Which Microscope Has The Best Resolution?

which microscope achieves the highest magnification and resolution

when it comes to superiority in resolution, the simplest question to answer is which type of microscope achieves the highest magnification and resolution. hands down, that is the electron microscope.

• in fact, the guinness world record for the highest resolution is held by an innovative, algorithm-driven version of the electron microscope that visualized single atoms of oxygen, scandium, and praseodymium.
• electron microscopes shoot a concentrated beam of electrons at a target object.
• an image is produced as the electrons pass through the specimen and are detected.
• because the electron wavelength is several thousand times shorter than that of light, the resolving power of an electron microscope is a thousand times greater than that of a light microscope.

transmission electron microscope with aberration correction has the highest resolution. they can go upto pm resolution. although aberration corrected microscopes are highly expensive.

• one important remark before i answer - magnification does not mean resolution, and by itself useless.
• current resolution record is slightly below 50 pm (!!!), achieved on double corrected s/tem.
• corrected tems such as the fei titan, can resolve .07 nm or so. (.7 angstroms)
• i think state of the art microscopes are getting down to .5 ang or so.

what is resolution

in microscopy, the term “resolution” is used to describe the ability of a microscope to distinguish details of a specimen or sample. in other words, the minimum distance between 2 distinct points of a specimen where they can still be seen by the observer or microscope camera as separate entities.

• resolution is intrinsically linked to the numerical aperture (na) of a microscope’s optical components, like the objective lens, as well as the wavelength of light used.
• the numerical aperture (na) is related to the refractive index (n) of a medium through which light passes as well as the angular aperture (α) of a given objective (na = n sinα).
• the resolution of an optical microscope is not solely dependent on the na of an objective, but the na of the whole system, taking into account the na of the microscope condenser.
• more image detail will be resolved in a microscope system in which all of the optical components are correctly aligned, have a relatively high na value and are working harmoniously with each other.
• resolution is also related to the wavelength of light which is used to image a specimen; light of shorter wavelengths are capable of resolving greater detail than longer wavelengths.

there are 3 mathematical concepts which need to be taken into consideration when dealing with resolution: abbe’s diffraction limit, airy discs, and the rayleigh criterion.

electron microscope and live or unfixed samples

• but does “highest” always equal “best”?
• not only size but also time and context matter.
• clearly, the hostile environment of an electron microscope precludes working with live or unfixed samples.
• so, if you’re interested in the movement, changes, and context that constitute life, a slight downgrade in resolution is likely your best bet.
• enter the world of super-resolution microscopy.

below. the characterization of nuclear pore architecture and mitochondrial protein patterns are just two examples of the technology’s spatial resolving power. this capacity grants a completely new perspective on molecular structure in biological context, revealing the architecture of biomolecules and their interactions.

• and yes, you can work with living cells.
• tracking of a kinesin-1 molecule walking along microtubules, including the corresponding configurational changes occurring at each step.
• the movement of kinesin-1 has never been tracked in a living cell before.

super-resolution microscopy options

research that does not require characterizing individual molecules but rather their spatial relation to others has a broader range of microscopy technologies at its disposal.

• another step down on the resolution scales makes widefield, confocal, sted, and palm/storm microscopy all options.
• as super-resolution technologies, sted and palm/storm outperform the spatial resolution of diffraction-limited confocal and widefield microscopy by a factor of 10.
• commonly discriminating objects at 20nm, sted is also fast, which has enabled, for example, visualizing the fission and fusion of mitochondria with exceptional clarity.
• approximate temporal and spatial resolution range of microscopy methods.

abbe’s diffraction limit

abbe recognized that specimen images are composed of a multitude of overlapping, multi-intensity, diffraction-limited points (or airy discs).

• in order to increase the resolution, d = λ/(2na), the specimen must be viewed using either a shorter wavelength (λ) of light or through an imaging medium with a relatively high refractive index or with optical components which have a high na (or, indeed, a combination of all of these factors).
• however, even taking all of these factors into consideration, the possibilities with a real microscope are still somewhat limited due to the complexity of the whole system, transmission characteristics of glass at wavelengths below 400 nm, and the challenge to achieve a high na in the complete microscope system.
• lateral resolution in an ideal optical microscope is limited to around 200 nm, whereas axial resolution is around 500 nm.

the rayleigh criterion

the rayleigh criterion defines the limit of resolution in a diffraction-limited system, in other words, when two points of light are distinguishable or resolved from each other.

• using the theory of airy discs, if the diffraction patterns from two single airy discs do not overlap, then they are easily distinguishable, ‘well resolved’ and are said to meet the rayleigh criterion.
• when the center of one airy disc is directly overlapped by the first minimum of the diffraction pattern of another, they can be considered to be ‘just resolved’ and still distinguishable as two separate points of light.
• if the airy discs are closer than this, then they do not meet the rayleigh criterion and are ‘not resolved’ as two distinct points of light.

full width at half maximum

• this value is relatively easy to measure with a microscope and has become a generally accepted parameter for comparison purposes.
• the theoretical value for the fwhm is rfwhm = 0.51λ/(na) which is approximately λ/(2na).
• so the fwhm as a resolution parameter is very close to abbe’s diffraction limit, but also can be measured from microscope image data.
• for calibration or resolution-limit measurements, often beads or colloids of various diameters are imaged and measured.

these theoretical resolution values, derived from physical and mathematical assumptions, are estimates. they assume perfect imaging systems and a point light source in a vacuum or a completely homogeneous material as the sample or specimen. of course, this assumption is almost never the case in real life, as many samples or specimens are heterogeneous. because there is only a finite amount of light transmitting through the sample or reflecting from its surface, the measurable resolution depends significantly on the signal-to-noise ratio (snr).

how to calculate the resolution of a microscope

taking all of the above theories into consideration, it is clear that there are a number of factors to consider when calculating the theoretical limits of resolution. resolution is also dependent on the nature of the sample.

• na = n(sinα)
• where n is the refractive index of the imaging medium and α is half of the angular aperture of the objective.
• the maximum angular aperture of an objective is around 144º.
• the sine of half of this angle is 0.95.
• if using an immersion objective with oil which has a refractive index of 1.52, the maximum na of the objective will be 1.45.
• if using a ‘dry’ (non-immersion) objective the maximum na of the objective will be 0.95 (as air has a refractive index of 1.0).

abbe’s diffraction formula

• abbe’s diffraction formula for lateral (xy) resolution is:
• d = λ/(2na)
• where λ is the wavelength of light used to image a specimen.
• if using a green light of 514 nm and an oil-immersion objective with an na of 1.45, then the (theoretical) limit of resolution will be 177 nm.

• abbe’s diffraction formula for axial (z) resolution is:
• d = 2λ/(na)2
• and again, if we assume a wavelength of 514 nm to observe a specimen with an objective having an na value of 1.45, then the axial resolution will be 488 nm.

rayleigh criterion formula

• the rayleigh criterion is a slightly refined formula based on abbe’s diffraction limits:
• r = 1.22λ/(naobj + nacond)
• where λ is the wavelength of light used to image a specimen.
• naobj is the na of the objective.
• nacond is the na of the condenser.
• the value ‘1.22’ is a constant.

taking the na of the condenser into consideration, air (with a refractive index of 1.0) is generally the imaging medium between the condenser and the slide. assuming the condenser has an angular aperture of 144º then the nacond value will equal 0.95.

• if using a green light of 514 nm, an oil-immersion objective with an na of 1.45, condenser with an na of 0.95, then the (theoretical) limit of resolution will be 261 nm.
• as already mentioned, the fwhm can be measured directly from the psf or calculated using:
• rfwhm = 0.51λ/(na).
• again using a light wavelength of 514 nm and an objective with an na of 1.45, then theoretical resolution will be 181 nm.
• this value is very close to the lateral resolution calculated just above from the abbe diffraction limit.

shorter wavelength and maximum theoretical resolution

• as stated above, the shorter the wavelength of light used to image a specimen, then the more the fine details are resolved.
• so, if using the shortest wavelength of visible light, 400 nm, with an oil-immersion objective having an na of 1.45 and a condenser with an na of 0.95, then r would equal 203 nm.
• to achieve the maximum theoretical resolution of a microscope system, each of the optical components should be of the highest na available (taking into consideration the angular aperture).
• in addition, using a shorter wavelength of light to view the specimen will increase the resolution.
• finally, the whole microscope system should be correctly aligned.