Confocal Microscopy: Principles, Techniques, and Applications
Confocal Microscopy: Techniques and Applications
Samples can be fixed or unfixed.
Results can be correlated with DIC (Nomarski) imaging.
Oriented to:
- Study of thick samples in fluorescence (fixed or unfixed).
- Study of samples marked with 1, 2, 3, or 4 fluorescent molecules during in vivo experiments in cultured cells with variable time lengths.
- Three-dimensional reconstruction of thick specimens studied in fluorescence.
- In vivo experiments in cultured cells requiring complex handling areas for stimulation techniques to perform advanced fluorescence microscopy.
Basic Imaging Applications: Imaging samples marked with two fluorochromes. Possibility of using the digital zoom function (especially useful for applications such as FRAP, very fast image acquisition). Used to electronically adjust the magnification and spatial resolution.
Important Note: The amount of light that radiates to the sample is proportional to the square of the zoom. Visualization of fluorescent cells with DIC (Nomarski) contrast for observation of cellular responses.
Scanning Systems
There are two types of scanning systems:
- A system with a filter (which works with wavelength and cannot use multiple fluorochromes). This is a system of buckets, used to work with more traditional fluorophores. It is high efficiency.
- A scanning system with a spectral detector version: that measures a diffraction grating, offering greater flexibility to observe small or large events, or complicated fluorochromes (which cause it to open or close the spectrum). It uses a diffraction grating to linearly analyze the spectrum. It allows you to choose the range of detection that we use and to work with separate fluorophores without a complicated filter system.
Olympus FV1000 Confocal Microscope
Characteristics of the FV1000: Twin Scanner, MEASUREMENT AND STIMULATION, simultaneously, excitement in specified area: With the screen while doing FV1000 We stimulation
Confocal Microscopy for Rotating Doctor: for real-time observation of rapid cellular events in vivo, minimizing photobleaching and phototoxicity.
Spinning Disk Confocal Microscopy
Types of Technologies Used in spinning disk confocal microscopy: Nipkow disk, disk ranirado (Olympus) using laser or fluorescent lamps of high efficiency
Slotted Disc (Slit Disk Confocal): The disc contains a grooved pattern that creates a series of virtual pinholes. While the disc spins at high speed (3000 rpm), it rejects unfocused light by positioning a rotating disk with alternating vertical and horizontal slots at the confocal pinholes of the microscope, creating virtual pinholes.
Why turn the disc? Because static images produce a bright image. Turning ensures that the scan occurs to generate a confocal image.
Two-Photon Microscopy
Two-photon microscopy is a process found in nature (except on stars). It has reduced photodamage and reaches a much greater distance than 600 or 800 microns. One uses a fluorescence microscope, reducing the excitation. There is no up or down lighting, not because it need not confocal pin hole, the larger wavelengths penetrate much more coming in tissue ls
Advantages of Two-Photon Microscopy
It collects photons, has pinholes, and reduces photodamage, which focuses only on the spatial resolution is lower at longer wavelengths.