FV1000-IX81 Confocal Microscope From Olympus

In our laboratory, we routinely use fluorescent labels for visualization and localization of different protein targets, using laser scanning confocal microscopy. We most often use double or triple combinations of labels so it is important for us to have good equipment that provides the most sensitive visualization of each fluorescent probe, precise discrimination of different fluorescent labels, and reproducibility of our quantitative results. Also, the cross-talk necessarily present in multi-label experiments should be limited as much as possible. That is why I am constantly monitoring technological advances in the field of confocal imaging, looking for a state of the art microscope adapted to our needs.

During a long period, I explored the capabilities of a new spectral Olympus model, the FV1000-IX81, equipped with four diode lasers: 405 nm, 473 nm, 559 nm and 635 nm, and with four spectral detection channels and one transmitted light detection channel. The system was coupled to the Olympus IX81 inverted microscope.

I was impressed with how this generation of Olympus laser confocal microscope has progressed in comparison to the previous generation, that of the FV500. First, this confocal microscope comes equipped with a complete set of diode lasers, which are considerably less cumbersome and noisy than gas lasers, and moreover, emit much less heat, which is important when you have limited room for the system.

Specificity of signal recovery is guaranteed by the presence of an independent photomultiplier tube (PMT) into each detection channel, and spectral scanning is assured by diffraction grating. This method is highly efficient and assures 2 nm spectral resolution (one of the best available), so that cross-talk from two fluorescent labels can be separated. Also, acquisition of the ë (lambda) series (a series of different “portions” taken with a fixed interval across the emission spectrum) allows spectral unmixing with or without previously recorded spectral data for the markers used. Nevertheless, for optimal unmixing, fluorescence intensities should be as similar as possible. To amend the sensitivity of the system, a special coating was introduced for the emission filters and dichroic mirrors, allowing a better signal transmission, which is important when the fluorescence is weak.

What is really worth noting is the new scanner for FRET; this new tornado scanning makes the bleaching procedure much more efficient. However, it is only available for the SIM scanner version (much more expensive than a conventional scanner).

The only regrettable point of the hardware configuration is the presence of a single pinhole, whose diameter cannot be adjusted to best fit each fluorescence spectrum during sequential acquisition (although this is possible on some models).

A feature that I really like is that realizing the XZ series (as well as XZT, XZëT) does not require a special Z-motorized stage. The same stage also allows XY motorization, so it is possible to automatically create a mosaic of an entire region of interest via a dedicated mosaic wizard. The acquisition software is very complete and even includes the Imaris program for 3D reconstruction, but it is also really expensive. The interface is very friendly and you do not have to think about the hardware configuration (dichroic mirrors assembly) necessary for your particular fluorescent marker combination. Images can be recorded with resolution up to 4096 x 4096 pixels and exported under OIB/OIF image format (format proper to Olympus, it conserves all acquisition tags), or under conventional formats: JPEG, BMP, TIFF, AVI.

Overall, this is a really versatile and competitive state of the art confocal microscope.