BIO230H1 Lecture Notes - Microscopy, Fluorescein, Green Fluorescent Protein

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Bio230 Lab: Live Cell Imaging
Introduction
Microscopy: techniques used to view live cells continues to be an invaluable tool
provides images/insights into the mechanisms (Ex. Cell and organelle movement).
Biochemical processes: used to view cell signaling and cell-cell interaction.
Learn how to adjust microscope (Koehler illumination)
Understand neglected concepts in optical microscopy with proper configuration
Intensity and wavelength spectrum of light emitted through various places
Viewing living cells including intracellular movements
Organelles and other cytoplasmic components of cells are capable of movements
Plant cells part of the cytoplasm may show cytoplasmic streaming, which may
result in broad cytoplasmic strands
Individual organelles, mitochondria and chloroplast, may position themselves
within streaming or exhibit short periods of rapid movement
Recent studies show that in many cells, layer of cytoplasm and organelles close to
plasma membrane
Nucleus commonly maintains its position in the cell regardless of cytoplasm
movement
Fluorescence Microscopy
Fluorescence diacetate (FDA): uncharged and non fluorescent derivative of a
fluorescence molecule, fluorescein
FDA is uncharged can passively cross the plasma membrane but once inside the
cell esterases (enzymes) convert FDA to the fluorescent, polar molecule,
fluorescein, which cannot easily diffuse back to the plasma membrane and escape
the cell
Dead cells don’t have esterase and do not fluorescein
FDA can be used to determine the dead cells from the live ones
Fluorescein can turn yellow or green (depends on pH) and turns blues under light
Florescence Microscopy
Advent and development of optical probes such as green fluorescent protein
(GFP) used with live cells
Use of genetically coded fluorescent proteins allow to tag and track range of
proteins
Without application of exogenous probes and fixation of tissues
Technological developments, cell biologists can study a range of cellular function
Importance of florescence
Procedures
A. Dissecting Microscope
Dissecting microscope is sometimes referred to as a stereo-microscope b/c it is
like 2 microscopes set to focus on one viewing a 3-D
Plug in microscope and adjust power
B. Compound Microscope: Koehler illumination and Cslibration of your microscope (see
figure in appendix 2)
C. Compound Microscope: Observation of living cells
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Document Summary

Microscopy: techniques used to view live cells continues to be an invaluable tool provides images/insights into the mechanisms (ex. Biochemical processes: used to view cell signaling and cell-cell interaction. Learn how to adjust microscope (koehler illumination) Understand neglected concepts in optical microscopy with proper configuration. Intensity and wavelength spectrum of light emitted through various places. Organelles and other cytoplasmic components of cells are capable of movements. Plant cells part of the cytoplasm may show cytoplasmic streaming, which may result in broad cytoplasmic strands. Individual organelles, mitochondria and chloroplast, may position themselves within streaming or exhibit short periods of rapid movement. Recent studies show that in many cells, layer of cytoplasm and organelles close to plasma membrane. Nucleus commonly maintains its position in the cell regardless of cytoplasm movement. Fluorescence diacetate (fda): uncharged and non fluorescent derivative of a fluorescence molecule, fluorescein.

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