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Table of Contents
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Location: [Oeiras] 0B06 |
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Microscope 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 (Evident now!) 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 |
|---|---|---|---|---|---|
| Green | - | 525/50 nm | blue | > 515 nm | GFP, FITC, Alexa488 |
| Orange | - | 5xx/xx nm | green | > xxx nm | TRITC, Alexa568 |
| Blue | - | 460x/xx nm | violet | > xxx nm | DAPI |
Emission Filters (multi-photon)
| Detector | Filter | Transmission | Fluorochromes |
|---|---|---|---|
| CH1 (PMT) | Red | 660/40 nm | Alexa647, Cy5 |
| Ch2 (GaAsP) | Green | 595/50 nm | mCherry, Alexa568, Cy3 |
| Ch3 (GaAsP) | Far Red | 525/50 nm | GFP, Alexa488, FITC |
| Ch4 (PMT1) | Blue | 460/50 nm | DAPI, CFP or BS 2nd Harmonic |
Upon request:
(requires changing filters and the dichroic beamsplitter)
| Detector | Filter | Transmission | Fluorochromes |
|---|---|---|---|
| Ch1 | - | - nm | Cy5 |
| Ch2 | - | - nm | Alexa568 |
| Ch3 | - | - nm | Alexa488 |
| Ch4 | - | - nm | DAPI |
| DODT | - | - nm | BF |
UNDER CONSTRUCTION!!!
System Turn On Procedures
- 1. Check that the cooling and power supply for the laser are ON
- 2. Turn on the fluorescence lamp
* 3. …
System turn Off procedures
If there is another user for this microscope in the next hour:
- Leave the fluorescent lamp and lasers on
- Clean up immersion objectives
Else:
- Clean up objective(s)
- Set the laser to
standby - …
