Reflected Light Microscopy

With epifluorescence microscopy, the entire specimen is flooded evenly in excitation light. All parts of the specimen are excited simultaneously and the resulting fluorescence (emission) is detected by the detector or camera. Epifluorescence microscopy is a powerful tool for visualizing sub-cellular structures, however, is prone to image distortion due to unfocused light (emanating from above and below the plane of focus) reaching the detector.

Compared to conventional fluorescence microscopy, out of focus blur, resulting from emission above and below the focal plane, can be overcome by 'optically sectioning' the specimen at the plane of focus. Examples of optical sectioning include: confocal laser scanning microscopy, multi-photon confocal microscopy, stimulated emission depletion microscopy, and structured illumination.

A post-acquisition procedure which re-assigns light associated with widefield fluorescence imaging. The distorted signal is mathematically deconvolved using the point spread function. The result is a high quality image with substantially less noise, better definition and higher resolution in 3D.

Optical sections can be obtained in epifluorescence microscopy using structured illumination. A plate with an imprinted grid is inserted into the field diaphragm plane in the illumination pathway and projected onto the specimen. The projected grid image is translated over the specimen by tilting the glass plate back and forth. At least three raw images of the specimen are acquired with the grid superimposed in different positions. These images are subsequently processed automatically by the microscope software to create an optical section. The software determines grid contrast as a function of location and removes out-of-focus image information before assembling the three images into a final single image.

A major limitation of widefield fluorescence microscopy arises from the fact that the sample is illuminated throughout its volume regardless of the focus plane, and fluorescence is collected not only from the focus plane, but also from planes above and below it. As a result of image distortion by out-of-focus light, widefield fluorescence images often demonstrate compromised specimen detail and contrast. Confocal microscopy largely eliminates out-of-focus haze by incorporating a pinhole near the detector that resides in a plane conjugate with the focal plane, allowing only in-focus-light through to the detector. The result is an image of fluorescence signal arising from the plane of focus, or an optical section at the plane of focus, with out-of-focus light eliminated by the pinhole.

Stimulated emission depletion (STED) creates super resolution images by altering the effective point spread function of the excitation beam using a second laser, in the shape of a doughnut surrounding the excitation beam, which suppresses fluorescence emission from fluorophores outside the hollow center of the 'doughnut'. Therefore, only fluorophores located in the very center of the 'doughnut' will be excited by the excitation beam. An image is created by raster-scanning the specimen with the excitation/depletion laser combination.

Multiphoton (MP) microscopy uses pulsed long-wavelength light as an excitation source. The fluorophores must absorb enough energy for excitation, which requires two photons of long-wavelength light (compared to just one photon of short wavelength). The MP microscope does not use a pinhole to produce optical sections, rather, depends on concentrating the excitation lasers to the plane of focus, where the density of long-wavelength photons will be high enough to achieve 2-photon excitation. Therefore, only fluorophores at the plane of focus will be excited, and emission light detected will be only that of in-focus-light. The long-wavelength, low energy excitation lasers of MP microscopes possess low photo-cytotoxicity, photobleaching, and photodamage effects compared to the short-wavelength, high energy excitation with traditional confocals. As a result, MP microscopy is well suited for live cell imaging, where cells may be observed for longer periods with fewer toxic effects. As well, long-wavelength light can penetrate deeper into the specimen, allowing for the visualization of thick living tissue samples.