Location: [Oeiras] 0B06
Manufacturer: Prairie (now Bruker!)
Model: Prairie Ultima
Nickname: "Prairie"
Software: PrairieView 4.5
Year: 2008
SN: N/A
 
Data older than 3 months is automatically deleted!  
 
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===== Microscope overview ===== {{ :confocal_vs_2photon.png?0x300}} A multi-photon, or 2-photon, microscope uses modules similar to those of a laser scanning confocal microscope: A laser source for sample excitation, a scanhead with galvo mirrors to scan the excitation beam, and photo-multiplier tubes to detect fluorescence signals. 2-photon imaging has advantages over confocal: the use of a near-infrared laser allows deeper penetration into tissues, the radiation is less energetic and potentially less phototoxic, and because the excitation effect is confined to the focal plane, de-scanning and pinholes are not necessary, so there are less light looses on the detection side. This Prairie Ultima system is integrated with a custom modified fixed stage ready for intra-vital imaging, and 2photon is ideal for studies in thick tissue slices, in-vivo, or imaging of small organisms. {{:info.png?25|}} Click on the image on the right to see it in higher detail {{:warning.png?25|}} Data files older than **3 months** will be automatically deleted on this system, please copy your data to the institutional server using the desktop link. ===== System components ===== ==== LASERs ==== ^ Laser Unit ^ Wavelength ^ | Coherent - Chameleon | 650 - 1050 nm | ==== Objectives ==== ^ Magnification ^ Model ^ Immersion ^ NA ^ WD (mm) ^ Reference ^ | 20x | Olympus (now Evident!) XLUM FL Plan | Water (dipping) | 1.00 | 2.00 | [[https://www.thorlabs.com/thorproduct.cfm?partnumber=N20X-PFH]] | ==== Filtersets (Ocular) ==== ^ Filterset ^ Reference ^ Excitation ^ Dichroic ^ Emission ^ Fluorochromes ^ | 2p (pos1) | - | mirror | - | - | all | | Green (pos3) | - | blue | - | ~525 nm | GFP, FITC, Alexa488 | | Orange (pos4) | - | green | - | ~580 nm | TRITC, Alexa568 | | Blue (pos5) | - | violet | - | ~460 nm | DAPI | ==== Emission Filters (multi-photon) ==== {{::xxfiltersetDiagram.png |}} \\ ^ Detector ^ Filter ^ Transmission ^ Fluorochromes ^ | CH1 (PMT) | Red | 640/680 nm | Alexa647, Cy5 | | Ch2 (GaAsP) | Green | 570-620 nm | mCherry, Alexa568, Cy3 | | Ch3 (GaAsP) | Far Red | 500-550 nm | GFP, Alexa488, FITC | | Ch4 (PMT)* | Blue | 435-485 nm | DAPI*, CFP or SHG | *Ch4 can be used either in fluorescence mode, or alternatively as a BF ("DODT") channel or for SHG - ask staff \\ **Upon request:** \\ (requires changing filters and the dichroic beamsplitter) UNDER CONSTRUCTION!!! ===== System Turn On Procedures ===== * Check the cooler. The temperature should be 20 Cº. {{::img.png?100|}} * Turn on the fluorescence lamp {{1_img.png?100|}} * Switch the laser from "stand by" to "on" by turning the key clockwise (unit under the table) * Make sure the workstation is ON * Turn on the pre-amps for the detectors you will use (Ch2 and Ch3 pre-amp unit in shelf above monitor, and Ch1 and Ch4 below the keyboard. Note that any switching of the Ch1&2 pre-amps should be preceded by "override" for protetion). * Ask staff to change the Ch4 for DODT (BF) detector or fluorescence/SHG ===== System turn Off procedures ===== * Transfer your data to the institutional server (data is deleted every 3 months) * Clean up immersion objective(s) * Remove all your samples and accessories and leave the working area ready for the next user If there is another user for this microscope in the __next hour__: * Leave the fluorescent lamp and LASER "ON" * Inform staff you are leaving the system "ON" Else: * Set the LASER to "Standby" (LASER key to the left) * Turn "OFF" all pre-amps (make sure to protect 1st for Ch1&2! * Turn "OFF" the fluorescence lamp * Turn "OFF" monitor, lights, and close the microscope curtains * Make sure you turn "OFF" any life support unit used (heating, CO2, O2...) ---- [[resources|Back to the Equipment page]]