Accessories of FV1000MPE |
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 Optimal for in vivo observation
Deep tissue penetration
Minimised photodamage/phototoxicity
Reduced photobleaching |
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Unique MPE objective: XLPLN25xW-MP
Most Olympus objective lenses used for multiphoton imaging had been designed for visible und UV light microscopy and in the meantime tehir optical properties habe been improved towards the IR. This means that a lot of compromises have to be made to achieve good quality both for confocal as for multiphoton imaging, which pose very different requirements on an objective lens.Olympus decided to take a different approach and designed an objective lens optimised for the needs of multiphoton microscopy.
Optical paramters optimised for multi-photon excitation
Apart from improving the coating both in the IR as well as the visible light to approximately 90% transmission, the vignetting has been reduced - allowing for a more even illumination in the focal plane. In MPE everything is about the probability of two or more photons being absorbed simultaneously - the excitation efficiency. As this depends on the density of photons a very small focus is crucial. The XLPLN25xW-MP has a numerical aperture (NA) of 1.05. The field number - which is a measure for the field of view (FOV) - for laser scanning is 18, which fits perfectly to the possibilites of the FV1000MPE, whereas the field number for fluorescence detection is much larger: 27.5. This is important as in multiphoton imaging excitation can only happen in the focus, but the fluorescence emitted from the focal area will be scattered multiple times on its way back out of the sample. The larger the FOV is, the more of these scattered fluorescence photons will be detected.
Special correction collar for spherical aberrations
Every objective lens is designed for one immersion medium - air, oil, water etc. - creating a perfect focus for the appropriate medium. A change in the refractive index of the medium, for example using a glass cover slip with a water immersion objective, will result in spherical aberration - mainly stretching the focus in z. Correction collars for spherical aberrations are well known in confocal imaging. Olympus has taken this even a step further. As the aim of multiphoton imaging is to image deep within the sample, this means the focussed beam will have to pass through several 100µm of tissue instead of water or oil. Biological tissue consists has a different refractive index than plain water, leading to spherical aberrations. The XLPLN25xW-MP objective has a correction collar to correct for these aberrations by tissue.
Figure: fluorescent beads, embedded in high-refractive index medium (n~1.37), imaged in different depth with the excitation wavelenght 950nm. With the current state-of-the-art objectives, the focus gets elongated along z, when imaging deeper within the sample. When using the correction collar on the XLPLN25xW-MP objective, the size of the focus stays the same, even at depth of ~1mm.
Good Accessibility to the specimen
Multiphoton imaging is mainly a topic when working with live-tissue preparations or whole animals. In this case good acces to the sample is very important. Therefore the XLPLN25xW-MP was designed with a working distance of 2.0mm and an acces angle of 35°.
Analog Module for integration of external signals
This analog unit allows the read-out of two independent voltage signals by the FluoView software - thus creating two additional acquisition channels. The FluoView software displays the acquired data as images, e.g. if the voltage signal is from detectors other than the integrated ones, or as time-traces, e.g. if the signal is an electrical signal measured during a patch clamp experiment. The supplied analog signal will be digitised with the same sampling speed as the FluoView detectors. Additionally the analog module has four output lines delivering TTL-signals for the pixel sampling clock, the line active, the scan active and the complete imaging time - making synchronization with external devices straightforward.
Multi-Point Stimulation Software
This additional software module allows the design of fast optical stimulation experiments. For one single point, several individual points or even a whole field of points (32x32) a time protocol with one or several stimulations can be set up, using all lasers attached to the system. The experiment protocol will be carried out with high temporal precision, minimizing the traveling time from on defined point to the next. The Multi-Point Stimulation software is a very powerfull tool, especially in the field of electrophysiology. With its flexibility it well allows the creation of interesting experiments in other fields of research.
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