System and method for simultaneous detection of secondary electrons and light in a charged particle beam system

摘要

A method and system for the imaging and localization of fluorescent markers such as fluorescent proteins or quantum dots within biological samples is disclosed. The use of recombinant genetics technology to insert “reporter” genes into many species is well established. In particular, green fluorescent proteins (GFPs) and their genetically-modified variants ranging from blue to yellow, are easily spliced into many genomes at the sites of genes of interest (GoIs), where the GFPs are expressed with no apparent effect on the functioning of the proteins of interest (PoIs) coded for by the GoIs. One goal of biologists is more precise localization of PoIs within cells. The invention is a method and system for enabling more rapid and precise PoI localization using charged particle beam-induced damage to GFPs. Multiple embodiments of systems for implementing the method are presented, along with an image processing method relatively immune to high statistical noise levels.

主权项

1. An image processing method for stored image data comprising:
directing a charged particle beam in a pattern across a region of a sample; detecting charged particles emitted from the sample due to the impact of the charged particle beam to form a charged particle imaging signal; forming a charged particle beam image using the charged particle imaging signal; detecting light emitted from the region of the sample, the amplitude of the detected light signal over time forming a curve; convolving the curve with a smoothing function to generate smoothed light signal data; combining the smoothed light signal data with a derivative function to generate derivative light signal data; determining values corresponding to local minima of the derivative light signal data and the position of the charged particle beam corresponding to the local minima of the derivative light signal data and; comparing the values of the minima with a threshold value; and displaying an indicator on the charged particle beam image if the value of an individual local minimum is lower than the threshold value, the indicator showing the position of the charged particle beam when the amplitude of the detected light signal changes.