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BIO230H1 (243)


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University of Toronto St. George
Jennifer Harris

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  In this part of the lab you will look at the tomato stem hair cells using bright field, phase contrast, and fluorescence microcopy to see cell structure and intracellular movement Appendix 1: Microscopy  Microscope magnify images of small objects  Nature of the images depends on the type of microscope and the way the specimen is prepared  “Light microscopy” uses visible light under a microscope  Magnification: main purpose of microscope  Resolution: Resolving power being the ability to distinguish between two close objects  Resolution is the minimum distance by which two objects are separated  Ex. 100 nm apart: resolution is 200 nm, then the image you will see one solid line instead of two  Limit of resolution (I.r) for a microscope is related to the shape of the cone light entering the objective lens (cone is called numerical aperture (N.A.) and wavelength of light  I.r = 0.67 wavelength/ N.A Light Microscopy  Magnify cells up to 1000 times  1. Bright light must be focused onto the specimen by lenses in the condenser  2. The specimen must be carefully prepared to allow light to pass through it  3. An appropriate set of lenses (Objective and eyepiece or ocular) must be arranged to focus on an image of the specimen in the eye  Compound microscope used in conventional bright light microsc
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