Keep reading...this page will present to you basic theory and techniques of confocal microscopy. Don't worry, it's easy, and fun! Just relax, and remember, the core staff is here to help you with any questions you might have!
What is a Confocal Microscope?
A confocal microscope is a device that utilizes a pinhole aperture to block incoming light of certain angles. This light-limiting system allows imaging of specimens that are thicker than the focal plane of the objective by restricting the out-of-focus light that comes from above and below the focal plane.
By only imaging the light in a particular focal plane (optical sectioning), a researcher can create a 3-dimensional reconstruction of a specimen. This has become a very powerful tool in modern life science.
Anatomy of a Confocal Microscope
There are a few terms that need addressing before we dig to deep into the technical world of microscopy.
Microscope: An instrument that enables researchers to view things that are too small to be seen by the naked eye.
Objective: The primary optical element that gathers light from the specimen and focuses the rays to produce a real image.
Nosepiece: A part of the microscope where the objectives are placed. Most modern microscopes can accomodate multiple objectives on the nosepiece and are motorized. The movement can include both switching objectives and axial (Z-direction) movement for focal plane adjustment.
Eyepiece: The lens group that is closes to the eyes on the microscope.
Stage: A part of the microscope where the speciment is placed. Stages can be manual or motorized. Motorized stages allow for a great deal of software control and automation in experiments.
Laser: An acronym for Light Amplification by Stimulated Emission of Radiation, a laser is a collimated, coherent light source that is highly wavelength-specific and bandwidth limited to provide targeted excitation of fluorophores.
PMT: A photomultiplier tube (PMT) is a vacuum tube that is an extremely sensitive detector and amplifier of light. PMTs convert photons into electrons and can amplify that current by as much as 160dB.
Dichroic Mirror: From the Greek word for "two-colored," a dichroic mirror is amirror that will pass a certain wavelength (or wavelengths) of light and reflect all others.
Filter: Much like a dichroic mirror, a filter is an optical device that passes only light from a certain range of wavelengths. There are three principal types of filters you will encounter in microscopy: shortpass, bandpass, and longpass. Shortpass filters pass light that is shorter than a prescribed wavelength, bandpass filters pass light that falls between two wavelengths, and longpass filters pass light that is longer than a prescribed wavelength.
Pinhole: A small aperture in a confocal microscope that is optically conjugate with the image-forming plane. The pinhole eliminates out-of-focus light and creates the "optical section."
Refraction, Reflection, Diffraction: Light interacts with its environment in different ways. Refraction is when light bends at the interface of two media of different refractive indices. Reflection is when light bounces of of a surface. Diffraction is the spreading out of light beams as they pass througha very small aperture. Al of these processes can result in loss of signal if not properly accounted for.
PSF: The Point Spread Function is a function that describes the system's respons to a point source of light. Whenever a perfect point source is focused in the system, it is convolved with the PSF to form the image that we see. The point spread function can be used (as it is a model of the Optical Transfer Function in the spatial domain) to determine the overall quality of the optical system. A smaller PSF generally results in less convolution of the latent image, thus a better final image and an overall better imaging system.
Airy Disk: The Airy Disk is the best-focused spot of light formed by an objective lens, limited by difraction. The smallest point of light that the system can create is the same diameter as the Airy Disk. This is important when considering our limitations of resolution. The size of the Airy Disk is determined by the wavelength of light and the aperture of the objective.
Resolution: Defined as the ability to discern between two closely spaced objecgts, the resolution of a confocal microscope is primarily determined by the objective numerical aperture (NA). A higher numerical aperture reduces the size of the Airy Disk, thus reducing the size of the smallest resolvable point. The NA also influences axial resolution by decreasing the optical section thickness. A higher NA objective will have higher lateral and axial resolution.
Nyquist-Shannon Sampling Theorem: Since maximum resolution is a function of the NA of the objective, we can set our minimal pixel size based on the Nyquist-Shannon sampling theorem. The theorem states that in order to adequately reconstruct a signal, we need to smaple that signal at a rate of at least twice its cutoff frequency. As applied to imaging, we need to ensure that our pixel size is at least two times smaller than our smalest resolvable object. NIS-Elements sets the Nyquist sampling rate to 2.3x smaller than the resolving power of the objective.
Fluorescence: The radiation of light from a material that has been exicted with a more energetic (higher frequency) light.
These videos will provide a basic foundation for image acquisition in NIS-Elements.
Familiarization with NIS-Elements and Basic Image Acquisition
Image Optimization: Gains, Laser Powers, and Noise