1 Confocal Microscopy David Kelly November 2013 book of Biological Confocal Microscopy. Ed. J. Pawley, Plenum Press amentals of Light Microscopy and Electronic Imaging. D. B. Murphy, Wiley-Lis Confocal design: CLSM microscope Pinhole Optical Sectioning Spinning Disk Confocal Photon Multiplier Tube CCD Confocal principles: Scan speed Optical resolution Pinhole adjustment Digitisation: sampling as opposed to imaging xy sampling: pixel size and zoom choices Photomultiplier tubes, noise, digitisation of intensity Multichannel imaging, crosstalk Colour Look Up Tables Recap of principal factors affecting image quality Imaging Thick Specimens Multiphoton Microscopy
56
Embed
1 Confocal Microscopy David Kelly November 2013 Handbook of Biological Confocal Microscopy. Ed. J. Pawley, Plenum Press Fundamentals of Light Microscopy.
This document is posted to help you gain knowledge. Please leave a comment to let me know what you think about it! Share it to your friends and learn new things together.
Transcript
1
Confocal MicroscopyDavid Kelly November 2013
Handbook of Biological Confocal Microscopy. Ed. J. Pawley, Plenum Press
Fundamentals of Light Microscopy and Electronic Imaging. D. B. Murphy, Wiley-Liss Inc.
Confocal design: CLSM microscopePinholeOptical SectioningSpinning Disk ConfocalPhoton Multiplier TubeCCD
Confocal principles: Scan speedOptical resolutionPinhole adjustmentDigitisation: sampling as opposed to imagingxy sampling: pixel size and zoom choicesPhotomultiplier tubes, noise, digitisation of intensityMultichannel imaging, crosstalkColour Look Up Tables
Recap of principal factors affecting image qualityImaging Thick SpecimensMultiphoton Microscopy
2
What to get out of this lectureHave an understanding of how a modern confocal microscope works
Become familiar with the principal factors affecting image quality in the CLSM
Begin to have an idea when and how to manipulate these factors for your purposes
This often means knowing when and where to make compromises (e.g. light collection versus spatial resolution)
3
Benefits of Confocal Microscopy
• Reduced blurring of the image from light scattering
• Increased effective resolution• Improved signal to noise ratio• Clear examination of thick specimens• Z-axis scanning• Depth perception in Z-sectioned images• Magnification can be adjusted
Scan speed: t resolutionOn modern confocals this is measured in Hz usually from 1-1400Hz
Decreasing scan speed-more light collected (dwell time
increased)more chance of photobleaching and phototoxicitylimits temporal resolution
Increasing scan speed- has opposite effect but often results in poor image qualityNote: Some types of confocal specifically optimised for fast scanning. Eg spinning disk, line scanner and resonant scanner
14
Pinhole adjustment
Airy disc
0.5 Maximum optical sectioning and resolution. Discard much in-focus light
1 xy resolution approaches that of conventional microscopy, but still retain good rejection of out-of-focus information. Still lose some in-focus photons.
>1 Maximise light collected. But this mostly comes from adjacent out-of-focus planes - lose z resolution. xy resolution not badly affected
xy
z
xy
z
Open pinhole
Close pinhole
15
Confocal Pinhole
16
210 nm
60 nm
z = 0 z = 2 z = 4
z = 6 z = 8
Fluotar 20x/0.5Zoom = 3Pinhole = 0.7
17
210 nm
60 nm
z = 0 z = 2 z = 4
z = 6 z = 8
Fluotar 20x/0.5Zoom = 3Pinhole = 3.0
18
Pinhole Summary
• In practise, pinhole size is mainly used to control optical section thickness other than to achieve highest lateral or Z-resolution
• Occasionally, pinhole size can be used to adjust amount of photon received by PMT to change the signal intensity and increase SNR. In addition to the "optimal" 1 AU, Pinhole 1-3 AU is the range of choice. Bigger pinhole give you stronger signal but with the compromised confocal effects.
19
Sampling
Scanning involves digitisation in x, y, z, intensity, and t
Resolution is affected by sampling during the digitisation process
0 0 0 0 0 0 0 0 0 0 0 0 0
0 0 0 22 45 66 11 0 0 0 0 0 0
0 0 0 0 0 0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 65 12 0 0 0 0
0 0 0 0 0 0 0 0 99 0 0 0 0
0 0 0 0 0 0 0 7 0 0 0 0 0
0 0 6 5 0 0 0 2 8 21 5 2 0
0 0 0 3 0 0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0 0 0 0 0 0
pixels(voxels)
20
Pixel choices
512x5121024x10242048x2048
More pixels—smoother looking image - more xy informationmore light exposure of specimenlarger file sizeslower imaging (less temporal resolution)
—250 kbyte (1 channel)—3 Mbyte (3 channel)
21
Digitisation can lose information
Correct choice of pixel size can minimise this
intensity
scan line
22
Pixel undersampling
Specimen
Large pixels
Small pixels, lucky
alignmentSmall pixels, unlucky
alignmentVery small
pixels
23
Nyquist sampling (xy)
Optimum pixel size for sampling the image is at least 1/2 spatial resolution
“If you must sample below the Nyquist limit, then spoil the resolution [to match better the pixel size]!”ie. Open the pinhole.
29
Digitisation of PMT voltage
3 bit8 levels of brightness
0
7
1 bit: 2 levels (black + white)
(Eye is a 6 bit device (~50 levels of brightness))x
Level
Voltage is sampled at regular intervals and converted into a digital pixel intensity value by the analogue-digital converter (ADC)
12 bit
3 bit
30
NoiseNoise: any variability in measurement that is not due to
signal changesS/N ratio determines the lower limit of the ability to
distinguish true changes in the measurement (dynamic range)
Photon sampling variability (shot noise):Statistical fluctuations in photons hitting PMT.
Electronic noise:Variability in PMT generated current.
These things are exacerbated at high gain settings
Reduce noise by sampling more photons:Reducing scan rate (increasing pixel dwell time), or opening pinhole.
Frame averagingNoise is reduced (dynamic range increased) with square root of number of framesSample exposure to light is increased
31
High gain
1 scan 16 scans
Apo 63x lens
Laser 488nm 10%PMT 1000V
Laser 488nm 80%PMT 800V
Medium gain
32
Digitisation of intensity
Normally 8 bit (256 brightness levels)Extended dynamics 12 bit (4095 brightness levels)
But useful dynamic range is degraded by noise
Why need so many bits?1. Spare dynamic range for exploring
intensity details during image processing2. Probably helps to smooth out noise
problems (e.g. capture in 12 bit and save in 8 bit)
Quantitation/physiology
33
Multi-channel imaging
34
Multi-channel imagingUse a fluorochrome combinationMultiple laser lines and PMTsComplicated filter sets needed to separate lightAlternatives: AOTF, AOBS, spectrophotometric detection
Advantages of 2 Photon Longer observation times for live cell studies Increased fluorescence emission detection Reduced volume of photobleaching and phototoxicity. Only the focal-plane
being imaged is excited, compared to the whole sample in the case of confocal or wide-field imaging.
Reduced autofluorescence of samples Optical sections may be obtained from deeper within a tissue that can be
achieved by confocal or wide-field imaging. There are three main reasons for this: the excitation source is not attenuated by absorption by fluorochrome above the plane of focus; the longer excitation wavelengths used suffer less Raleigh scattering; and the fluorescence signal is not degraded by scattering from within the sample as it is not imaged.
All the emitted photons from multi-photon excitation can be used for imaging (in principle) therefore no confocal blocking apertures have to be used.
It is possible to excite UV fluorophores using a lens that is not corrected for UV as these wavelengths never have to pass through the lens.
53
Limitations of 2-Photon
Slightly lower resolution with a given fluorochrome when compared to confocal imaging. This loss in resolution can be eliminated by the use of a confocal aperture at the expense of a loss in signal.
Thermal damage can occur in a specimen if it contains chromophores that absorb the excitation wavelengths, such as the pigment melanin.
Only works with fluorescence imaging.
54
Multi Photon3 photon
• Use of near-infrared wavelengths (down to 720 nanometers) 3 photon excitation extend the fluorescence imaging range into the deep ultraviolet.
ExampleSingle, dual, and triple photon excitations of tryptophan, Single photon excites at 280nm with emission of fluorescence at 348 nanometers (UV). Two-photon excites with greenish-yellow light centered at 580nm.Three-photon excites with near-infrared light at 840nm