Additional online resources, including general information and tools, for fluorescence microscopy and image analysis are available using the following links and supplementary reading. Please feel free to contact us with any other references to share.
- Fluorescence SpectraViewer (Invitrogen)
- Fluorescence Spectrum Viewer (BD Biosciences)
- Fluorescence Dye and Filter Database (Zeiss)
- Cell Staining Simulation Tool (Life Technologies)
- Focus/Alignment of Mercury Arc Lamps (Nikon)
- Optical Microscopy Primer (Molecular Expressions)
- MicroscopyU (Nikon)
- Microscopy Resource Center (Olympus)
- Microscopy Online Campus (Zeiss)
- SVI-wiki (SVI)
- Andor Learning Center (Oxford Instruments)
- ImageJ User Guide (NIH)
- Imaris Learning Center (Bitplane)
- Bioimage Data Analysis (Wiley)
- Lightsheet Analysis Pipeline (Janelia)
- SciCom Software via GitHub (Janelia)
Overview of Widefield Microscopy
Widefield microscopy is any technique that illuminates the entire field of view simultaneously and collects the resulting image on a camera. Widefield techniques can be further classified as either 'epi' or 'trans', depending on whether the illumination and signal collection occur on the same (epi) or opposite (trans) sides of the sample. Trans-illumination techniques require a lens on each side of the sample: one termed the condenser, which focuses the illuminating light, and one termed the objective, which forms the image. In epi-illumination techniques, a single lens acts as both the condenser and objective. Widefield techniques are highly sensitive and so capable of high frame rate (100+ fps) as well as very long term time-lapse imaging. However, these techniques also collect signal coming from all depths in the sample, and so out-of-focus light can greatly reduce image contrast.
Widefield: Bright-field Microscopy
Bright field is the oldest of all microscopic techniques. The image is produced via absorption as light passes through the sample. Bright-field images of unstained biological samples tend to have low contrast, because most cells are not strongly light-absorbing.
Widefield: Phase-contrast Microscopy
Phase contrast generates an image based on abrupt changes in a sample's refractive index (RI), a measure of 'optical density'. These optical edges cause light to diffract (bend) in many directions, where the amount of bending depends on the degree and abruptness of the RI change. Simply speaking, phase contrast measures how much light is bent at each location in the sample relative to how much light was not bent. Physically, this comparison occurs via an induced interference between the diffracted and undiffracted light. This technique enhances contrast in many biological samples, but also results in a 'halo' effect where large changes in RI also cause scatter.
Widefield: Differential Interference Contrast Microscopy
Like phase contrast, differential interference contrast (DIC) (a.k.a. Nomarski interference contrast) generates an image based on changes in the sample's refractive index (RI). However, DIC employs a very different mechanism: DIC simultaneously acquires two images of the sample using distinct polarities, with one image being slightly offset relative to the other. The images are then re-aligned and converted back to the same polarity where they are compared via interference. DIC gives even better contrast than phase and without the 'halo' effect. It also gives an interesting 'shadow' impression, though these 'shadows' are due to the direction of the offset between the two intermediate images and have no relationship to the sample's vertical topography.
Widefield: Epifluorescence Microscopy
Epifluorescence microscopy is an extremely popular way to visualize fluorescent probes in biological samples. As the name suggests, epifluorescence employs epi-illumination and so a single lens (the objective) both illuminates the sample and collects the fluorescent emissions. Filters restrict the excitation light to a small range of wavelengths suitable to excite fluorophores in the sample, while other filters sort out the longer wavelength fluorescence emissions before they reach the camera. A major drawback of epifluorescence microscopy is out-of-focus background emissions, or 'flare', which greatly degrades image contrast (and therefore resolution).
Overview of Point-Scanning Microscopy
A main drawback of widefield microscopy is that out-of-focus light greatly degrades image contrast and thus effective resolution. The ability to remove out-of-focus light is a tremendous imaging advantage, and many technologies have been developed to create optical sections. Point-scanning techniques illuminate only one diffraction-limited spot (~200 nm diameter) in the focal plane, while rotating mirrors traverse this point back and forth in a raster pattern across the sample to create an image. Light emitted (or reflected) from each point then travels back through the objective and mirrors before reaching a detector.
Point-Scanning: Confocal Microscopy
Because the illuminating spot of light is created by focusing, the light spreads into a cone above and below the focal plane. Thus, out-of-focus sample regions are illuminated and contribute (unwanted) emissions, though their intensity is less than in widefield mode. A second effect completes the optical section: before reaching the detector, all fluorescence emissions pass through a very small aperture located in a conjugate image plane (i.e. a location outside the sample where light from the sample is also in focus). Said in an abbreviated way, the focal plane within the sample and the pinhole outside the sample are 'confocal'. Thus, only emissions from the focal plane can pass through the pinhole, while out-of-focus (i.e. spatially diffuse) emission can not fit through the aperture. Together, the combination of reduced out-of-focus illumination and blocking of out-of-focus emissions produces a crisp optical section.
Point-Scanning: Multiphoton Microscopy
Like confocal, multiphoton microscopy also creates an optical section, but does so using entirely different mechanisms. Multiphoton microscopy relies on very short (100 fs), but very intense bursts of light to induce an effect called multiphoton absorption. In the most likely scenario, two photons interact with a dye's electron simultaneously to trigger fluorescence emissions. Such events are highly improbable and so occur to an appreciable degree within the spot of light in the focal plane, where the light is most concentrated. The result is that zero out-of-focus emissions are generated and so all emissions can be detected directly (i.e. no pinhole is needed). Scattered emissions (non-ballistic) can also usefully contribute to the image, because their point of origin is known. An additional benefit is that since two photons impart their energy to an electron simultaneously, each needs only one half of the energy typically required to trigger fluorescence emissions. Thus, infrared light (700-1000 nm) can trigger fluorescence from visible wavelength dyes. This property is useful because tissue is more transparent to infrared light. Together, these effects contribute to multiphoton's ability to image far (10x) deeper into scattering tissues than is possible using confocal microscopy.