, NA 0.7); Super Apochromats UPLSAPO 4x (NA 0.13), 10x (NA 0.4), 20xo (NA 0.85), 40x (NA 0.95), 60xw (NA 1.2), 100xo (NA 1.4) and TIRF APON 60x (NA 1.49).
Focus drive (combines a piezo-element having 250 µm of fine travel (resolution 50 nm) with 25 mm of coarse movement).
Motorized beamsplitter changer (for up to 3 octo-cube beamsplitters).
Dichrotome Level A – Dual emission Imaging Extension – single beam splitter separate the emission light into two independent color/polarization channels and each channel is simultaneously projected onto half of the CCD-chip.
Dichrotome Level B – extend the Dichrotope Level A with possibility to additionally split the emission light into two independent channels using Polarizing Beam splitter (for wavelength 400-700 nm) and each channel could be simultaneously projected onto own CCD-chip. For such type applications two identical CCD Clara cameras (Andor) available upon request (default position of those cameras is on STED-FLCS microscope).
TuCam (Andor) – two camera adapter that in default configuration coupled to one output port of Dichrotome Level B. Used for switching between two cameras: EMCCD 885 (Andor) – used for single molecule experiments and ICCD Li2CAM (Lambert Instruments) – used for FLIM/TGL.
The Prior stage is equipped with adapters for a wide range of sample holders: multiwell plates, slides, petri dishes, Chamlide magnetic multiwell chambers (available WL-2460-10 and CM-S22-4) and closed Flow Cell Systems (Focht Chamber System FCS2 from Bioptech and Magnetic closed chamber CF-T12 from Chamlide).
The iMIC stand is equipped with:
Objective carousel (up to 4 Olympus objectives). Available objectives: UPLFLN 4x (WD 17, NA 0.13), LUCPLFLN 20x (WD 6.6-7.8, NA 0.45) and 60x (WD 1.5-2.2, NA 0.7); Super Apochromats UPLSAPO 4x (NA 0.13), 10x (NA 0.4), 20xo (NA 0.85), 40x (NA 0.95), 60xw (NA 1.2), 100xo (NA 1.4) and TIRF APON 60x (NA 1.49).
Focus drive (combines a piezo-element having 250 µm of fine travel (resolution 50 nm) with 25 mm of coarse movement).
Motorized beamsplitter changer (for up to 3 octo-cube beamsplitters).
Dichrotome Level A – Dual emission Imaging Extension – single beam splitter separate the emission light into two independent color/polarization channels and each channel is simultaneously projected onto half of the CCD-chip.
Dichrotome Level B – extend the Dichrotope Level A with possibility to additionally split the emission light into two independent channels using Polarizing Beam splitter (for wavelength 400-700 nm) and each channel could be simultaneously projected onto own CCD-chip. For such type applications two identical CCD Clara cameras (Andor) available upon request (default position of those cameras is on STED-FLCS microscope).
TuCam (Andor) – two camera adapter that in default configuration coupled to one output port of Dichrotome Level B. Used for switching between two cameras: EMCCD 885 (Andor) – used for single molecule experiments and ICCD Li2CAM (Lambert Instruments) – used for FLIM/TGL.
The Prior stage is equipped with adapters for a wide range of sample holders: multiwell plates, slides, petri dishes, Chamlide magnetic multiwell chambers (available WL-2460-10 and CM-S22-4) and closed Flow Cell Systems (Focht Chamber System FCS2 from Bioptech and Magnetic closed chamber CF-T12 from Chamlide).
For the illumination there are different options available.
- Trans-illumination – Green LED. Supplied through long working distance condenser IX-LWUCD NA 0.55 (Olympus).
- Widefield Epifluorescence illumination:
- High-quality modulating LEDs in Multi-LED excitation source (Lambert Instruments). Digital modulation up to 120 MHz. Integrated into LIFA-X system (Lambert Instruments) for widefield Frequency Domain Fluorescence Lifetime Imaging (FD-FLIM) and widefield Time-Gated Luminescence imaging (TGL). Four lines with peak light intensities at wavelengths 368, 432, 477, 532 nm are available.
- Ultra-fast (< 3ms) filter switching Xenon Flash lamp – Oligochrome(Till Photonics/FEI) with up to 5 excitation filters capacity in the wavelength range from 320 nm to 700 nm. Suitable for high-speed multi-color and ratio widefield imaging. Combined with laser TIRF through Polytrope Imaging-Mode Switch.
- Objective TIRF illumination – MultiLaser/SOLE-6 (Lambert Instruments/Omicron) – High-Speed digital and/or analogue modulation laser light engine fiber coupled through Polytrope and position controlled by Yanus Scan Head. Contains 5 PhoxX laser diodes for CW and modualated applications (lines: 405, 445, 488, 515, 638 nm) and 1 DPSS line modulated by AO-modulator (Cobolt Jive 561 nm). Laser diodes are also integrated into LIFA system for TIRF- FD-FLIM measurements. Polytrope and Yanus combination for all MultiLaser lines provides variable angle with azimuthal scan (2D) TIRF functionality and FRAP and photoactivation possibilities.
Software: Live Acquisition 2.6 (FEI/Till Photonics), Li-FLIM (Lambert Instruments), ImageJ/FIJI, Matlab.
iMIC STED-FLCS and FCCS
The microscope is custom build around the iMIC stand (Till Photonics/FEI) with Motorized TILL stage.
The iMIC stand is equipped with:
Objective carousel (up to 4 Olympus objectives). Available objectives: UPLFLN 4x (WD 17, NA 0.13), LUCPLFLN 20x (WD 6.6-7.8, NA 0.45) and 60x (WD 1.5-2.2, NA 0.7); Super Apochromats UPLSAPO 4x (NA 0.13), 10x (NA 0.4), 20xo (NA 0.85), 40x (NA 0.95), 60xw (NA 1.2), 100xo (NA 1.4) and TIRF APON 60x (NA 1.49).
Focus drive (combines a piezo-element having 250 µm of fine travel (resolution 50 nm) with 25 mm of coarse movement).
Motorized beamsplitter changer (for up to 3 octo-cube beamsplitters).
Dichrotome Level A – Dual emission Imaging Extension – single beam splitter separate the emission light into two independent color/polarization channels and each channel is simultaneously projected onto half of the CCD-chip. By default used one of two available Clara CCD cameras (Andor). The second Clara CCD camera is used for STED donut alignment.
The TILL stage is equipped with adapters for a wide range of sample holders: slides, small petri dishes, Chamlide magnetic multiwell chambers (available WL-2460-10 and CM-S22-4) and closed Flow Cell System (Magnetic closed chamber CF-T12 from Chamlide).
For the illumination there are different options available.
The iMIC stand is equipped with:
Objective carousel (up to 4 Olympus objectives). Available objectives: UPLFLN 4x (WD 17, NA 0.13), LUCPLFLN 20x (WD 6.6-7.8, NA 0.45) and 60x (WD 1.5-2.2, NA 0.7); Super Apochromats UPLSAPO 4x (NA 0.13), 10x (NA 0.4), 20xo (NA 0.85), 40x (NA 0.95), 60xw (NA 1.2), 100xo (NA 1.4) and TIRF APON 60x (NA 1.49).
Focus drive (combines a piezo-element having 250 µm of fine travel (resolution 50 nm) with 25 mm of coarse movement).
Motorized beamsplitter changer (for up to 3 octo-cube beamsplitters).
Dichrotome Level A – Dual emission Imaging Extension – single beam splitter separate the emission light into two independent color/polarization channels and each channel is simultaneously projected onto half of the CCD-chip. By default used one of two available Clara CCD cameras (Andor). The second Clara CCD camera is used for STED donut alignment.
The TILL stage is equipped with adapters for a wide range of sample holders: slides, small petri dishes, Chamlide magnetic multiwell chambers (available WL-2460-10 and CM-S22-4) and closed Flow Cell System (Magnetic closed chamber CF-T12 from Chamlide).
For the illumination there are different options available.
- Trans-illumination – Green LED. Supplied through long working distance condenser IX-LWUCD NA 0.55 (Olympus).
- Widefield Epifluorescence illumination: Ultra-fast (< 1.6 ms) switching monochromator Xenon Flash lamp – Polychrome V (Till Photonics/FEI) variable wavelength range from 320 nm to 680 nm. Suitable for high-speed multi-color and ratio widefield imaging.
- Confocal Epifluorescence illumination:
- Picosecond Pulsed supercontinuum laser Solea (Picoquant). Spectral range 480-900 nm (variable slit width from 3 till 15 nm). Internal oscillator 2.5 – 40 MHz. Triggered for TCSPC applications (FLIM, FCS) and syncronised with VISIR-765-STED laser for STED-FLCS applications and LLC for FCCS applications. Directly coupled through Yanus Scan Head. Yanus provides Beam Park, line, raster and orbital scan modes.
- CW laser line combiner (LLC) with two lines – Cobolt Calypso Dual 488/532. Lines are selectable with AOTF (AA Optoelectronics). Used as the second line for cross-correlation with Solea laser.
- Picosecond Pulsed laser module VISIR-765-STED. Average power 1.5 W. Syncronised with Solea laser through variable picosecond delayer (MicroPhotonDevices). Used for STED “donut” formation by Vortex Phase Plate.
For confocal detection Two High Speed Hybrid Detectors with GaAsP cathodes HPM-100-40 are used (Becker&Hickl). For Gated-STED applications syncronisation of detectors with Solea illumination is realized with USB-Controlled Delay Switch Box. For acquisition of FLIM images and correlation and cross correlation applications 16 Channel Photon Correlator DPC-230 is used (Becker&Hickl).
Software: IQ 2.7 (Andor), SPCImage 5.2 and Burst analyzer 2.0 (Becker&Hickl), ImageJ/FIJI, Matlab
Nikon PALM (under construction)
The microscope is custom build around the Eclipse TE2000-U stand (Nikon) with Motorized Prior stage H117 controlled with ProScan III.
The TE2000-U stand is equipped with:
Objective carousel (up to 6 Nikon objectives). Available objectives: Plan Fluor 10x (NA 0.3), Plan Fluor 40x (NA 0.6) and TIRF APO 100x (NA 1.49).
Remote focus controller.
Manual beamsplitter changer (for up to 6 beamsplitter cubes).
The Prior stage is equipped with adapters for a wide range of sample holders: multiwell plates, slides, petri dishes, Chamlide magnetic multiwell chambers (available WL-2460-10 and CM-S22-4) and closed Flow Cell Systems (Focht Chamber System FCS2 from Bioptech and Magnetic closed chamber CF-T12 from Chamlide).
For the illumination there are different options available.
- Trans-illumination – Halogen Lamp.
- Widefield Epifluorescence illumination: Mercury Lamp
- Objective TIRF illumination: Laser diodes: 405 nm and 561 nm. Used for PALM
- Confocal Epifluorescence illumination:Gas lasers Argon (488 nm) and Helium-Neon (543 nm). Gas lasers are fiber coupled to Nikon C1 Confocal System unit
Emission detection. For confocal measurements detection unit with 3 PMTs is available. For widefield/PALM – EMCCD Ultra 897 camera (Andor) with Oasis chilled system.
Software: Nikon Confocal Microscope C1 EZ-C1 Software, MicroManager, LabView, ImageJ/FIJI, Matlab.
