Multiphoton Laser Scanning Microscope. Deep Imaging
The FV1000MPE provides cutting-edge technology for various areas of scientific research such as neuroscience and cell biology and is available in three configurations.
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Features of FluoView FV1000MPE
Newly designed multiphoton objective: XLPLN 25x W MP Especially for deep-tissue multiphoton imaging, Olympus designed a new objective lense, taking into account all the MPE-related requirements. It has very high transmission both in the IR as well as in the visible range - to get as much IR light to the sample as possible and to efficiently collect vivible light fluorescence. With the NA of 1.05 the excitation volume will be very small and the enhanced large field-of-view allows efficient collection of the scattered fluorescence signal. The working distance of 2mm and the ceramic tip make this water immersion lens the optimum tool for in-vivo imaging. Additionally, this objective is equipped with a correction collar to allow the compensation of spherical aberrations induced by the sample tissue when imaging deep within a specimen.
Use of a femtosecond pulsed IR laser with pulse pre-compensation In multiphoton microscopy, a pulsed laser is required to achieve sufficient excitation efficiency of the fluorophores. As laser pulses (typically ~100fs) are stretched when travelling through microscope optics (dispersion), fluorophore excitation and fluorescence detection will become less efficient. By using a femtosecond pulsed laser with pulse-precompensation (negative chirp) this effect can be strongly reduced and the original pulse-width almost completely restored. Either a Mai Tai® DeepSee (Spectra-Physics, a unit of Newport Corporation) or Chameleon™ Vision (Coherent, Inc.) can be adapted to the FV1000MPE. Both lasers exist as special Olympus versions which are designed to achieve a perfect marriage of laser and multiphoton microscope.
Motorized beam expander optics As the probability for multiphoton excitation is very low it is crucial to have a very small exciation volume (requiring objectives with high NA) and to guide as much IR light to the sample as possible - that means the back opening (backfocal aperture) of the objective should be filled completely by the excitation laser beam. Having the beam larger than the backfocal aperture would mean loss of excitation laser light. The backfocal apertures of objectives sometimes differ by more than a factor of two - either leading to losses or to reduced NA. With implementing motorized beam expander optics Olympus ensures that for all the objectives and different wavelength used the size of the IR beam fits to the backfocal aperture of the objective.
Efficient fluorescence acquisition via a flexible non-descanned detector system With multiphoton microscopy, efficient detection of fluorescence is a crucial topic, especially as this fluorescence has been emitted from deep within a specimen and scattered multiple times on its way towards the detectors. The FV1000MPE provides very efficient detection with minimal loss using an external non-descanned detection (NDD) system placed close to the specimen. The detection system can be equipped with two or four PMT detectors in the reflective pathway (fluorescence collection via the objective lens). Additionally, the emitted fluorescence can by collected via a special condensor lens in the transmission pathway and directed onto two PMT detectors. This detection pathway can prove extremely useful for SHG imaging or just additional collection of fluorescence from deep in the sample close to the lower surface. Dichromatic mirrors are used in the light path to separate the emitted fluorescence; the mirrors and filters can be changed easily according to the specimen’s fluorescence emission characteristics.
Simultaneous MPE imaging and MPE stimulation Analogue to the confocal system FV1000 the FV1000MPE can incorporate two laser scanners in a single compact design for simultaneous fluorescence imaging and independent laser light sitmulation - in all imaginable ways: confocal imaging with pinpoint MPE stimulation or MPE imaging with visible light stimulation or MPE imaging with MPE stimulation. Any region of interest can be specified for stimulation and scanning independently, with unrestricted control of variations in timing, duration and intensity. With this setup Olympus takes a variety of applications, including FRAP, FLIP, photoactivation, photoconversion, uncaging, laser ablation and many others deep into living tissue.