Image processing for drift compensation in fluorescence microscopy

Steffen B. Petersen; Thiagarajan Viruthachalam; Isabel Coutinho; Gnana Prakash Gajula; Maria Teresa Neves-Petersen

doi:10.1117/12.2004273

22 February 2013 Image processing for drift compensation in fluorescence microscopy

Steffen B. Petersen, Thiagarajan Viruthachalam , Isabel Coutinho, Gnana Prakash Gajula, Maria Teresa Neves-Petersen

Proceedings Volume 8587, Imaging, Manipulation, and Analysis of Biomolecules, Cells, and Tissues XI; 85871H (2013) https://doi.org/10.1117/12.2004273
Event: SPIE BiOS, 2013, San Francisco, California, United States

Abstract

Fluorescence microscopy is characterized by low background noise, thus a fluorescent object appears as an area of high signal/noise. Thermal gradients may result in apparent motion of the object, leading to a blurred image. Here, we have developed an image processing methodology that may remove/reduce blur significantly for any type of microscopy. A total of ~100 images were acquired with a pixel size of 30 nm. The acquisition time for each image was approximately 1 second. We can quantity the drift in X and Y using the sub pixel accuracy computed centroid location of an image object in each frame. We can measure drifts down to approximately 10 nm in size and a drift-compensated image can therefore be reconstructed on a grid of the same size using the “Shift and Add” approach leading to an image of identical size as the individual image. We have also reconstructed the image using a 3 fold larger grid with a pixel size of 10 nm. The resulting images reveal details at the diffraction limit. In principle we can only compensate for inter-image drift – thus the drift that takes place during the acquisition time for the individual image is not corrected. We believe that our results are of general applicability in microscopy and other types of imaging. A prerequisite for our method is the presence of a trackable object in the image such as a cell nucleus.

Citation Download Citation

Steffen B. Petersen, Thiagarajan Viruthachalam , Isabel Coutinho, Gnana Prakash Gajula, and Maria Teresa Neves-Petersen "Image processing for drift compensation in fluorescence microscopy", Proc. SPIE 8587, Imaging, Manipulation, and Analysis of Biomolecules, Cells, and Tissues XI, 85871H (22 February 2013); https://doi.org/10.1117/12.2004273

ACCESS THE FULL ARTICLE

INSTITUTIONAL
Select your institution to access the SPIE Digital Library.

SELECT YOUR INSTITUTION

PERSONAL
Sign in with your SPIE account to access your personal subscriptions or to use specific features such as save to my library, sign up for alerts, save searches, etc.

PERSONAL SIGN IN

No SPIE Account? Create one

PURCHASE THIS CONTENT

SUBSCRIBE TO DIGITAL LIBRARY

50 downloads per 1-year subscription

Members: $195

Non-members: $335 ADD TO CART

25 downloads per 1 - year subscription

Members: $145

Non-members: $250 ADD TO CART

PURCHASE SINGLE ARTICLE

Includes PDF, HTML & Video, when available