Microscopy
The development of microscopy revolutionized biology, gave rise to the field of histology and so remains an essential technique in the life and physical sciences.
[7][8] Antonie van Leeuwenhoek developed a very high magnification simple microscope in the 1670s and is often considered to be the first acknowledged microscopist and microbiologist.
A more recent technique based on this method is Hoffmann's modulation contrast, a system found on inverted microscopes for use in cell culture.
Oblique illumination enhances contrast even in clear specimens; however, because light enters off-axis, the position of an object will appear to shift as the focus is changed.
Dark field can dramatically improve image contrast – especially of transparent objects – while requiring little equipment setup or sample preparation.
They include brightfield Becke line, oblique, darkfield, phase contrast, and objective stop dispersion staining.
The ring in the objective has special optical properties: it, first of all, reduces the direct light in intensity, but more importantly, it creates an artificial phase difference of about a quarter wavelength.
This method is of critical importance in the modern life sciences, as it can be extremely sensitive, allowing the detection of single molecules.
Most fluorescence microscopes are operated in the Epi-illumination mode (illumination and detection from one side of the sample) to further decrease the amount of excitation light entering the detector.
In wide-field multiphoton microscopy the high intensities are best achieved using an optically amplified pulsed laser source to attain a large field of view (~100 μm).
[27][28][29] The image in this case is obtained as a single frame with a CCD camera without the need of scanning, making the technique particularly useful to visualize dynamic processes simultaneously across the object of interest.
[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.
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.
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.
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.
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.
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.
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.
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.
[40][41] These include IR Near-field scanning optical microscope (NSOM),[42] photothermal microspectroscopy and atomic force microscope based infrared spectroscopy (AFM-IR), as well as scattering-type Scanning Near-field Optical Microscopy (s-SNOM)[43] & nano-FTIR that provide nanoscale spatial resolution at IR wavelengths.
In digital holographic microscopy (DHM), interfering wave fronts from a coherent (monochromatic) light-source are recorded on a sensor.
In transmission mode, the phase shift image provides a label-free quantitative measurement of the optical thickness of the specimen.
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.
[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] A microscopy technique relying on the photoacoustic effect,[61] i.e. the generation of (ultra)sound caused by light absorption.
Collectors of minerals, insects, seashells, and plants may use microscopes as tools to uncover features that help them classify their collected items.
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.
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."
In ink markings, blood stains or bullets, no specimen treatment is required and the evidence shows directly from microscopical examination.
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.
