The CRIFF system provides for a very high level of focus stability, allowing a specimen to remain accurately focused for hours at a time with less than 0.1 micron drift. The system compensates for focus changes caused by temperature variations as well as mechanical drifts of the microscope mechanisms. CRIFF promises to be a solution to the focus drifts that plague time-lapse experiments at high magnification.

The CRIFF substantially eliminates focus drift in high power microscopy by effectively monitoring the distance between the objective lens and the specimen's cover slip. Minute changes in the distance between the specimen and the objective lens are sensed using an optical detector. This detector provides a feed back signal for the closed-loop positioning system which actively controls the focus of the microscope. The Z-axis positioning mechanism can be our standard MFC-2000 focus controller system, our 3-axis MS-2000 and MS-4000 XYZ stage systems, or our PZ-2000 and PZU-2000 piezo-Z top-plate stage systems as well as our manual PZM-2000 and PZMU-2000 versions.

The CRIFF module fits on the camera port of most microscopes. The unit contains a dichroic beam splitter that allows visible light to go to a camera while reflecting the 780 nm laser light. The CRIFF module has provision for a blocking filter in front of the camera. See the CRIFF Manual for further information about appropriate filter sets.

Features

• Closed-loop positioning system with feedback sensing at the sample
• Maintain long-term precise nanometer-scale continuous focusing – perfect for TIRF Microscopy
• Easily retrofits to camera port of most microscopes
• Stand-alone operation or control via RS232 serial communication
• Auto-calibration and programmable loop gain

Specifications

Background

Providing stable focus of a high power microscope over long periods of time can be extremely difficult. Several factors combine to frustrate the microscopist. The very narrow depth of focus of the microscope objective (>300 nm for high NA objectives) means that focus position must be held to less than 100 nm for optimum stability. Focusing mechanisms have inherent mechanical limitations at this scale. Minute rotation of the focus shaft or the compression of grease layers on the gears of the rack and pinion mechanism generates enough motion to defocus the microscope. Equally important are minute dimensional changes in the microscope caused by thermal expansion of the microscope components. Researchers needing to maintain accurate focus for more than a few minutes can be plagued by such focus drifts.

The CRIFF uses a laser with about 1 mW of intensity as the illumination source for the position-sensitive detector. The laser is focused into the back pupil of the objective lens, and introduced parallel to its centerline axis, offset by an amount d as shown in the figure on the right. The positioning system will make use of the small geometric shift in the position of the beam that is reflected off the cover slip / sample interface. Ordinary cover slips are used with TIRF objectives, while reflective-coated cover slips are required for objectives with an NA of less than 1.4.

CRIFF in Action

Our thanks to Dr. Clare Waterman-Storer, Scripps Research Institute, La Jolla, CA for providing this movie clip (923KB MPG) showing CRIFF holding an hour-long focus on fluorescent beads side-by-side with a similar sample without focus feedback.
Click here to play movie.