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

• US 9,040,909 B2
• Filed: 11/20/2012
• Issued: 05/26/2015
• Est. Priority Date: 01/30/2011
• Status: Active Grant

• Current Assignee: FEI Company (Thermo Fisher Scientific Incorporated)
• Original Assignee: FEI Company (Thermo Fisher Scientific Incorporated)
• Inventors: Parker, N. William, Utlaut, Mark W.
• Primary Examiner(s): Logie, Michael
• Assistant Examiner(s): MCCORMACK, JASON L
• Application Number:
  US13/681,746
• Publication Number:
  US 20140131573A1
• Time in Patent Office: 917 Days
• Field of Search: 250/305, 250/306, 250/307, 250/311
• US Class Current: 250/306
• CPC Class Codes: G01N 21/6428 Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" in vivo A61B5/00; immunoassay G01N33/53
• CPC Class Codes: G01N 21/6458 Fluorescence microscopy fluorescence microscopes per se G02B21/0076 and G02B21/16
• CPC Class Codes: G01N 21/6486 Measuring fluorescence of biological material, e.g. DNA, RNA, cells G01N21/6428 takes precedence
• CPC Class Codes: G01N 23/225 using electron or ion
• CPC Class Codes: H01J 2237/206 Modifying objects while observing
• CPC Class Codes: H01J 2237/2443 Scintillation detectors
• CPC Class Codes: H01J 2237/24455 Transmitted particle detectors
• CPC Class Codes: H01J 2237/2448 Secondary particle detectors
• CPC Class Codes: H01J 2237/2803 characterised by the imaging method
• CPC Class Codes: H01J 2237/2857 Particle bombardment induced emission
• CPC Class Codes: H01J 37/228 whereby illumination and light collection take place in the same area of the discharge
• CPC Class Codes: H01J 37/28 with scanning beams H01J37/268, H01J37/292, H01J37/2955 take precedence
• CPC Class Codes: H01J 37/285 Emission microscopes, e.g. field-emission microscopes

Abstract

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.

Claims

1. A charged particle system, comprising:

a charged particle optical column for directing a charged particle beam onto a sample;

a secondary electron detector for collecting secondary electrons emitted from the sample upon impact of the charged particle beam;

a light detector for detecting light emitted by the sample; and

an electrically conductive mirror, the mirror configured to;

reflect toward the light detector the light emitted from the sample; and

provide an electric field to deflect the secondary electrons from the sample into the secondary electron detector.

2. The system of claim 1 in which the mirror is a paraboloidal mirror for collecting light from an area of the sample, the focal point being in proximity to the impact point of the charged particle beam.

3. The system of claim 1 in which the light detector detects light emitted by the impact of the charged particle beam on the sample.

4. The system of claim 1, further comprising a beam splitter located between the mirror and the light detector;

the beam splitter being configured to;

transmit the light reflected by the mirror into the light detector; and

reflect the light from a light source towards the mirror.

5. The system of claim 4, further comprising a color filter located between the beam splitter and the light detector, the transmissivity of the color filter being adjusted to largely block transmission of light from the light source, while passing light emitted by the sample.

6. The system of claim 1 in which the solid collection angle for both light emitted from the sample and for secondary electrons is greater than π

/2 steradians.

7. The system of claim 1 in which the light detector comprises a multiplicity of detectors, each detector in the multiplicity of detectors being configured to collect light of a different frequency.

8. The system of claim 1 in which the light detector and mirror are positioned above the sample, and further comprising:

a transmitted electron detector located below the sample;

a second light detector for detecting light emitted by sample; and

a second mirror positioned between the sample and the transmitted electron detector, the second mirror having a focal point positioned in proximity to the impact point of the charged particle beam with the sample, the second mirror being configured to reflect the light emitted by the sample toward the second light detector.

9. A charged particle system, comprising:

a charged particle beam source for generating charged particles;

a charged particle beam optical column for focusing a beam of charged particles from the charged particle beam source onto a sample;

a sample stage for holding a sample in a sample position, a sample plane through the sample position dividing space into a region above the sample plane including the charged particle beam source and a region below the sample plane;

a secondary electron detector for collecting secondary electrons emitted from the sample due to the impact of the charged particle beam with the sample, the secondary electron detector positioned above the sample plane;

a light detector for detecting light emitted by the sample upon impact of the charged particle beam; and

an electrically conductive mirror positioned above the sample plane, the mirror configured to deflect light emitted by the sample onto the light detector without the light passing completely through the sample, and to deflect secondary particles from the sample.

10. The charged particle beam system of claim 9 in which the mirror comprises an electrically conductive paraboloidal mirror.

11. The charged particle beam system of claim 10 in which the mirror has a focal point for light, the focal point positioned in proximity to the impact point of the charged particle beam with the sample.

12. The charged particle beam system of claim 11 in which the mirror is configured to reflect the light emitted by the sample toward a light detector.

13. The charged particle beam system of claim 10 further comprising an electrode having a hole through which the charged particle beam passes and configured to provide an electric field between the electrode and the electrically conductive paraboloidal mirror configurable to deflect the secondary electrons from the sample into the secondary electron detector.

14. The charged particle beam system of claim 13 in which the electrode is biased to approximately the potential of the sample.

15. A method for examining a sample, comprising the steps of:

directing a charged particle beam in a pattern onto the sample surface, the charged particle impacting the sample in a region;

detecting light emitted from the region, the light from the sample being reflected by an electrically conductive first mirror toward a detector, the first mirror having an opening therein for passage of the charged particle beam;

moving the charged particle beam across the surface of the sample in a pattern;

deflecting away from the first mirror and towards the detector charged particles from the sample caused by the impact of the charged particle beam on the sample; and

determining from the detected light the composition of the sample.

16. The method of claim 15 in which the charged particle beam comprises an electron beam or a focused ion beam.

17. The method of claim 15 in which the charged particle detector is positioned on the same side of the sample as the mirror and below the level of the mirror.

18. The method of claim 15 in which deflecting away from the first mirror and towards the detector charged particles from the sample comprises providing an electrical bias on the mirror.

19. The method of claim 15 in which the light emitted from the sample is collected by a second mirror on the opposite side of the sample from the first mirror.