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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 cell 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 biological 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 filter blue , an emission filter green , and 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 illuminated point in the sample and to detect light from only that point. The lenses that image the pinholes onto the sample have been omitted for simplicity.

The scan mirror scans the illuminated spot across the sample; because the scan mirror is in 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 shown , is used to form a thin sheet of light that illuminates the sample.

Fundamentals of Light Microscopy and Electronic Imaging

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.


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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 referred to by terms such as Achromat and Plan Apochromat. The NA is defined as the sine of the largest angle of 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 axis , the theoretical resolution is given by 0.

A quick guide to light microscopy in cell biology

Although the resolution of the objective lens is set by its NA, the objective magnification is important to ensure that the image is 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 similar 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.

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However, for thick, densely stained samples 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 out-of-focus 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 image 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 scans over every point in the sample during a single exposure Toomre and Pawley, Spinning-disk confocal microscopes combine ease of use, high speed up to hundreds of frames per second , and 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. Maybe you have knowledge that, people have look hundreds times for their favorite books like this fundamentals of light microscopy and electronic imaging, but end up in harmful downloads. Rather than enjoying a good book with a cup of coffee in the afternoon, instead they juggled with some infectious bugs inside their laptop.

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