DirectOverlay™ combines AFM and optical microscopy precisely and easily
The optical calibration and import feature DirectOverlay™ set a milestone in AFM. Invented by JPK Instruments in 2006, it revolutionized the way AFM and optical microscopy could be combined. Using AFM simultaneously with optical microscopy provides complementary information from the sample. Interesting locations on the sample can be identified with a wide variety of contrast methods such as optical phase contrast, DIC or VAREL contrast. Additionally fluorescence techniques such as epi-fluorescence, confocal microscopy, TIRF, FRET, FCS, FLIM, STORM/PALM, STED etc. give insights about the behaviour or location of particular molecules.
The DirectOverlay™ feature is designed for most precise correlation of AFM and optical data while it is easy-to-use regardless of the user’s experience level.
The image shows Alexa555-labeled Rad51 proteins bound to DNA (click on the image for details).
A tip-scanning design is crucial for perfect optical integration
Combining AFM imaging or force measurements with these optical methods on the same spot at the same time is not straight forward. First of all the optical image acquisition must be independent from the AFM image recording. A tip scanning AFM design is crucial for this.
A simple overlay of AFM image data and optical images afterwards shows a mismatch of both data sets, caused by errors in the optical lens systems. These are not made for metrology measurements. To overcome this, JPK developed the patented DirectOverlay™ software feature.
- Perfect overlay of optical and AFM data with sub-diffraction limit precision
- Direct in optical image navigation and selection of AFM measurements (imaging and force curves) in the graphical user interface
- Optical image navigation to specific regions of interest, even without AFM scanning. This protects functionalized tips for molecular recognition, avoiding tip passivation from image scanning before the force measurements.
- Dramatic reduction of overview image scanning in AFM, giving faster results and lower tip contamination
Details of the DirectOverlay™ procedure can be found in the following technical notes:
Click on the images to see examples of DirectOverlay™ / AFM combined with advanced optics.