Zeiss LSM780 CLSM with inverted stand
Zeiss LSM780 CLSM with inverted stand at UPSC.
This confocal has laser excitation lines at 405, 458/488/514, 561 and 633 nm and a 32-channel GaAsP spectral detector and two additional channels + transmission light (DIC) detector. The confocal is mounted on an inverted microscope with motorized XYZ stage and there are four objectives of 10x 0.45 dry, 25x multiimmersion, 40x/1.2 water and 63x/1.4 oil. This confocal can be used for FRAP, FRET, colocalization studies, 3D imaging, time lapse studies with up to 8 fps at 512x512 pixel, spectral range scans, tile scans, multiposition scanning, linear unmixing, online fingerprinting, photon counting, etc.
The confocal is located in room KB.K2 (B2.18.51). This instrument is operated on an hourly fee basis. You are allowed to use this confocal only after passing a mandatory introduction! A basic introduction to this instrument usually takes 4 h and each introduction is maximized to two participants. Due to heavy usage, expect to wait 3 weeks or more for an introduction. For introduction or other questions regarding this instrument, please contact Anna Gustavsson.
Zeiss LSM 800 with airyscan
Zeiss LSM 800 with airyscan at UPSC.
Arabidopsis hypocotyl stained with propidium iodine.
The confocal is mounted on an upright Axioimager.Z2 microscope with motorized XYZ stage. There are five objectives of 10x/0.45 Plan-apo dry, 20x/0.5 water-dipping N-Achroplan, 40x/1.0 water-dipping N-Achroplan, 40x/1.2 water and 63x/1.4 oil and many of them are adjusted for DIC. This confocal has laser excitation lines at 405, 488, 561 and 640 nm, 2 single GaAsP detectors, airyscan detector optimal for 40x objectives and transmission light (ESID) detector. It is also equipped with an axiocam 506 color camera and LED lamps for brightfield and fluorescence imaging.
The confocal can be used for FRAP, colocalization studies, 3D imaging, time lapse studies with up to 8 fps at 512x512 pixel, tile scans, multiposition scanning, fast panorama overview of whole sample with pyramidal imaging, setting up of complex acquisition experiments with experimental designer, imaging of both non-fluorescent dyes in combination with fluorescence etc.
The confocal is located in room KB.K2 (B2.18.51). The instrument is operated on an hourly fee basis. You are allowed to use this confocal only after passing a mandatory introduction! A basic introduction to this instrument usually takes 4 h and each introduction is maximized to two participants. Due to heavy usage, expect to wait 3 weeks or more for an introduction. For introduction or other questions regarding this instrument, please contact Anna Gustavsson.
Zeiss LSM 880 with airyscan and PicoQuant Fluorescence Lifetime Imaging (FLIM), Fluorescence Correlation Spectroscopy (FCS) and FCCS
Zeiss LSM 880 with airyscan.LSM 880:
The confoc
Arabidopsis apical hook stained with propidium iodine.al is fully motorized and built on an Axio Observer 7 inverted microscope with a scanning stage and 13 mm Z travel range. There are adjustable holders for slides, 24-98 mm Petri dishes and for standard multiwell plates. It has 6 objective lenses where most have DIC: 10x/0.45 dry Plan-Apo, 20x/0.8 dry Plan-Apo, long working distance 32x/0.85 water corr C-achroplan, 40x/1.2 water corr (good for FCS), 40x/1.2 Imm autocorr Plan-Apo for water, silicone oil or glycerine immersion, 63x/1.3 imm autocorr Plan-Neofluar for water,silicone oil or glycerine immersion.
For laser excitation there are continuous laser lines for 405, 440 (PicoQuant cw or pulsed), 458/488/514, 561 and 640 nm.
The detector unit is a QUASAR spectral detection system (2 PMT detectors and a 32-channel GaAsP spectral detector) and one airyscan detector for high sensitivity and resolution detection.
PicoQuant FLIM and FCS:
There are pulsed (also cw) lasers of 440, 485, 510 and 561 nm. Two sensitive HyD detectors for FLIM, FLIM-FRET, FCS and FCCS detection with time detection MultiHarp 150 4N. The software SymPhoTime 1+2 for analysis and data acquisition.
The Zeiss LSM 880 and PicoQuant systems are connected and synchronized.
This confocal can be used for FRAP (fluorescence recovery after photobleaching), FRET (fluorescence resonance energy transfer), colocalization studies, 3D imaging, time lapse studies with up to 8 fps at 512x512 pixel, spectral range scans, tile scans, multiposition scanning, linear unmixing, online fingerprinting, photon counting, ~2x higher resolution than standard confocal with airyscan, FLIM, FLIM-FRET, FCS, FCCS etc.
The confocal is located in room KB.K2 (B2.18.51). This instrument is operated on an hourly fee basis. You are allowed to use this confocal only after passing a mandatory introduction! A basic introduction to this instrument usually takes 4-8 h and each introduction is maximized to two participants. Due to heavy usage, expect to wait 3 weeks or more for an introduction. For introduction or other questions regarding this instrument, please contact Anna Gustavsson.
Stellaris 8 DIVE
Multiphoton microscope Leica Stelaris 8 at UPSC.
The confocal and multiphoton microscope is fully motorized and built on a DMi8 inverted microscope with a scanning stage. It has a Z motion range and a Galvo stage with up to 1.5 mm moving range (Super Z range). It is a spectral system for both confocal and multiphoton imaging. There are adjustable holders for slides, 24-98 mm Petri dishes and for standard multiwell plates. As alternative to standard stage there is a cooling/heating stage from 10°C below room temperature up to 37°C for 35 mm Petri dishes. Fluorescence metal halide light source with filter cubes FITS LP, GFP BP, Y3 (red) BP. The system stands on an active antivibration table.
Objective lenses:
- HC Plan APO 10x/0.4 air, WD 2.56 mm (0.17 mm cover slip)
- HC Fluotar L 25x/0.95 W (water), LWD 2.5 mm (0.17 mm cover slip)
- HC Plan APO 40x/1.25 Glycerol with motorized correction collar - adjusted inside software, WD 0.35 mm.
Available lasers:
- 405 nm laser
- White light laser (WLL): Tunable in the range of 440-790 nm in increment of 1 nm, maximum 8 lines at a time with a minimum output power of 1.1 mW. Pulse frequency 78 MHz.
- Multiphoton laser: Tunable in the range of 690-1040 nm, 1 line at a time, 2.1 W power average, > 100 fs pulse width, adjustable diameter of laser beam, internally operated in software for easy alignment and tuning. Note that the multiphoton laser is very strong and in the invisible range, do not look directly on the laser beam (which you cannot see, but it may damage your eyes)!!!
Beam splitting system:
Acusto-optical tunable filter (AOTF) sets the correct wavelengths of the WLL and acusto-optical beam splitter (AOBS) reflects the correct laser wavelengths to the sample and transmits the rest to the detector.
Scanning mirrors:
Scanning can be done with precision using X2Y scanning mirrors or fast with resonance scanner.
The field of view can be rotated 270 degrees. Is done with an Abbe-König rotator; which allows a scan of smaller field of view compared to digital rotation that some other confocal systems have.
Detectors:
3 HyD-S detectors (can be up to 5) for confocal imaging, detection range: 410-850 nm with 1 nm precision. Highly sensitive detectors can be used in analog, reflection or photon counting (1 detected photon=1 grey level) mode.
1 HyD-S and 1PMT spectral detector for multiphoton imaging (non-descanned detectors (NDD); can add up to 4 NDD detectors), detection range: 380-800 nm.
Other features:
- Lambda scan: scan for the emission spectra with given laser line.
- LAMDA scan: scan for the excitation spectra for WLL and MP lasers.
- LAMBDA-lambda scan: scan both laser excitation and emission spectra
- Z intensity compensation during Z stack acquisition and 3D image analysis software
- Rolling average during time lapse imaging
- LAS X navigator: spiral scan overview, imaging of large samples, multiposition imaging, change of focus over image plane
- Fluorescence lifetime information can be used with TauContrast for Intensity, TauSeparation, TauGating, GateScan or TauScan to separate information based on fluorescence lifetime.
- LiveData Mode
- FRAP wizard for setting up bleaching or laser ablation experiments
- Lightning is used for high resolution imaging with direct adaptable deconvolution.
Multiphoton:
Multiphoton laser is in the infrared spectra so it cannot be seen by human eyes but is very strong and dangerous upon exposure! If the laser safety box is on you are safe, but if that is removed one must take great caution to avoid the laser beam. Use laser safety glasses or cover the laser with a laser-safe cover (not any cover, it must stop the IR laser and not catch fire).
Multiphoton needs two (2-photon) or more laser pulses to converge very close in time on the same fluorophore for excitation to occur. This only happen in a very small volume of the sample (femtoliter) in the focal plane. Hence, no need for pinhole with multiphoton as it is very unlikely to get excitation from other planes than the focus plane.
The IR light generate a lot of heat so great care must be taken with laser intensity, but bleaching is very low other than in the femtoliter area where excitation occur. The small volume and heat generation is why laser ablation, bleaching and photoswitching can be done in a specific cell or compartment (confocal shine and excite fluorophores in the whole sample causing blech in several layers).
As one does not need any pinhole it is possible to use detectors that are going directly from the objective, so called non-descanned detectors (NDD), which then conserve a lot more of the photons and thus one can get higher sensitivity (around 4x better than confocal detectors) besides that one can get light from several airy unit orders of light to somewhat increase resolution. We have one PMT and one HyD (supersensitive) detector that are NDD (two more can be added if needed and somebody wants to pay for it). Due to the sensitivity, a dark room is needed to not induce noise.
Variable beam expander:
For multiphoton laser one can adjust the laser beam to maximize laser intensity or resolution.
To maximize laser intensity one underfill objective with light leading to deeper penetration but lower resolution.
To optimize resolution one overfill objective with light (b) left).
This feature is found in Beam routing - VBE, drag the bar to the left for high power and to the right for high resolution.
Second harmonics:
You can also look at second harmonics with MP, which is another form of light that is emitted at half the wavelength of the laser (e.g., with an 800 nm laser the second harmonics will be at 400 nm). This signal is usually weaker than fluorescence so using the HyD NDD detector is advisable. Only 20-30% of the second harmonics is going backwards the most efficient detector would be a forward detector, but we don’t have that. To know if it is second harmonics or autofluorescence etc. one can test bleaching; second harmonics does not bleach whereas fluorescence does or, move the emission detector slightly away from the half laser spot and second harmonics disappear as it has a very narrow detection window whereas fluorescence has a broader. For second harmonics one might need to increase the gain to higher than 100% (like 180%) as the MP may fry the sample if one uses too much laser.
Using it:
The Leica Stellaris 8 DIVE is located in room KB.K2 (B2.20.51). This instrument is operated on an hourly fee basis. You are allowed to use this confocal only after passing a mandatory introduction! A basic introduction to this instrument usually takes 4-8 h and each introduction is maximized to two participants. Due to heavy usage, expect to wait 3 weeks or more for an introduction. For introduction or other questions regarding this instrument, please contact Anna Gustavsson.
Macroconfocals
Macroconfocals are systems built for confocal imaging of larger specimens such as insects or plant materials. They are equipped with macroobjectives which allow sharp wide-field confocal imaging.
Nikon vertical macroconfocal (AZ-C2 vertical)
Front side of vertica macroconfocal Nikon AZ-C2 at UPSC.
Sample holder.
Arabidopsis root cell walls stained with propidium iodine and nuclei with DAPI.
This macroconfocal is specially built for imaging of plants in gravity on 12 x 12 cm agar plates or standard slides. The system is equipped with a AZ100 horisontally mounted macroscope with option of 2x/0.2 WD 45 mm or 5x/0.5 WD 15 mm DIC macroobjectives, a zoom range of 1-8x and diascopic or epifluorescent light (CFP, GFP, YFP and RFP) options. The samples are placed in a humidifying chamber to minimize sample drying. In addition the stage is motorized in XYZ and there is a macromanipulation holder attached to the stage. The macroscope is attached to a C2 confocal that is equipped with 405, 458/488/514, 561 nm laser lines. In addition, the system is equipped with both a filter detection system and a spectral detection system in addition to a transmission detector. This macroconfocal can be used for multicolor imaging, linear unmixing, 3D imaging, tile scans, time lapse studies, multiposition scanning, colocalization studies etc.
The confocal is located in room KB.K2 (B2.18.51). This instrument is operated on an hourly fee basis. You are allowed to use this confocal only after passing a mandatory introduction! A basic introduction to this instrument usually takes two sessions of 2-3 h or more and each introduction is maximized to two participants. For introduction or other questions regarding this instrument, please contact Anna Gustavsson.
Analysis computers:
To alleviate the using time at the microscopes and allow data analysis with the software available at the microscopes, there are two off-site image analysis computers in the room inside the confocals. These computers are connected to the image servers so that the acquired images can be easily reached.
Computer #1: This computer has software for Zeiss ZEN black 2010, Image J, Leica LAS, photoshop and windows office.
Computer #2: This computer has software for PicoQuant SymPhoTime 1+2 for data analysis of FLIM, FLIM-FRET, FCS and FCCS. Full version of Arivis vision 3D and 4D for fast visualization and analysis of multi-dimensional images. Computers are located in room KB.K2 (B2.18.51).
Data storage on microscope server:
The data must not be stored on any of the microscopes or the analysis computers for long time as space is limited on these systems. Please move all data to the dedicated UPSC microscope server. For access to this server, contact Simon Birve. Do not contaminate the computers with viruses (high risk when using USB sticks or external hard drives).