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Light microscopy is a key tool in modern cell biology. Light microscopy has several features that make it ideally suited for imaging biology in living cells: Here I provide a brief introduction to using light microscopy in cell biology, with particular emphasis on factors to be considered when starting microscopy experiments. Most broadly, light microscopy techniques can be divided into two categories: In brightfield microscopy, info cell light ing light source and detection objective are placed on opposite sides of the sample, and the sample is imaged by its effect on the light passing through it as the sample absorbs, scatters, or deflects the light.
Because most cells are thin and transparent, they do info cell light ing absorb much light and so are difficult to see without adding optics that allows the phase shift of light induced by the cells to be seen.
The two most commonly used techniques to visualize this phase shift are phase contrast, which causes cells to appear dark on a light background, and differential interference contrast DICwhich gives a pseudo—three-dimensional 3D shaded appearance to cells Murphy and Davidson, Brightfield without phase contrast or DIC is usually info cell light ing to see the general outlines of cells, but phase contrast or DIC is necessary to achieve detailed, high-contrast brightfield images.
Fluorescence microscopy uses fluorescent dyes fluorophoreswhich are molecules that absorb one wavelength of light the excitation wavelength and emit a second, longer wavelength of light the emission wavelength. Most molecules in the info cell light ing are not very fluorescent, so fluorescent labels to be imaged are typically introduced by the experimenter.
This allows the labels to be face changer jar launcher to the molecule s of interest, either by genetically encoding a fluorescent protein or by binding a fluorescently labeled antibody.
Multiple different fluorescent molecules can be distinguished simultaneously and can be detected at very low abundance single molecules can be imagedmaking this a very powerful technique. Fluorescence microscopy is typically done using epifluorescence, in which the fluorescence excitation light illuminates the sample through the same objective that is used to detect the emission from the sample.
A fluorescence filter cube separates the light by wavelength so that the emitted light can be imaged without interference from the excitation light Murphy and Davidson, The two major techniques for introducing fluorescent labels into cells are immunofluorescence, in which fluorescently labeled antibodies that bind to specific proteins in cells are introduced, and genetic introduction of a fluorescent protein. In immunofluorescence, the cells are first fixed to cross-link proteins in the cell and then permeabilized to allow antibodies access to the cellular milieu.
Typically, primary antibodies, which recognize the proteins of interest in the cell, are first introduced. After unbound antibodies are washed off, fluorescently labeled secondary antibodies, which bind to the primary antibodies, are added.
This is known as indirect immunofluorescence and makes it easy to switch primary antibodies, as they do not need to be labeled, and the secondary antibodies typically have broad specificity e. Genetic introduction of a fluorescent protein involves fusing a fluorescent protein to a target of interest, which is then either introduced into the genome of the cell or expressed from a plasmid. This allows imaging of proteins in live cells and, if done by genomic introduction, means that the protein is expressed from its endogenous promoter at its endogenous level.
For both immunofluorescence and fluorescent protein imaging, it is straightforward to image four colors in a cell. Most commonly, filter sets with,and nm excitation wavelengths are used. Particularly in the nm-excitation channel, new fluorescent proteins are being developed rapidly, and better combinations may emerge in the near future; for reviews of fluorescent proteins see Day and Davidson and Dean and Palmer Most info cell light ing biology imaging is done with widefield microscopy, in which the microscope simply forms an image of the sample on the camera, without any additional optical manipulation.
Live cells are most commonly imaged on an inverted epifluorescence microscope Figure 1. In such a microscope, the objective images the sample from below. Inverted microscopes are popular for cell info cell light ing imaging because they allow imaging through a glass coverslip to see cells grown above. This means that cells can be grown in coverslip-bottom Petri dishes or multiwell plates containing growth media, which can be left open at the top.
Alternatively, an upright microscope can be used with a water-dipping objective, which is immersed into the medium in which the cells are grown, but this is less convenient and less common. Schematic drawings of common microscopy techniques. A An inverted epifluorescence microscope. The sample sits between the slide and coverslip.
The condenser lens delivers illumination for viewing light transmitted through the sample; the objective lens collects light from the sample and delivers excitation light for fluorescence microscopy. The filter cube consists of an excitation info cell light ing bluean emission filter greenand a dichroic mirror gray.
The excitation and emission filters select the wavelengths that will illuminate the sample and be recorded on the camera, respectively, and the dichroic mirror reflects the excitation light to the sample while transmitting the emission light to the camera.
B A confocal microscope. The excitation and emission pinholes are imaged onto the sample to define an info cell light ing point in the sample and to detect light from only that point. The lenses that image info cell light ing pinholes onto the sample info cell light ing been omitted for simplicity. The scan mirror scans the illuminated spot across the sample; because the scan mirror is info cell light ing both the excitation and emission paths, the position of the spot detected from the sample is scanned in parallel with the excitation spot.
C A light sheet microscope. An illumination objective, along with additional optics not shownis used to form a thin sheet of light that illuminates the sample.
A detection objective images the light emitted from this sheet onto a camera. Most of the key properties of the microscope are dictated by the choice of objective lens. The objective determines the magnification and resolution of the image; it also determines how much light will be collected from the sample and hence the sensitivity of the microscope.
The objective performance is largely determined by its magnification and numerical aperture NA. In addition, objectives come in multiple classes according to how well aberrations have been corrected; these are info cell light ing to by terms such as Achromat and Plan Apochromat. The NA is defined as the sine of the largest angle info cell light ing light emitted by the specimen that the objective can collect multiplied by the sample refractive index for which the objective is designed.
This controls both the light-gathering power of the objective collecting a larger range of angles collects more light; this scales as the square of the NA and the resolution limit of the objective. In the XY -plane the plane perpendicular to the focus axisthe theoretical resolution is given by 0. Although the resolution of the objective lens is set by its NA, the objective magnification is important to ensure that the image info cell light ing magnified sufficiently on the camera to capture that resolution the rule is that there must be at least two camera pixels per resolvable element to capture the full resolution of the microscope objective; Jonkman et al.
Most objectives are designed to image through glass coverslips 0. To acquire the best images, it is important to grow cells on these coverslips. If cells must be grown on plastic, specialized objectives for imaging through plastic are available, although they typically have poorer performance than objectives designed for imaging through coverslips.
Alternatively, specialized plastic dishes can be purchased that have optical properties info cell light ing to 0. A major limitation of conventional epifluorescence microscopy is that the illuminating light excites fluorophores in a cone throughout the sample, and the detection camera cannot distinguish this out-of-focus light from the light emitted by the focal plane of the sample.
Hence the in-focus information that we seek to image is obscured by blurred images of the out-of-focus regions of the sample. For samples that are not too thick and not too densely labeled with fluorophores, this out of focus light is not a major problem.
However, for thick, densely stained info cell light ing or in cases in which we wish to achieve well-resolved 3D images, this out-of-focus light can obscure valuable information. Many techniques have been developed to eliminate this info cell light ing light.
The most commonly used is confocal microscopy, in which the sample is illuminated by a focused laser beam at a single point in the sample focal plane Figure 1. Light from this point is detected after passage through a pinhole, such that only light emitted from the focal plane makes it through the pinhole and is recorded on the detector.
Light from out-of-focus planes is blocked by the pinhole, and so the confocal only records light from the focal plane of the sample. Because laser-scanning confocal microscopes record an info cell light ing point by point, they do not use cameras, but instead use a point detector, which tend to be less sensitive than cameras. To overcome this limitation, systems that scan multiple focus spots across the sample simultaneously and image the resulting emission on a camera have been designed.
The most common of these is the spinning-disk confocal, which uses a disk of pinholes that sweep across the sample such that a revolution of the disk dialogues of bajirao mastani over every point in the sample during a single exposure Toomre and Info cell light ing, Spinning-disk confocal microscopes combine ease of use, high speed up to hundreds of frames per secondand high sensitivity, so they have become widely used in cell biology.
Spinning-disk confocal microscopy is believed to be more live-cell—friendly than widefield or laser-scanning confocal microscopy, but definitive evidence of this is lacking. Spinning-disk confocal microscopy is widely used for imaging protein and organelle dynamics in single cells—for example, imaging mitochondrial inheritance in yeast Rafelski et al.
Another microscopy technique widely used in cell biology is total internal reflection fluorescence TIRF microscopy Axelrod, This technique relies on total internal reflection of a laser beam at the interface between the coverslip and the aqueous sample above it.
The reflected laser beam sets up an evanescent light field at the coverslip interface; the evanescent field penetrates only a few hundred nanometers into the sample.
This allows excitation of fluorophores within a few hundred nanometers of the coverslip but nowhere else in the sample. This highly localized excitation makes TIRF microscopy one of the most sensitive microscopy techniques, as there is essentially no background from the rest of the sample, but also limits its use to imaging samples that are immediately adjacent to the coverslip. This has made TIRF a very powerful technique for imaging membrane dynamics and trafficking e.
Finally, a revolution in microscopy has been occurring with the development of light sheet microscopes Weber and Huisken, ; Keller and Ahrens, These are microscopes that illuminate the sample from a info cell light ing orthogonal to the imaging plane Figure 1. This eliminates the problem of out-of-focus light because only light from the focal plane or very close to it is excited.
This selective illumination also reduces the total light exposure of the sample, info cell light ing in turn reduces photobleaching and phototoxicity. If identical objectives are used to produce the light sheet and detect the fluorescence, their roles can be interchanged, with orthogonal views of the sample being captured through the two objectives. These images can then be fused to produce a 3D image of the sample with isotropic resolution, eliminating the poorer Z -resolution add document wordpress template other forms of microscopy Wu et al.
Alternatively, sophisticated optical design can be used to produce microscopes that capture high-resolution 3D images at high speed Chen et al. Although these microscopes are being commercialized, they are not yet widely available.
I expect that these and other light sheet designs will become widely available in the near info cell light ing, heralding a revolution in 3D microscopy of live cells. Although beyond the scope of this introduction, another recent revolution in light microscopy is the development of superresolution techniques that allow resolution beyond that of conventional light microscopy Huang et al.
With these techniques, resolution as high as 20 nm in XY and 50 nm in Z can be achieved, although these often require the use of special sample preparation techniques, special microscopes, or special fluorophores and can be difficult to use in live cells. Although superresolution microscopes are commercially available, adoption of these techniques has been slow. However, it is likely that these techniques will be increasingly important for resolving cellular ultrastructure in the future.
When planning a microscopy experiment, the first thing to do is to determine the systems to which you have access and how they are configured; check the systems both in your lab and in core facilities. The relative performances of different imaging modalities are compared in Table 1.
In general, for routine imaging, widefield epifluorescence is a good starting point: Laser-scanning confocal microscopy is excellent for rejecting out-of-focus light and acquiring 3D image data, but in general it is less sensitive and more phototoxic than spinning-disk confocal microscopy. For live specimens for which 3D information is required, spinning-disk confocal microscopy should be considered.
For membrane imaging, or for any other sample that is within a few hundred nanometers of the coverslip, TIRF microscopy should be considered. Finally, should you have access to a light sheet microscope or other specialized microscope, learn its capabilities and consider whether it is a good fit for your experiments. Maximum sample thickness is a guideline, and with careful sample preparation, thicker samples can often be imaged. The performance of a microscope can depend heavily on the details of its components and how it is configured.
For this reason, it often makes sense to try multiple systems when trying a new imaging experiment to determine which one will best meet your needs. Local microscopy experts, such as a core director, can often help you choose an appropriate microscope.