Optical or light microscopy involves passing
visible light transmitted through or reflected from the sample through a single lens or multiple
lenses to allow a magnified view of the sample. The resulting image can be detected directly by the eye, imaged on a
photographic plate, or
captured digitally. The single lens with its attachments, or the system of lenses and imaging equipment, along with the appropriate lighting equipment, sample stage, and support, makes up the basic light microscope. The most recent development is the
digital microscope, which uses a
CCD camera to focus on the exhibit of interest. The image is shown on a computer screen, so eye-pieces are unnecessary.
Limitations Limitations of standard optical microscopy (
bright field microscopy) lie in three areas; • The technique can only image dark or strongly refracting objects effectively. • There is a
diffraction-limited resolution depending on incident wavelength; in visible range, the resolution of optical microscopy is limited to approximately 0.2
micrometres (
see: microscope) and the practical magnification limit to ~1500x. • Out-of-focus light from points outside the focal plane reduces image clarity. Live cells in particular generally lack sufficient contrast to be studied successfully, since the internal structures of the cell are colorless and transparent. The most common way to increase contrast is to
stain the structures with selective dyes, but this often involves killing and
fixing the sample. Staining may also introduce
artifacts, which are apparent structural details that are caused by the processing of the specimen and are thus not features of the specimen. In general, these techniques make use of differences in the refractive index of cell structures. Bright-field microscopy is comparable to looking through a glass window: one sees not the glass but merely the dirt on the glass. There is a difference, as glass is a denser material, and this creates a difference in phase of the light passing through. The human eye is not sensitive to this difference in phase, but clever optical solutions have been devised to change this difference in phase into a difference in amplitude (light intensity).
Techniques To improve specimen
contrast or highlight structures in a sample, special techniques must be used. A huge selection of microscopy techniques are available to increase contrast or label a sample. Image:Paper_Micrograph_Bright.png|
Bright field illumination, sample contrast comes from
absorbance of light in the sample Image:Paper_Micrograph_Cross-Polarised.png|
Cross-polarized light illumination, sample contrast comes from rotation of
polarized light through the sample Image:Paper_Micrograph_Dark.png|
Dark field illumination, sample contrast comes from light
scattered by the sample Image:Paper_Micrograph_Phase.png|
Phase contrast illumination, sample contrast comes from
interference of different path lengths of light through the sample
Bright field Bright field microscopy is the simplest of all the light microscopy techniques. Sample illumination is via transmitted white light, i.e. illuminated from below and observed from above. Limitations include low contrast of most biological samples and low apparent resolution due to the blur of out-of-focus material. The simplicity of the technique and the minimal sample preparation required are significant advantages.
Oblique illumination The use of oblique (from the side) illumination gives the image a three-dimensional appearance and can highlight otherwise invisible features. 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. This limitation makes techniques like optical sectioning or accurate measurement on the z-axis impossible.
Dark field Dark field microscopy is a technique for improving the contrast of unstained, transparent specimens. Dark field illumination uses a carefully aligned light source to minimize the quantity of directly transmitted (unscattered) light entering the image plane, collecting only the light scattered by the sample. Dark field can dramatically improve image contrast – especially of transparent objects – while requiring little equipment setup or sample preparation. However, the technique suffers from low light intensity in the final image of many biological samples and continues to be affected by low apparent resolution. under Rheinberg illumination
Rheinberg illumination is a variant of dark field illumination in which transparent, colored filters are inserted just before the
condenser so that light rays at high aperture are differently colored than those at low aperture (i.e., the background to the specimen may be blue while the object appears self-luminous red). Other color combinations are possible, but their effectiveness is quite variable.
Dispersion staining Dispersion staining is an optical technique that results in a colored image of a colorless object. This is an optical staining technique and requires no stains or dyes to produce a color effect. There are five different microscope configurations used in the broader technique of dispersion staining. They include brightfield Becke line, oblique, darkfield, phase contrast, and objective stop dispersion staining.
Phase contrast of undecalcified
hyaline cartilage showing
chondrocytes and
organelles,
lacunae and
extracellular matrix :
In electron microscopy: Phase-contrast imaging More sophisticated techniques will show proportional differences in optical density.
Phase contrast is a widely used technique that shows differences in
refractive index as difference in contrast. It was developed by the Dutch physicist
Frits Zernike in the 1930s (for which he was awarded the Nobel Prize in 1953). The nucleus in a cell for example will show up darkly against the surrounding cytoplasm. Contrast is excellent; however it is not for use with thick objects. Frequently, a halo is formed even around small objects, which obscures detail. The system consists of a circular annulus in the condenser, which produces a cone of light. This cone is superimposed on a similar sized ring within the phase-objective. Every objective has a different size ring, so for every objective another condenser setting has to be chosen. 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. As the physical properties of this direct light have changed, interference with the diffracted light occurs, resulting in the phase contrast image. One disadvantage of phase-contrast microscopy is halo formation (halo-light ring).
Differential interference contrast Superior and much more expensive is the use of
interference contrast. Differences in optical density will show up as differences in relief. A nucleus within a cell will actually show up as a globule in the most often used
differential interference contrast system according to
Georges Nomarski. However, it has to be kept in mind that this is an
optical effect, and the relief does not necessarily resemble the true shape. Contrast is very good and the condenser aperture can be used fully open, thereby reducing the depth of field and maximizing resolution. The system consists of a special prism (
Nomarski prism,
Wollaston prism) in the condenser that splits light in an ordinary and an extraordinary beam. The spatial difference between the two beams is minimal (less than the maximum resolution of the objective). After passage through the specimen, the beams are reunited by a similar prism in the objective. In a homogeneous specimen, there is no difference between the two beams, and no contrast is being generated. However, near a refractive boundary (say a nucleus within the cytoplasm), the difference between the ordinary and the extraordinary beam will generate a relief in the image. Differential interference contrast requires a
polarized light source to function; two polarizing filters have to be fitted in the light path, one below the condenser (the polarizer), and the other above the objective (the analyzer). Note: In cases where the optical design of a microscope produces an appreciable lateral separation of the two beams we have the case of
classical interference microscopy, which does not result in relief images, but can nevertheless be used for the quantitative determination of mass-thicknesses of microscopic objects.
Interference reflection An additional technique using interference is
interference reflection microscopy (also known as reflected interference contrast, or RIC). It relies on cell adhesion to the slide to produce an interference signal. If there is no cell attached to the glass, there will be no interference. Interference reflection microscopy can be obtained by using the same elements used by DIC, but without the prisms. Also, the light that is being detected is reflected and not transmitted as it is when DIC is employed.
Fluorescence . This is a
confocal laser scanning fluorescence micrograph of
thale cress anther (part of
stamen). The picture shows among other things a nice red flowing collar-like structure just below the anther. However, an intact thale cress stamen does not have such collar, this is a fixation artifact: the stamen has been cut below the picture frame, and
epidermis (upper layer of cells) of stamen stalk has peeled off, forming a non-characteristic structure. Photo: Heiti Paves from
Tallinn University of Technology. When certain compounds are illuminated with high energy light, they emit light of a lower frequency. This effect is known as
fluorescence. Often specimens show their characteristic
autofluorescence image, based on their chemical makeup. This method is of critical importance in the modern life sciences, as it can be extremely sensitive, allowing the detection of single molecules. Many fluorescent
dyes can be used to stain structures or chemical compounds. One powerful method is the combination of
antibodies coupled to a fluorophore as in
immunostaining. Examples of commonly used fluorophores are
fluorescein or
rhodamine. The antibodies can be tailor-made for a chemical compound. For example, one strategy often in use is the artificial production of proteins, based on the genetic code (DNA). These proteins can then be used to immunize rabbits, forming antibodies which bind to the protein. The antibodies are then coupled chemically to a fluorophore and used to trace the proteins in the cells under study. Highly efficient fluorescent
proteins such as the
green fluorescent protein (GFP) have been developed using the
molecular biology technique of
gene fusion, a process that links the
expression of the fluorescent compound to that of the target protein. This combined fluorescent protein is, in general, non-toxic to the organism and rarely interferes with the function of the protein under study. Genetically modified cells or organisms directly express the fluorescently tagged proteins, which enables the study of the function of the original protein
in vivo. Growth of
protein crystals results in both protein and salt crystals. Both are colorless and microscopic. Recovery of the protein crystals requires imaging which can be done by the intrinsic fluorescence of the protein or by using transmission microscopy. Both methods require an ultraviolet microscope as proteins absorbs light at 280 nm. Protein will also fluorescence at approximately 353 nm when excited with 280 nm light. Since
fluorescence emission differs in
wavelength (color) from the excitation light, an ideal fluorescent image shows only the structure of interest that was labeled with the fluorescent dye. This high specificity led to the widespread use of fluorescence light microscopy in biomedical research. Different fluorescent dyes can be used to stain different biological structures, which can then be detected simultaneously, while still being specific due to the individual color of the dye. To block the excitation light from reaching the observer or the detector,
filter sets of high quality are needed. These typically consist of an
excitation filter selecting the range of excitation
wavelengths, a
dichroic mirror, and an
emission filter blocking the excitation light. 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. See also:
total internal reflection fluorescence microscope Neuroscience Confocal Confocal laser scanning microscopy uses a focused
laser beam (e.g. 488 nm) that is scanned across the sample to excite
fluorescence in a point-by-point fashion. The emitted light is directed through a pinhole to prevent out-of-focus light from reaching the detector, typically a
photomultiplier tube. The image is constructed in a computer, plotting the measured fluorescence intensities according to the position of the excitation laser. Compared to full sample illumination, confocal microscopy gives slightly higher lateral resolution and significantly improves
optical sectioning (axial resolution). Confocal microscopy is, therefore, commonly used where 3D structure is important. A subclass of confocal microscopes are
spinning disc microscopes which are able to scan multiple points simultaneously across the sample. A corresponding disc with pinholes rejects out-of-focus light. The light detector in a spinning disc microscope is a digital camera, typically
EM-CCD or
sCMOS.
Two-photon microscopy A two-photon microscope is also a laser-scanning microscope, but instead of UV, blue or green laser light, a
pulsed infrared laser is used for excitation. Only in the tiny focus of the laser is the intensity high enough to generate fluorescence by
two-photon excitation, which means that no out-of-focus fluorescence is generated, and no pinhole is necessary to clean up the image. This allows imaging deep in scattering tissue, where a confocal microscope would not be able to collect photons efficiently. Two-photon microscopes with wide-field detection are frequently used for functional imaging, e.g.
calcium imaging, in brain tissue. They are marketed as
Multiphoton microscopes by several companies, although the gains of using 3-photon instead of 2-photon excitation are marginal.
Single plane illumination microscopy and light sheet fluorescence microscopy Using a plane of light formed by focusing light through a cylindrical lens at a narrow angle or by scanning a line of light in a plane perpendicular to the axis of objective, high resolution optical sections can be taken. Single plane illumination, or light sheet illumination, is also accomplished using
beam shaping techniques incorporating multiple-prism beam expanders. The images are captured by CCDs. These variants allow very fast and high signal to noise ratio image capture.
Wide-field multiphoton microscopy Wide-field multiphoton microscopy refers to an
optical non-linear imaging technique in which a large area of the object is illuminated and imaged without the need for scanning. High intensities are required to induce non-linear optical processes such as
two-photon fluorescence or
second harmonic generation. In
scanning multiphoton microscopes the high intensities are achieved by tightly focusing the light, and the image is obtained by beam scanning. 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). 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: s(x,y) = PSF(x,y) * o(x,y) + n Where is the additive noise. Knowing this point spread function means that it is possible to reverse this process to a certain extent by computer-based methods commonly known as
deconvolution microscopy. 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 and
Her2, target of the
breast cancer drug
Trastuzumab, within a cancer cell. 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.
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 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.
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. 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.
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,
X-ray microscopy has also been developed since the late 1940s. The resolution of X-ray microscopy lies between that of light microscopy and electron microscopy. == Electron microscopy ==