Perfect Focus System (PFS) | Perfect Focus System (PFS)

Key Words: Dynamic Focus, Fluorescence, Perfect Focus, Time-Lapse, TIRF

Definition:The Perfect Focus System (PFS) is a system that automatically maintains focus so that the point of interest within a specimen is always kept in sharp focus no matter what mechanical or thermal changes take place.

TECHNOLOGY:

Long-term time-lapse imaging of live cells is complex and dependent on a number of variables, especially those related to maintaining the focal plane of interest. Nikon's Perfect Focus System (PFS) provides real time focus correction to overcome focus drift caused by thermal and mechanical effects for dramatically increased quality of long-term time-lapse imaging data in live cells.

APPLICATIONS:

In recent years, live cell imaging has become an essential tool in many cell biology laboratories because of the wealth of information it can provide into the fundamental nature of cellular function. Many of biology's most interesting questions, including those governing the growth, division and apoptosis of living cells, can best be answered by microscopic observations of the cells over time using long-term time-lapse imaging techniques.

The benefits of long-term live cell time-lapse imaging, however, are inevitably accompanied by many challenges, namely those associated with keeping the cells alive and in focus throughout the course of the experiment, which may be hours, days or even weeks. Not only are living cells sensitive to photodamage and to environmental factors such as temperature, pH and CO2 levels, maintaining the precise focal plane of interest, which can easily shift during imaging with vibrations, changing temperature or addition of media, can also be extremely challenging.

The Perfect Focus System (PFS) (Figure 1) is perhaps the most significant advance in live cell imaging in recent history. Designed to combat focus drift during long-term experiments caused by thermal and mechanical effects, the System eliminates the need for the researcher to constantly readjust the focus during the course of the experiment, thereby enabling imaging experiments to be conducted for longer than ever before possible, with increased accuracy.

PFS works by reflecting a semicircle of infrared light from an 870 nm LED off of the coverslip and projecting that reflection onto a linear CCD sensor in order to track the position of the coverslip (Figure 2). By combining this highly sensitive feedback system with the accurate Z-axis control of the TiE microscope, focusing precision of less than 1/3 the focal depth of the objective is easily achieved. Furthermore, the Nikon Perfect Focus System contains proprietary optical offset technology, which allows the researcher to focus at the desired height above the coverslip while simultaneously tracking the focus of the coverslip interface. This technology allows focus correction to be achieved at a 5 ms sampling rate, making the PFS orders of magnitude faster, and considerably less prone to error than other manufacturer's systems that must continuously shift between the coverslip interface and the focal plane of interest.

Tested and proven in the TE2000-PFS system, Nikon's Perfect Focus Technology has been further improved in the new TiE-PFS. Now seamlessly incorporated into the microscope's nosepiece and operable via controls on the front panel of the microscope, the TiE-PFS is fully integrated and no longer takes up a level of the infinity space. Additionally, the range of usable fluorescence wavelengths has been expanded to 340-750 allowing far red fluorophores such as Cy5 and calcium dyes like Fura-2 to be used. IR transmission (e.g. 1064 nm) has also been improved, making the new TiE-PFS compatible with laser trapping and laser tweezer applications. Finally, the TiE-PFS can be used with over 52 objectives, including oil immersion and dry lenses, a selection of phase contrast lenses, and several long working distance options.

Nikon's PFS system is ideal for a variety of applications including TIRF. In most commercial applications of TIRF microscopy, a laser beam undergoes total internal reflection at the interface between the specimen and the coverslip, and fluorescence is excited with resulting evanescent wave. Since this wave only illuminates ~50 nm into the sample, tight control of focus is essential for effective long-term time-lapse TIRF imaging. Additionally, many live cell imaging applications require the addition of media or a drug solution during the course of the experiment. As demonstrated in the accompanying images of fluorescent microspheres (Figure 3), taken at the indicated times with and without the Perfect Focus System engaged, the change in temperature caused by adding media (indicated by the arrow) causes the focus to drift if PFS is not used; while engaging the PFS system completely eliminates this problem. A final example of an application benefiting from the TiE-PFS is multipoint imaging. As shown in the accompanying DIC images of cells taken at multiple stage positions, it is possible to change the XY position of the microscope stage without losing focus, making true 6D multipoint imaging possible and truly revolutionizing long-term live cell imaging.

The Nikon Instruments Perfect Focus System greatly increases the ease and success with which long-term time-lapse live cell imaging experiments can be performed. Utilizing unique optical offset technology, the Nikon PFS system is extraordinarily fast and precise, while exhibiting extreme stability over any other system available to biological researchers. Additionally, Nikon PFS allows the microscope to remain in focus for days or even longer without any user intervention, even with addition of media or movement of the microscope stage. The Nikon TiE-PFS is having a profound effect on the quality of imaging data obtainable to researchers performing long-term time-lapse imaging of living cells.