In vivo imaging: reaching new depths

15th December 2014

Posted By Paul Boughton

Neuronal plasticity is visualised 2mm deep within the brain with the FVMPE-RS. z-stacking of neuronal tissue is cleared with the Scaleview reagent
Tail muscles of a zebra fish visualised with the FVMPE-RS via second harmonic generation (SHG; green)

A high-speed, high-precision, deep-imaging multiphoton system presents new possibilities in deep, in vivo imaging. By Bülent Peker.

Multiphoton excitation microscopy combines laser-scanning microscopy with multiphoton fluorescence to create high-resolution, three-dimensional images. It can be used to probe deep within living tissues without damaging the sample, presenting a powerful tool within life science research, thereby achieving fundamental insights into the intricate and complex workings of biological systems, in vivo. 

Olympus has responded to the need for a high-performance in vivo imaging system with the FluoView FVMPE-RS. Dedicated to multiphoton microscopy, the system delivers rapid and accurate performance, enabling sharp image acquisition and measurement deep within the sample, even when working under the most demanding conditions. The system is ideal for a host of advanced imaging applications, from multicolour in vivo imaging to combined optogenetics (a technique using light to selectively activate ion channels in living specimens) and electrophysiology studies. Its flexible and modular design also offers adaptability for researchers designing their own custom-built systems.

The FVMPE-RS achieves exact timing of multiple imaging and stimulation protocols, as well as extended and complex time-lapse experiments. Furthermore, through advanced programming functions, sophisticated multi-position imaging and optogenetic stimulation protocols can now be effortlessly accomplished, unravelling the mysteries of brain function.

It has the ability to capture 438 fps at a resolution of 512x32 and at field number 18. Researchers can now track in great detail the most rapid processes taking place within the cell, tissue or organism – from the transport of mitochondria through neurones to blood flow analysis, or even Ca2+ signalling – all in real time.

Observing larger areas is critical for many of these functional imaging studies, and the system also captures full-frame, 512x512 images at 30 fps without compromising the field of view. This is made possible by the hybrid scanning unit, which builds on the capabilities of the high-speed resonant scanner.

A galvanometric scanner is also available for maximising the signal to noise ratio during deep imaging. With the ability to seamlessly switch between the two, rapid and smooth imaging with excellent definition is now realised. An additional galvanometric scanner module is also available for simultaneous 3D stimulation and image acquisition in the sample, catching the fastest responses.

Synchronising excitation and imaging with this SIM scanner setup is ideal for quantitative photomanipulation experiments and also enables advanced optogenetics studies. This neuromodulation technique controls and monitors neuronal activity using light sensitive proteins channelrhodopsin and halorhodopsin, and with the FVMPE-RS, laser light stimulation of these optogenetic ‘switches’ is achieved alongside simultaneous real-time imaging of neuronal cell activity.

Also of importance in many biological processes are the interactions of cells within tissues or organs. Measuring rapid fluctuations in groups of cells with a high signal-to-noise ratio is now achieved with both precision and speed using multipoint mapping, an approach where dedicated scanning patterns allow measurements of fluorescence and electrical responses without crosstalk from neighbouring pixels – ideal for electrophysiology, optogenetics or studies with a systems biology focus. For example, this capability can generate information into how specific cell types contribute to the overall network function of an organ such as the brain.  

Precise multicolour excitation and imaging

Providing further insights into cell connectivity and how different cellular structures interact, the FVMPE-RS is optimised for tracking multiple molecular species simultaneously. Through multi-wavelength excitation, crosstalk is minimised to produce a clearer definition between multiple fluorophores. Moreover, with the new system, Olympus has introduced a four-axis auto-alignment system for precise alignment of multiple laser beams, eliminating pixel shifts caused by excitation beam angle mismatches. This results in clear separation of multiple fluorophores, enabling multiphoton excitation multicolour experiments of unmatched quality. 

Deep-tissue observation has revolutionised many areas of life science, for example enabling in vivo studies of brain function – reaching depths of up to 1.3mm under in vivo conditions. Depths of 8mm can also be reached in non-living tissue treated with the Scaleview tissue transparency reagent or other methods (Fig. 1. shows visualisation of neuronal plasticity at a depth of 2mm). Such investigations have been made possible not only by the deep penetration of IR light through tissue, but also by improvements in detection sensitivity.

From dedicated multiphoton objectives to a high-sensitivity GaAsP detector, optical efficiency is prioritised at every point of the FVMPE-RS’ light path, achieving bright, high-resolution images deep within the sample. Allowing the use of low laser power protects living cells against phototoxicity, and precisely adapting the laser to match demanding sample conditions is also realised in ‘Deep Focus Mode’, which is ideal for in vivo samples with heavy scattering.

Expanding the IR range to accommodate a wider range of fluorophores, the FVMPE-RS now offers optimal multiphoton excitation up to 1300nm, with the ability to support wavelengths of 1600nm. These longer wavelengths are also well suited to studies utilising third- and second-harmonic generation techniques (Fig. 2). Such label-free imaging allows researchers to visualise structures such as collagen and haemoglobin in their natural state – a powerful technique when combined with in vivo imaging.

Uniting speed, precision and sensitivity with an extended IR range, the FVMPE-RS system is ideally suited to a variety of deep-observation, in vivo imaging applications.

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Bülent Peker is product manager of Laser Scanning Microscopy, Olympus Europa.





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