BIOS 312 EXAM 2: Fluorescence microscopy
(1) Composite image (2) Grayscale (3) Multi-bandpass (4) Confocal microscopy
(1) Composite image: combined collected mages (2) Grayscale: the digital cameras used with fluorescence microscopes usually record images in grayscale since color detectors are less sensitive (3) Multi-bandpass: filters that selectively allow more than one excitation and emission wavelength to pass. (4) Confocal microscopy: solution to epifluorescence; background fluorescence is limited by the use of a pinhole aperture (at the expense of signal intensity, resulting in longer exposure times) and excitation of fluorophores by tightly focused lasers that scan through the specimen. Therefore, only a particular portion of the specimen is exposed to the excitation wavelength and a virtual section is created. If structures are truly co-localized, they will appear on the same virtual section.
(1) Epifluorescence (2) Dichroic mirror (3) Emission filters (4) Channels
(1) Epifluorescence: When excitation light is directed onto the specimen from the viewing side (2) Dichroic mirror: allows emitted wavelengths but not excitation wavelengths to pass is placed in the light path (keeps reflected excitation light from entering the optical system) (3) Emission filters: enhances sensitivity of epifluorescence; which only allow emitted light of the desired wavelength range to pass through (4) Channels: collected images
(1) Fluorescence Microscopy (2) Fluorophore (3) FITC
(1) Fluorescence microscopy: cellular structures are visualized based on light emission by the specimen (2) Fluorophore: chemical structure on antibody temporarily captures electromagnetic radiation of one wavelength (the excitation wavelength) and releases it as radiation of another, lower energy, wavelength (the emission wavelength.) (3) FITC: fluorescent dye - stains cilia green
Parts of the epifluorescence microscopy
(1) eyepiece lens (2) emission filter: allows only desired wavelengths of emitted light to pass (3) dichroic mirror: reflects incident or scattered excitation light but allows the emitted wavelengths to pass (4) excitation filter: allows only desired wavelengths of excitation to pass - in epifluorescence microscopy, illuminating light comes through objective from above
Fluorescence is... - How do you produce a fluorescence image? (the dyes)
- Fluorescence is a phenomenon in which the chemical structure of a fluorophore temporarily captures electromagnetic radiation of one wavelength (the excitation wavelength) and releases it as radiation of another, lower energy, wavelength (the emission wavelength.) -> For example, when light at 488nm (in the blue region of the visible spectrum) is absorbed by the fluorescent dye fluorescein isothiocyanate (FITC), electrons in the outer orbital of certain atoms are excited to a higher energy state. When they return to their normal energy level, they emit photons of light at 525 nm, which is in the green region of the visible spectrum - To produce a fluorescence image, a specimen is illuminated with light at a wavelength that excites natural fluorescent pigments or synthetic fluorescent dyes that have been used to stain the material. -> These dyes can either bind directly to structures in the specimen (a form of histochemical staining) or they can be attached to antibodies that specifically recognize antigens in cells or tissues (immunofluorescent staining.) -> When excitation light is directed onto the specimen from the viewing side, the method is known as epifluorescence. -> Emitted light is passed through the optics of the microscope to the eyes or a digital camera. -> To keep reflected excitation light from entering the optical system, a dichroic mirror that allows emitted wavelengths but not excitation wavelengths to pass is placed in the light path
What happens transmitted-light microscopy/light microscopy? - what about in fluorescence microscopy?
- In transmitted-light microscopy, visible light passes through a specimen mounted on a slide and continues through the optical system to the eyes or a camera. - Structures in the specimen will interfere slightly with the passage of the light, absorbing some wavelengths and transmitting others to produce color, or subtly bending it to reveal shapes. - The structures visible by light microscopy can be enhanced by staining with colored dyes that bind differentially to components in the specimen. - In fluorescence microscopy, cellular structures are visualized based on light emission by the specimen.
What's a limitation of epifluoroescence?
- One limitation of epifluorescence microscopy is the difficulty in determining if a combined signal indicates that two fluorophores are in very close proximity within the specimen (co-localization.) - For example, if light emissions from a green fluorophore and a red fluorophore appear at the same two-dimensional position in a specimen, the combined image is yellow at that point. - This could be because the structures the fluorophores are attached to directly contact one another, an important consideration when using fluorescence microscopy to investigate interactions between proteins. - Cells, however, are three dimensional objects with a reasonably large depth on a molecular scale. The two fluorophores, therefore, might actually be far apart but positioned so that their signals overlap one another
What's the solution to the limitation in epifluoroescence?
- One solution to this problem is confocal microscopy. In this method, background fluorescence is limited by the use of a pinhole aperture (at the expense of signal intensity, resulting in longer exposure times) and excitation of fluorophores by tightly focused lasers that scan through the specimen. - Therefore, only a particular portion of the specimen is exposed to the excitation wavelength and a virtual section is created. -> If structures are truly co-localized, they will appear on the same virtual section, whereas if they are distant but the signals are overlapping, only one will appear on the section. -> Another benefit to confocal microscopy is that sequential sections can be stacked on top of one another to create a three-dimensional image (a "Z stack"). -> Due to the lack of background fluorescence, confocal images appear clearer than epifluorescence images
(1) How do you enhance sensitivity? (2) Why can traditional fluorescence microscopes only image one fluorophore at a time? What happens when a specimen contains multiple fluorophores?
- Sensitivity is enhanced by using emission filters, which only allow emitted light of the desired wavelength range to pass through. Fluorescence microscopes have multiple adjustable filters so that a range of excitation and emission wavelengths can be selected (2) Because of the arrangement of their excitation and emission filters, traditional fluorescence microscopes can only image a single fluorophore at a time. - When a specimen contains multiple fluorophores with different excitation and emission wavelengths, two or more images must be acquired. The microscope is set for one fluorophore, and an image is recorded before the system is set for the next fluorophore. The collected images, called channels, can then be combined digitally to create a composite image.
Why is grayscale used?
- The light emitted by the fluorophores is often very weak or at a wavelength that is difficult to detect with the human eye. -> Therefore, the digital cameras used with fluorescence microscopes usually record images in grayscale since color detectors are less sensitive. - Before the channels are combined, each one is assigned a false color that matches the color of the emitted light. - Note that some newer microscopes use single-color LED excitation sources and multi-bandpass filters that selectively allow more than one excitation and emission wavelength to pass. These microscopes can image several fluorophores simultaneously, although there is sometimes "bleed-through" between channels - Retaining the individual color channel images can be useful since the grayscale format does a better job of showing fine details than the composite image, and comparing separate channels makes it easier to identify different fluorophores that could be very close to one another in a specimen. -> As a result, scientists often present grayscale images of the blue, green, and red channels along with a composite color image when including immunofluorescence micrographs in lectures and publications