Fluorescence Microscopy Flashcards
How does refractive index affect NA?
Higher the refractive index of the medium between the from lens of the objective, the higher the NA. Oil (1.51)> water (1.33) > air (1).
Numerical Aperture
Measure of a microscope objectives ability to gather light and resolve fine specimen detail at a fixed object distance. The higher the NA, the higher the resolution. In general, the higher the mag, the higher the NA. The better corrected the objective, the higher the NA.
Why is a camera a better image acquisition device than an eye?
Can be more sensitive, records images, can be used in grayscale or RGB.
What are some advantages of using grayscale over RGB for DAPI signals?
More sensitive, grayscale cameras are sensitive to infrared signals, they can be higher resolution, can be faster.
Why use fluorescence microscopy?
Multiple labels can be discriminated within the same tissue (I.e. DAPI and GFP).
Living cells or tissues can be labeled and imaged using fluorescent dyes.
Fluorescent markers can be expressed in living cells.
What are some limitations of fluorescence?
Out-of-focus light contributions. Photobleaching and quenching. Spectral separation. Phototoxicity to living cells. Concerns with chromatic and spherical aberration.
What is microscopy?
Using a microscope to generate a representation of a sample.
What are some factors that influence the representation of an image?
Quality of sample and sample prep. Illumination and optics of the microscope. The technique of the microscopist. The image acquisition device.
What is fluorescence?
Light emitted by a substance after a short period of time (0.5-20 nanoseconds) following excitation with another light. For example: Alexa488 emits green light when excited by blue. Emitted photons have less energy and are shifted to longer wavelengths than the light used for excitation (stokes shift describes the physics of this).
What are 3 components a microscope relies on?
Illumination source, magnifying lens, image acquisition device.
What is fluorescence microscopy?
Microscopy adapted to fluorescent samples. Similar to brightfield microscopy. The optical paths are similar, but a fluorescence microscope must maximize the collection of emitted fluorescent light and minimize the collection of the incident excitation light.
What are filters and what is a filter cube?
Filters are optical devices that limit the passage of light or reflect light. A filter cube is a set of filters (excitation, barrier, and dichromatic mirror) packaged together to both maximize the collection of emitted fluorescent light and minimize the collection of the incident excitation light.
Total mag
Mag of objective lens x mag of eyepiece x tube factor
What is magnification?
Amount or degree to which the object observed is enlarged in the image.
What is resolution?
Smallest distance between two points on a specimen that can still be distinguished as two separate entities.
