Imaging Resource
Standard Operating Procedure
BAIR Confocal Microscope Training
Microscope Resolution
The resolution of an optical microscope is defined as the shortest distance between two points on a specimen that can still be distinguished by the observer or camera system as separate entities.
The limit of resolution of a microscope objective refers to its ability to distinguish between two closely spaced Airy disks in the diffraction pattern (noted in the figure). Three-dimensional representations of the diffraction pattern near the intermediate image plane are known as the point spread function, and are illustrated in the lower portion of Figure 1. The specimen image is represented by a series of closely spaced point light sources that form Airy patterns and is illustrated in both two and three dimensions.
Resolution is a somewhat subjective value in optical microscopy because at high magnification, an image may appear unsharp but still be resolved to the maximum ability of the objective. Numerical aperture determines the resolving power of an objective, but the total resolution of the entire microscope optical train is also dependent upon the numerical aperture of the sub stage condenser. The higher the numerical aperture of the total system, the better the resolution.
The resolving power of an objective determines the size of the Airy diffraction pattern formed, and the radius of the central disk is determined by the combined numerical apertures of the objective and condenser. When the condenser and objective have equivalent numerical apertures, the Airy pattern radius from the central peak to the first minimum is given by the equation:
r(Airy) = 1.22λ/2NA(Obj)
where r(Airy) is the Airy radius, λ is the wavelength of illuminating light, and NA(Obj) is the objective (and condenser) numerical aperture.
Microscope lens resolution equation.( NA=Numerical aperture)
* Fluorescence use (2*NAobj )
Fluorescent Application: FITC Emission 510mn
X60 objective 1.4NA 488nm =1.22x488/2x1.4=0.212um
X60 objective 0.7NA 488nm= 122x488/2.0.7=0.424um
X10 objective 0.3NA 488um= 122x488/2x0.3=0.992
The above is the theoretical distance that the lens can resolve. Two points in theory would have to be 0.992um apart when using the 10x dry lens in order to see then as two distinct points. From the above you can see that the x60 lens with the higher NA has the greater resolving power.
Put in some images of our beads taken with different lenses with different NAs
Pixel choice Scan Resolution
512x512 single channel 250kb
1024x1024 3 channels 3MB
More pixels in the image will produce a larger file size but the image will look smother as you have more XY information.You will also have more light exposure of the specimen and slower imaging.If you choose the correct size of pixel you can minimise this:
The images were taken at different scan resolutions 128x128 pixels and 1024x1024 pixels. The images were cropped in photoshop.
128x128 1024x1024
The image pixel size in XY is different at each scan resolution on the FV1000 confocal microscope. Using a x63 lens the sizes were as follows.
At 2040x2040 the image pixel size is 0.103um/pixel
At 1024x1024 the image pixel size is 0.207um/pixel
At 512x512 the image pixel size is 0.414um/pixel
At 128x128 the image size is 0.828um/pixel ( x2 zoom)
There is no point in choosing 2040x2040 if the lens resolution is 0.18um.
You also must be careful when using the zoom function.
At 1024x1024 zoom of 2 = image pixel size is 0.103um/pixel
At 1024x1024 zoom of 4 = image pixel size is 0.051um/pixel
Nyquest Sampling Equation
- 0.4xwavelength/NA=resolvable Distance for confocal ( 1.22x wavelegnth for widefield)
- 2 pixels is the smallest optically resolvable distance
- resolvable Distance/2= smallest resolvable point
Nyquest Sampling examples
•X10 Objective with 0.3 NA using GFP
•0.4 x 520 = 693nm
0.3
693 = 346.6nm smallest resolvable distance
2
•Actual image Scan Size = 1500µm
•Box Size/image pixel resolution = 1024 pixels
•1500 = 1464nm pixel size
1024
1464 = 4.2 zoom for nyquist in xy
346.6 objective resolving distance
Or a box size large enough to produce a pixel size of 346.6
Nyquest sampling and Z series
What distance between z steps?
Optimum z step for sampling the image is 1/2 the axial resolution
For high NA lens of 500nm z resolution, optimum z stepping is 250nm (assuming optimum pinhole size, etc).
In practice, this is often too many for a very thick specimen. 500nm-1um is often fine.Especially if pinhole opened.
Over and under sampling
Oversampling (pixels small compared with optical resolution)
Image smoother and withstands manipulation better
Specimen needlessly exposed to laser light
Image area needlessly restricted
File size needlessly large
Undersampling (pixels large compared with optical resolution)
Degraded spatial resolution
Photobleaching reduced
Image artefacts (blindspots, aliasing)
Objective Lens
Microscopes can be fitted with a wide range of objectives to meet the performance needs of the imaging methods you are using. They can be made to compensate forvariations in coverglass thickness and made with an increase in the effective working distance of the objective. Not all lenses with the same magnification perform and produce images of the same quality. How they perform will depend on what they have been made for. For example a x40 lens with phase will usually produce weaker fluorescent images that a x40 lens with no phase.The barrel of the lens gives you information on what the lens was made for.We have Dry, oil and water lenses for each of our confocal microscopes. We have lenses which have longer working distances than others. We have lenses that are better for fluorescence.
Dry lenses: lower magnification/lower resolution longer working distances.
Oil lenses: Higher magnifications/Higher resolutions/shorter working distances
Water lenses: Range from low to high magnifications. Some can be used as dipping lenses.
•CP-Achromat
Good colour correction – exactly for two wavelengths. Field flatness in the image center, refocusing also covers the peripheral areas. For fields of view up to dia. 18 mm. Versions for phase contrast.
•Achroplan
Improved Achromat objectives with good image flatness for fields of view with dia. 20 or even 23 mm. Achroplan for transmitted light and Achroplan Ph for phase contrast.
•Plan-Neofluar
Excellent colour correction for at least three wavelengths. Field flattening for the field of view with dia. 25 mm. Highly transmitting for UV excitation at 365 nm in fluorescence. All methods possible, specialhigh-quality variants are available for Pol and DIC.
•Plan-Apochromat
Perfect colour rendition (correction for four wavelengths!). Flawless image flatness for fields of view
Working distances
Working Distances of objective. The Higher magnification objectives haveshorter working distances so you cannot focus through plastic tissue culture dishes or plates.When using glass slides always focus through the glass coverslip and not the glass slide.
Widefield Fluorescent Microscopy
GFP excitation and emission spectra
All fluorophore and Fluorescent proteins have a characteristic exitation and emission spectra. It is very important that you know the excitation and emission spectra for the fluorescent marker you are using. Excitation and emission spectra can overlap. Microscope filter blocks are used to separate some of them but not all of them. To find spectra and check compatibility of your fluorophores check the Spectra Viewer on the BAIR website.
What can filters Separate?
NUA | DAPI DAPI | ex BP 330-385/emBA 420
WU | DAPI LP NUA2 | ex BP 360-370 /em BA 420-460
NB | FITC/GFP FITC | ex BP 470-490/em BA 520IF
WG | TRITC/TR TRITC | ex 510-550/em BA 590
Why Confocal Microscopy?
Confocal microscopy is an optical sectioning technique that produces high resolution images of samples without the out of focus light normally associated with wide field imaging. In confocal images the out of focus light is removed and only the light from the focal plane reaches the confocal detector. This is done by placing a pinhole in front of the detector which excludes the out of focus light. Consequently only in focus light passes through and the image collected is sharp and well resolved. Confocal microscopes allow optical sectioning through the sample by changing the pinhole width. This means that slices can be reassembled to produce 3D imaging of your cells or tissue. Confocal microscopes have much greater spatial resolution than non-confocal microscopes.
Wide-Field image Confocal Image
Confocal imaging is a method to get rid of out of focus light so the image looks less blurred.The whole sample isilluminated by the laser light but only light form the focal plane is passed through the pinhole to the detector.
•In practise, pinhole size is mainly used to control optical section thickness rather than to achieve highest lateral or Z-resolution
•Occasionally, pinhole size can be used to adjust amountof 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.
•
Fluotar x20/ 0.5NA/zoom3pinhole= 0.7 Fluotar x20/ 0.5NA Zoom 3 pinhole= 3
Confocal Photon Multiplier Tube ( PMT)
The signal detector is usually a photomultiplier tube (PMT). The detected signal is directly proportional to the quantum efficiency. The effective quantum efficiency of the photomultiplier tubes used in most confocal instruments drops from about 15 percent in the blue end to approximately 4 percent in the red end of the spectrum.
The PMT voltage determines the amplification of the PMT. An increase of 50 volts corresponds to a factor of about two more in gain. Beware that PMT black level or (offset) control permits the addition or subtraction of an arbitrary amount from the signal presented to the digitizer. The black level should be set so that the signal level in the darkest parts of the image is 5-10 digital units.
There are many possible sources of noise and all will distort the recorded value. Usually, PMTs produce dark current noise that is small compared to the signal level. Apart from any stray light that may inadvertently reach the PMT, the main remaining noise source is Poisson or statistical noise. This is equal to the square root of the number of photons recorded in a given pixel. The result is it becomes larger at higher signal levels, even though the ratio of signal to noise improves.
Digitization, usually using a computer (Figure 1(g)) should be linear. The electronic signals presented to the digitizer of "8-bit" microscopes must be of a size to be recorded between 1 and 255. Because of statistical noise, a value between 10 and 220 is safer. The digital conversion factor refers to the ratio between the number of photons detected and the number stored. This depends on the PMT voltage and other electronic gain, but is usually about 30 for normal specimens recorded on 8-bit instruments.
What is Noise?
Noise: any variability in measurement that is not due to signal changes
S/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 averaging
Noise is reduced (dynamic range increased) with square root of number of frames
Sample exposure to light is increased
1 scan medium gain laser power 80% PMT 800V 16 scans med gain laser power 80% PMT 800V
1 scan high gain laser power 10% PMT 1000V 16 scans high gain Laser power 10% PMT 1000V
Scan Speed: Resolution
On modern confocals this is measured in Hz or frames/second
Decreasing scan speed-
more light collected (dwell time increased)
more chance of photobleaching and phototoxicity
limits temporal resolution
Increasing scan speed- has opposite effect but often results in poor image quality
Some types of confocal are specifically optimised for fast scanning. Eg spinning disk and resonant scanner
scan speed 1 frame/second 1 frame/2 seconds 1 frame / 4 seconds
Image Bit Depth
Our confocal images can be captured at different bit depth depending on the system you are using. This is important for quantification!!!
8 bit (256 brightness levels)
12 bit (4095 brightness levels)
What bit depth do I need?
- Spare dynamic range for exploring intensity details during image processing
- Probably helps to smooth out noise problems (e.g. capture in 12 or 16 bit for quantification)
Benefits of Confocal Microscopy
More colour possibilities: Because the images are detected by a computer rather than by eye it is possible to detect more colors
Less Cross talk
In most applications, fluorochromes have overlapping emission spectra. Hence, the
emission signals cannot be separated completely into different detection channels
resulting in “bleed through” or cross talk.
However, if the fluorochromes have distinct excitation spectra, the fluorochromes
can be excited sequentially using one excitation wavelength at a time. This is only
possible with confocal systems that offer the multitracking feature.
Brain slices Nerve fibers FITC nuclei PI
Standard Widefield Confocal
Other features
•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 3D/4D imaging
•Magnification can be adjusted electronically
Applications
- Co localization
- Live Cell Imaging
- FRAP/FLIP
- FRET
Imaging FacilityPage 103/04/2019