6/3/15. The Anatomy of a Digital Image. Representative Intensities. Specimen: (molecular distribution)

Transcription

1 2015 LMIC Imaging Workshop Sidney L. Shaw Technical Director An introduction of concepts for Super-Resolution Light Microscopy The Anatomy of a Digital Image Representative Intensities Specimen: (molecular distribution) Optical Image: (collected light that interacted with or eminated from the molecules in the distribution) Digital Image: (a representation of the light energy quantized in space and for relative intensity) 1

2 The optical image (i.e. radiative energy + lenses) places a physical limit on resolving information from the specimen. You are Here How Do we Get here? Super-Resolution Light Microscopy Resolving information below the physical limits of the lens system Structured Illumination Microscopy (SIM) - No special probes - 3D to 100x100x300 nm resolution STimulated Emission Depletion (STED) - Special probes and lasers - 2D to 20x20x650 nm resolution - Massive photobleaching issues Point based methods (PALM/STORM/Etc.) - Special probes and mounting medium - 2D to 40x40x? Nm resolution - time-intensive acquisition 2

3 Image Formation: When light originating at a specimen is focussed on to a plane, the distribution of light intensities, together with positive and negative interference, yield an image. Constructive (everything in between) Destructive ( Image Formation: All lenses have a finite resolving limit related to the maximum angle of light collection or convergence. All objects smaller than that limit look about the same size. Very small objects form spots of a predictable (diffraction limited) size. Original Object Screen ( 3

4 Image Formation: All lenses have a finite resolving limit related to the maximum angle of light collection or convergence. All objects smaller than that limit look about the same size. Very small objects form spots of a predictable size. ( Image Formation: That minimum spot size for the lens leads to critical limitations for an optical system starting with the resolution limit. Diffraction Limited Spot Intensity Cross Section Converging set of Diffraction Limited Spots Airy Disk and part of a Point Spread Function When is this considered one spot or two spots? Recall the actual molecular size could be much smaller than the size of the Airy disk. 4

5 Image Formation: The resolution limit of the lens cannot be overcome by simply magnifying the spots. Image Formation: The minimum spot size also plays a significant role in generating image contrast. Let us now replace the set of spots with a set of parallel lines. Black and White High Contrast Gray Low Contrast Rela-ve Spot Size

6 Contrast and Resolution are related in the optical image: The Contrast Transfer Function Distance Contrast = 22% Contrast = 50% Contrast = 100% Ç Contrast = 100% Contrast = 50% Contrast = 22% Modula-on Transfer Func-on Ç Distance (Spacing) 1/Distance (Frequency) Stelzer, 1998 The Anatomy of a Digital Image Representative Intensities Specimen: (molecular distribution) Resolution is limited at the level of the optical image. Optical Image: (collected light that interacts with or eminates from the molecules in the distribution) Digital Image: (a representation of the light energy quantized in space and for relative intensity) 6

7 General Rule: you need minimally 2-3 pixels per Airy disk to capture the optical resolution to the digital image. e- Voltage e- e- e- Intensity Sampling: Each pixel in the digital camera collects photoelectrons and reads them out to the computer. 7

8 General Rule: you need minimally 2-3 pixels per Airy disk to capture the optical resolution to the digital image. General Rule: you need minimally 2-3 pixels per Airy disk to capture the optical resolution to the digital image. 8

9 General Rule: you need minimally 2-3 pixels per Airy disk to capture the optical resolution to the digital image. Does this spot represent only one molecule (e.g. your protein)? What if we knew this was only one molecule? 9