Active Microscope Stabilization in Three Dimensions Using Image Correlation.

Abstract

Background: Super-resolution microscopy techniques are often extremely susceptible to sample drift due to their high spatial resolution and the long time needed for data acquisition. While several techniques for stabilizing against drift exist, many require complicated additional hardware or intrusive sample preparations. We introduce a method that requires no additional sample preparation, is simple to implement and simultaneously corrects for x, y and z drift.

Results: We use bright-field images of the specimen itself to calculate drift in all three dimensions: x, y and z. Bright-field images are acquired on an inexpensive CCD. By correlating each acquired bright-field image with an in-focus and two out-of-focus reference images we determine and actively correct for drift at rates of a few Hertz. This method can maintain stability to within 10 nm for x and y and 20 nm for z over several minutes.

Conclusion: Our active drift stabilization system is capable of simultaneously compensating x, y and z drift through an image-based correlation method that requires no special sample treatment or extensive microscope modifications. While other techniques may provide better stability, especially for higher frequency drift, our method is easy to implement and widely applicable in terms of both sample type and microscopy technique.