Microscopy

However, blurring is not caused by random processes, such as light scattering, but can be well defined by the optical properties of the image formation in the microscope imaging system. If one considers a small fluorescent light source (essentially a bright spot), light coming from this spot spreads out further from our perspective as the spot becomes more out of focus. Under ideal conditions, this produces an "hourglass" shape of this point source in the third (axial) dimension. This shape is called the point spread function (PSF) of the microscope imaging system. Since any fluorescence image is made up of a large number of such small fluorescent light sources, the image is said to be "convolved by the point spread function". The mathematically modeled PSF of a terahertz laser pulsed imaging system is shown on the right.

The output of an imaging system can be described using the equation:

${\displaystyle s(x,y)=PSF(x,y)*o(x,y)+n}$

Where n is the additive noise.^[32] Knowing this point spread function^[33] means that it is possible to reverse this process to a certain extent by computer-based methods commonly known as deconvolution microscopy.^[34] There are various algorithms available for 2D or 3D deconvolution. They can be roughly classified in nonrestorative and restorative methods. While the nonrestorative methods can improve contrast by removing out-of-focus light from focal planes, only the restorative methods can actually reassign light to its proper place of origin. Processing fluorescent images in this manner can be an advantage over directly acquiring images without out-of-focus light, such as images from confocal microscopy, because light signals otherwise eliminated become useful information. For 3D deconvolution, one typically provides a series of images taken from different focal planes (called a Z-stack) plus the knowledge of the PSF, which can be derived either experimentally or theoretically from knowing all contributing parameters of the microscope.

Sub-diffraction techniques

A multitude of super-resolution microscopy techniques have been developed in recent times which circumvent the diffraction limit.

This is mostly achieved by imaging a sufficiently static sample multiple times and either modifying the excitation light or observing stochastic changes in the image. The deconvolution methods described in the previous section, which removes the PSF induced blur and assigns a mathematically 'correct' origin of light, are used, albeit with slightly different understanding of what the value of a pixel mean. Assuming most of the time, one single fluorophore contributes to one single blob on one single taken image, the blobs in the images can be replaced with their calculated position, vastly improving resolution to well below the diffraction limit.

To realize such assumption, Knowledge of and chemical control over fluorophore photophysics is at the core of these techniques, by which resolutions of ~20 nanometers are obtained.^[35][36]

Serial time-encoded amplified microscopy

Serial time encoded amplified microscopy (STEAM) is an imaging method that provides ultrafast shutter speed and frame rate, by using optical image amplification to circumvent the fundamental trade-off between sensitivity and speed, and a single-pixel photodetector to eliminate the need for a detector array and readout time limitations^[37] The method is at least 1000 times faster than the state-of-the-art CCD and CMOS cameras. Consequently, it is potentially useful for scientific, industrial, and biomedical applications that require high image acquisition rates, including real-time diagnosis and evaluation of shockwaves, microfluidics, MEMS, and laser surgery.^[38]

Extensions

Most modern instruments provide simple solutions for micro-photography and image recording electronically. However such capabilities are not always present and the more experienced microscopist may prefer a hand drawn image to a photograph. This is because a microscopist with knowledge of the subject can accurately convert a three-dimensional image into a precise two-dimensional drawing. In a photograph or other image capture system however, only one thin plane is ever in good focus.^{[citation needed]}

The creation of accurate micrographs requires a microscopical technique using a monocular eyepiece. It is essential that both eyes are open and that the eye that is not observing down the microscope is instead concentrated on a sheet of paper on the bench besides the microscope. With practice, and without moving the head or eyes, it is possible to accurately trace the observed shapes by simultaneously "seeing" the pencil point in the microscopical image.^{[citation needed]}

It is always less tiring to observe with the microscope focused so that the image is seen at infinity and with both eyes open at all times. ^{[citation needed]}

Other enhancements

Microspectroscopy:spectroscopy with a microscope

X-ray

As resolution depends on the wavelength of the light. Electron microscopy has been developed since the 1930s that use electron beams instead of light. Because of the much smaller wavelength of the electron beam, resolution is far higher.

Though less common,

Electron microscopy

Until the invention of sub-diffraction microscopy, the wavelength of the light limited the resolution of traditional microscopy to around 0.2 micrometers. In order to gain higher resolution, the use of an electron beam with a far smaller wavelength is used in electron microscopes.

• Transmission electron microscopy (TEM) is quite similar to the compound light microscope, by sending an electron beam through a very thin slice of the specimen. The resolution limit in 2005 was around 0.05 ^{[dubious – discuss]} nanometer and has not increased appreciably since that time.
• Scanning electron microscopy (SEM) visualizes details on the surfaces of specimens and gives a very nice 3D view. It gives results much like those of the stereo light microscope. The best resolution for SEM in 2011 was 0.4 nanometer.

Electron microscopes equipped for X-ray spectroscopy can provide qualitative and quantitative elemental analysis. This type of electron microscope, also known as analytical electron microscope, can be a very powerful tool for investigation of nanomaterials.^[39]

Scanning probe microscopy

This is a sub-diffraction technique. Examples of scanning probe microscopes are the

Ultrasonic force microscopy allows the local mapping of elasticity in atomic force microscopy by the application of ultrasonic vibration to the cantilever or sample. To analyze the results of ultrasonic force microscopy in a quantitative fashion, a force-distance curve measurement is done with ultrasonic vibration applied to the cantilever base, and the results are compared with a model of the cantilever dynamics and tip-sample interaction based on the finite-difference technique.

Ultraviolet microscopy

Ultraviolet microscopes have two main purposes. The first is to use the shorter wavelength of ultraviolet electromagnetic energy to improve the image resolution beyond that of the diffraction limit of standard optical microscopes. This technique is used for non-destructive inspection of devices with very small features such as those found in modern semiconductors. The second application for UV microscopes is contrast enhancement where the response of individual samples is enhanced, relative to their surrounding, due to the interaction of light with the molecules within the sample itself. One example is in the growth of protein crystals. Protein crystals are formed in salt solutions. As salt and protein crystals are both formed in the growth process, and both are commonly transparent to the human eye, they cannot be differentiated with a standard optical microscope. As the tryptophan of protein absorbs light at 280 nm, imaging with a UV microscope with 280 nm bandpass filters makes it simple to differentiate between the two types of crystals. The protein crystals appear dark while the salt crystals are transparent.

Infrared microscopy

The term infrared microscopy refers to microscopy performed at infrared wavelengths. In the typical instrument configuration, a Fourier Transform Infrared Spectrometer (FTIR) is combined with an optical microscope and an infrared detector. The infrared detector can be a single point detector, a linear array or a 2D focal plane array. FTIR provides the ability to perform chemical analysis via infrared spectroscopy and the microscope and point or array detector enable this chemical analysis to be spatially resolved, i.e. performed at different regions of the sample. As such, the technique is also called infrared microspectroscopy^[40][41] An alternative architecture called

IR versions of sub-diffraction microscopy also exist.

In

DHM can operate both in reflection and transmission mode. In reflection mode, the phase shift image provides a relative distance measurement and thus represents a topography map of the reflecting surface. In transmission mode, the phase shift image provides a label-free quantitative measurement of the optical thickness of the specimen. Phase shift images of biological cells are very similar to images of stained cells and have successfully been analyzed by high content analysis software.

A unique feature of DHM is the ability to adjust focus after the image is recorded, since all focus planes are recorded simultaneously by the hologram. This feature makes it possible to image moving particles in a volume or to rapidly scan a surface. Another attractive feature is The ability of DHM to use low cost optics by correcting optical aberrations by software.

Digital pathology (virtual microscopy)

Digital pathology is an image-based information environment enabled by computer technology that allows for the management of information generated from a digital slide. Digital pathology is enabled in part by virtual microscopy, which is the practice of converting glass slides into digital slides that can be viewed, managed, and analyzed.

Laser microscopy

Laser microscopy is a rapidly growing field that uses laser illumination sources in various forms of microscopy.^[44] For instance, laser microscopy focused on biological applications uses ultrashort pulse lasers, in a number of techniques labeled as nonlinear microscopy, saturation microscopy, and two-photon excitation microscopy.^[45]

High-intensity, short-pulse laboratory x-ray lasers have been under development for several years. When this technology comes to fruition, it will be possible to obtain magnified three-dimensional images of elementary biological structures in the living state at a precisely defined instant. For optimum contrast between water and protein and for best sensitivity and resolution, the laser should be tuned near the nitrogen line at about 0.3 nanometers. Resolution will be limited mainly by the hydrodynamic expansion that occurs while the necessary number of photons is being registered.^[46] Thus, while the specimen is destroyed by the exposure, its configuration can be captured before it explodes.^{[47][48][49][50][51][52][excessive citations]}

Scientists have been working on practical designs and prototypes for x-ray holographic microscopes, despite the prolonged development of the appropriate laser.^{[53][54][55][56][57][58][59][60][excessive citations]}

Photoacoustic microscopy

A microscopy technique relying on the photoacoustic effect,^[61] i.e. the generation of (ultra)sound caused by light absorption. A focused and intensity modulated laser beam is raster scanned over a sample. The generated (ultra)sound is detected via an ultrasound transducer. Commonly piezoelectric ultrasound transducers are employed.^[62]

The image contrast is related to the sample's absorption coefficient ${\displaystyle \alpha }$. This is in contrast to bright or dark field microscopy, where the image contrast is due to transmittance or scattering. In principle, the contrast of fluorescence microscopy is proportional to the sample's absorption too. However, in fluorescence microscopy the fluorescence quantum yield ${\displaystyle \eta _{fl}}$ needs to be unequal to zero in order that a signal can be detected. In photoacoustic microscopy, however, every absorbing substance gives a photoacoustic signal ${\displaystyle PA}$ which is proportional to

${\displaystyle PA\propto \alpha *\Gamma *((1-\eta _{fl})*E_{g}+(E_{laser}-E_{g}))}$

Here ${\displaystyle \Gamma }$ is the Grüneisen coefficient, ${\displaystyle E_{laser}}$ is the laser's photon energy and ${\displaystyle E_{g}}$ is the sample's band gap energy. Therefore, photoacoustic microscopy seems well suited as a complementary technique to fluorescence microscopy, as a high fluorescence quantum yield leads to high fluorescence signals and a low fluorescence quantum yield leads to high photoacoustic signals.

Neglecting non-linear effects, the lateral resolution dx is limited by the

${\displaystyle dx=\lambda /(2*NA)}$

where ${\displaystyle \lambda }$ is the wavelength of the excitation laser and NA is the numerical aperture of the objective lens. The Abbe diffraction limit holds if the incoming wave front is parallel. In reality, however, the laser beam profile is Gaussian. Therefore, in order to the calculate the achievable resolution, formulas for truncated Gaussian beams have to be used.^[63]

Amateur microscopy

Amateur Microscopy is the investigation and observation of

While microscopy is a central tool in the documentation of biological specimens, it is often insufficient to justify the description of a new species based on microscopic investigations alone. Often genetic and biochemical tests are necessary to confirm the discovery of a new species. A laboratory and access to academic literature is a necessity. There is, however, one advantage that amateurs have above professionals: time to explore their surroundings. Often, advanced amateurs team up with professionals to validate their findings and possibly describe new species.

In the late 1800s, amateur microscopy became a popular hobby in the United States and Europe. Several 'professional amateurs' were being paid for their sampling trips and microscopic explorations by philanthropists, to keep them amused on the Sunday afternoon (e.g., the diatom specialist A. Grunow, being paid by (among others) a Belgian industrialist). Professor John Phin published "Practical Hints on the Selection and Use of the Microscope (Second Edition, 1878)," and was also the editor of the "American Journal of Microscopy."

Examples of amateur microscopy images:

• 
  "house bee" Mouth 100X
• 
  Rice Stem cs 400X
• 
  Rabbit Testis 100X
• 
  Fern Prothallium 400X

Application in forensic science

Microscopy has applications in the forensic sciences.^[64] The microscope can detect, resolve and image the smallest items of evidence, often without any alteration or destruction. The microscope is used to identify and compare fibers, hairs, soils, and dust...etc.

In ink markings, blood stains or bullets, no specimen treatment is required and the evidence shows directly from microscopical examination. For traces of particular matter, the sample preparation must be done before microscopical examination occurs.^{[clarification needed]}

Light microscopes are the most use in forensics, using photons to form images, microscopes which are most applicable for examining forensic specimens are as follows:^[65]

1. The compound microscope

2. The comparison microscope

3. The stereoscopic microscope

4. The polarizing microscope

5. The micro spectrophotometer

This diversity of the types of microscopes in forensic applications comes mainly from their magnification ranges, which are (1- 1200X), (50 -30,000X) and (500- 250,000X) for the optical microscopy, SEM and TEM respectively.^[65]

See also

References

Further reading

• Pluta, Maksymilian (1988). Advanced Light Microscopy vol. 1 Principles and Basic Properties. Elsevier.
  ISBN 978-0-444-98939-0
  .
• Pluta, Maksymilian (1989). Advanced Light Microscopy vol. 2 Specialised Methods. Elsevier.
  ISBN 978-0-444-98918-5
  .
• Bradbury, S.; Bracegirdle, B. (1998). Introduction to Light Microscopy.
  ISBN 978-0-387-91515-9
  .
• Inoue, Shinya (1986). Video Microscopy. Plenum Press.
  ISBN 978-0-306-42120-4
  .
• Cremer, C.; Cremer, T. (1978). "Considerations on a laser-scanning-microscope with high resolution and depth of field" (PDF). Microscopica Acta. 81 (1): 31–44.
  PMID 713859. Archived from the original
  (PDF) on 2016-03-04. Retrieved 2009-12-22. Theoretical basis of super resolution 4Pi microscopy & design of a confocal laser scanning fluorescence microscope
• Willis, Randall C. (2007). "Portraits of life, one molecule at a time". Analytical Chemistry. 79 (5): 1785–8.
  PMID 17375393
  ., a feature article on sub-diffraction microscopy from the March 1, 2007 issue of Analytical Chemistry

External links

Wikimedia Commons has media related to Microscopy.

General

• Microscopy glossary, Common terms used in amateur light microscopy.
• Nikon MicroscopyU Extensive information on light microscopy
• Olympus Microscopy Microscopy Resource center
• Carl Zeiss "Microscopy from the very beginning", a step by step tutorial into the basics of microscopy.
• Microscopy in Detail - A resource with many illustrations elaborating the most common microscopy techniques
• Manawatu Microscopy - first known collaboration environment for Microscopy and Image Analysis.
• Audio microscope glossary

Techniques

• Ratio-metric Imaging Applications For Microscopes Examples of Ratiometric Imaging Work on a Microscope
• Interactive Fluorescence Dye and Filter Database Carl Zeiss Interactive Fluorescence Dye and Filter Database.
• New approaches to microscopy Eric Betzig: Beyond the Nobel Prize—New approaches to microscopy.