With the ever changing landscape of life science research, it is imperative that any instrument purchased not only fulfils the present requirements, but also any future ones too. This is even more important for micros-copy where new imaging techniques regularly appear and very quickly become mainstream processes ena-bling huge advances in our knowledge. To cope with the burgeoning protocols, Olympus has developed its research microscopes to act as ‘optical benches’ capable of incorporating many different modules. By using this modular approach, there is no need to compromise on any of the processes that need to be incorpo-rated. For example, the Olympus FluoView FV1000 confocal laser scanning microscope (cLSM) can be fitted with a number of advanced modules, such as the SIM scanner, a fully implemented 2nd laser scanner for in-dependent ‘photo-manipulation’ simultaneously to ongoing image recording, or a TIRFM unit using the main scanner’s lasers to provide comprehensive evanescent imaging. Optional features, like the laser based Z-drift compensation system and an on-stage CO2 incubator, ensure optimum conditions for long-term live cell imaging.

ments. Typically, photo-bleaching is car-ried out by the same scanner as the detection and therefore, there is a signifi-cant delay between bleaching and docu-mentation. As a result, early important re-covery events are missed. With the innovative SIM scanner module of the FV1000, photo-bleaching can be activated during ongoing image recording and therefore, even fast recovery reactions are not missed.

SIM scanner technology is also advan-tageous when using new fluorescent im-aging molecules that give researchers con-trol of the activation and deactivation of fluorescence by photo-manipulation. Photo Activatable Green Fluorescent Pro-tein (PA-GFP), for instance, will only fluo-resce once activated by 405 nm or 488 nm

light. PA-GFP can be used to mark tar-geted cells, organelles and proteins during time-lapse changes of fluorescent signals at different locations. More over, the fluo-rescence intensity of Dronpa can be re-duced by a strong 488 nm laser and read-ily restored with a faint 405 nm laser. As a result, repeated photoactivation experi-ments can be completed on the same cell providing excellent information about pro-tein diffusion.

Also, Kaede, a recombinant fluores-cent protein from Trachyphyllia geof-froyi, emits a green light under fluores-cent illumination. Violet or UV illumination converts the fluorescence and the molecules emit at a longer red wavelength. When violet/UV illumination is directed onto a Kaede-expressing cell,

Fig. 1: cLSM fluorescence images of Kaede-expressing HeLa cells recorded every three seconds. Simultaneous illumination with a 405 nm laser shows the photo-converted Kaede (red) progressively spreading through the cell.Courtesy of: Ms. Ryoko Ando & Dr. Atsushi Miyawaki, RIKEN Brain Science Institute

Advanced Modular Imaging for the 21st CenturyConfocal System for Extended Imaging Capabilities

Fluorescent Advances

Over the last ten years there has been a paradigm shift in microscope imaging. Techniques like Foerster Resonance En-ergy Transfer (FRET), Fluorescence Loss In Photobleaching (FLIP) and Fluores-cence Recovery After Photobleaching (FRAP), amongst others, have become common place. FLIP and FRAP both re-quire bleaching of fluorescent molecules in target areas, to assess intracellular pro-tein movements. For FLIP, bleaching is ap-plied throughout the experiment and the movement of molecules assessed by the resultant loss of fluorescence surrounding the target. In FRAP, the target is bleached for a fixed time and the recovery of fluo-rescence is used to track protein move-

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diffusion of the reddish Kaede can be monitored, providing an easy and accu-rate method for capturing cell labelling. In a similar process, the 405 nm laser can be used to activate (un-cage) caged derivatives of compounds such as ATP. Both of these protocols can be completed by a point-by-point or Region of Interest (ROI) operation using the second laser scanner (SIM scanner), while the main scanner captures images of the resultant changes.

Objective-based TIRFM

Total internal reflection fluorescence mi-croscopy (TIRFM) utilises a completely different illumination method to cLSM. The TIRFM module for the FV1000 di-rects the laser into the outer edge of the objective’s back aperture. This projects it down the edge of the objective to exit at an angle that enables total internal re-flection in the coverslip or vessel base. Therefore, this requires objectives with very high numerical apertures (1.45 and above). An electro-magnetic wave, known as an evanescent wave, is propa-gated into the sample but degrades very quickly, illuminating only fluorescent molecules within the first few hundred nanometres. The depth of penetration is determined in-part by the wavelength of the laser light used, but can also be al-tered by changing the angle at which the light enters the coverslip – this is achieved by controlling the galvanometer mirror in the main FV1000 imaging scanner. A minimum definable evanescent illumina-tion depth of 50 nm can be achieved us-ing the highest available NA 1.65 objec-tive from Olympus. For TIRFM image recording, the FV1000 platform is cou-pled with a synchronised CCD-based cell^M/^R imaging system.

By integrating the TIRFM unit with the FV1000, all laser lines can be used for both cLSM and TIRFM, enabling pre-cise multi-wavelength optical slices as well as thin layer TIRFM imaging. Com-plex time-lapse protocols such as se-quential, multi-wavelength TIRF imag-ing, and variable penetration depth recording, can be programmed by the user. The TIRF method is perfect for the investigation of a wide-spectrum of cell surface events such as endo/exocytosis, cell adhesion and the formation of junc-tions.

Remain Focused

For long-term time-lapse imaging, it is essential to keep samples in the same fo-cal plane. Unfortunately, factors such as

temperature changes can cause focus drifts. The new Olympus Z-drift compen-sation module uses a 785 nm laser to de-tect any movement in the Z-axis of a ref-erence plane that does not change with respect to the sample – e.g. coverslip. Us-ing a predefined offset distance to the sample plane of interest, a feedback loop ensures that all images are recorded in the same focal plane.

Conclusion

The modular ‘optical bench’ concept of the Olympus FluoView FV1000 cLSM en-ables the incorporation of a number of advanced units, adapting the system to a broad range of requirements. For exam-ple, the TIRFM module significantly ex-pands imaging capabilities and the Z-drift compensation device permits perfect focusing for long-term imaging. Addi-tionally, the Olympus UIS2 optics are key to the superior performance of the FV1000, with excellent transmission properties and superior aberration cor-rection from the UV to the IR ends of the spectrum. The combination of all these technologies with the advanced software ensures that both confocal and TIRFM imaging produces clear and bright im-ages with greatly reduced background noise.

Contact:Olympus Life and Material Science Europa GmbHMicroscopyHamburg, GermanyTel.: +49 40 2 37 73 5426Fax: +49 40 2 37 73 647microscopy@olympus-europa.comwww.olympus-europa.com

A

Fig. 2 A: Specimen: PA-GFP labeled HeLa cell, 488nm excitation, image acquisition every 1 second. Light stimulation: 405nm laser with intermittent stimulationB: Using caged ATP, it is possible to observe an increase in calcium ion concentration inside the cell in response to the release of caged ATP via a pulse from a 405nm laser.Courtesy of: Dr. Takeharu Nagai, Dr.Takayuki Miyauchi & Dr. Atsushi Miyawaki, RIKEN Brain Science Institute.

B

Fig. 3: Multiple fluorescence labelling of focal adhesion kinase (FAK, green fluorescence) and the F-actin cytoskeleton (red fluorescence). Compared to conventional epi-fluorescence illumination (top) of the F-actin cytoskeleton, TIRFM (centre) achieves an image of substantially higher contrast.Courtesy of Götz Pilarczyk, Fraunhofer Institut für Biomedizinische Technik, Berlin.

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