Evident/Olympus MPE
Evident/Olympus multiphoton microscope - funded by BBSRC ALERT
Multiphoton microscopy allows imaging at much greater depth than standard single-photon fluorescence imaging, and is the gold-standard for imaging in live thick samples. The multiphoton (MPE) comes with two broad-spectrum femtosecond pulsed lasers, four detectors, a piezo z-stage, and a resonant scanner, making it ideal for imaging multiple fluorophores at relatively high speed. It also has an additional scanner for simultaneous imaging and photo-manipulation/ablation.
The system comes with additional fluorescence lifetime imaging (FLIM) capabilities from PicoQuant for quantitative biosensing experiments.
The incubator, with temperature and CO2 control, make it excellent for imaging live samples, including embryos and tissue.
Located in the basement of IBRB, in room IBRB03.
Educational papers and resources
, old but nice.
on things to consider to get the most out of a multiphoton microscope especially when doing quantitative measurements and how to ensure quality control.
Tilt of your coverslip is important says 1.5 degree tilt can lose you 75% of your signal.
Does using the fast resonant scanner help with photo-bleaching/toxicity?
This seems unclear, and the best approach is to test with your sample. See the following for discussions on the , , and .
Absorption spectra of some dyes (no promises on the reliability of these):
See fpbase.org for other dyes and fluorescent proteins (make sure to verify yourself).
Opening files
The .oir files open in Fiji (not ImageJ) by just drag dropping. Sometimes there are problems and you have to install a separate plugin,
The FLIM data from the SymPhoTime software can be opened for quick and simple viewing with a Fiji plugin called PTU_reader, .
Please acknowledge the below funding in your publications, in addition to following the usual CAMDU acknowledgment policy.
BBSRC BB/T018070/1
Components overview (see Hardware details for further details)
| Objectives | 60x silicone, 60x oil, 30x silicone, 20x air, 10x air |
| Lasers | 2 x fs pulsed lasers tunable from 690-1300 nm |
| FRAP | All available wavelengths |
| Detectors | 2x PMTs, 2x GaAsPs, and 2x FLIM PicoQuant PMA Hybrid |
| Stage | motorised xyz |
| Incubation | Up to 37 degrees and CO2 (contact CAMDU if you want to use CO2) |
| Filters | Options available for GFP/RFP, CY5, SHG, NADH |
| Filters for FLIM |
Currently in microscope is a 488 dichroic between the FLIM detectors with 693/46 and 440/40 "cleanup filters". Also available are 483/40 and 550/49. |
| Autofocus | Hardware-based autofocus |
Located in room IBRB03 in the Interdisciplinary Biomedical Research Building.
If you are interested in using this system please contact camdu@warwick.ac.uk for further information.