Table of Contents
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Location: [Oeiras] 0B06
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XXX Quality Control
XXX Usage StatisticsMicroscope overview
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.
Click on the image on the right to see it in higher detail
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)
| 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.
- Turn on the fluorescence lamp
- 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…)
