BME 210 Lecture 6 Confocal Microscopy

BME 210 Lecture 6 Confocal Microscopy - 6. Confocal...

Info iconThis preview shows pages 1–5. Sign up to view the full content.

View Full Document Right Arrow Icon
6. Confocal Microscopy
Background image of page 1

Info iconThis preview has intentionally blurred sections. Sign up to view the full version.

View Full DocumentRight Arrow Icon
dichroic mirror image plane sample focal plane light source objective blurring out-of-focus light in-focus light Limitation of standard fluorescent microscope: Imaging of thick samples • A thick sample is illuminated by excitation light above and below the focal plane (blue region). All this region emits fluorescence • We want to image fluorescence coming from the focal plane and focused by the objective lens in the image plane (solid green lines) • However, the out-of-focus fluorescence (dashed lines) is also collected by the objective lens and focused above or below the image plane This out-of-focus light causes image blurring and loss of resolution Solution - confocal microscopy
Background image of page 2
pinhole 1 dichroic mirror sample focal plane Confocal microscope Design: Standard fluorescent microscope + Two pinholes Pinhole 1 is placed in the illumination path at the location, which is “confocal” to the sample focal plane : The objective lens creates point-like image of Pinhole 1 at the sample focal plane – within this plane, fluorescence is excited only at one point In addition, two cone regions (blue) within the sample are excited above and below the focal plane
Background image of page 3

Info iconThis preview has intentionally blurred sections. Sign up to view the full version.

View Full DocumentRight Arrow Icon
pinhole 1 dichroic mirror photodetector sample focal plane pinhole 2 (confocal) out-of-focus light in-focus light Confocal microscope 2 Pinhole 2 is placed in the image plane of the objective (it is also “confocal” relative to the sample focal plane): Light only from the illuminated point in the focal plane (solid green traces) passes Pinhole 2 and reaches a photodetector The out-of-focus light (dashed green traces) is blocked by the screen it doesn’t cause image blurring Fluorescence is measured at one point at a time
Background image of page 4
Image of page 5
This is the end of the preview. Sign up to access the rest of the document.

This note was uploaded on 04/28/2010 for the course BME eng. biolo taught by Professor Fast during the Spring '10 term at University of Alabama at Birmingham.

Page1 / 20

BME 210 Lecture 6 Confocal Microscopy - 6. Confocal...

This preview shows document pages 1 - 5. Sign up to view the full document.

View Full Document Right Arrow Icon
Ask a homework question - tutors are online