Optics Microscopes

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FJ
Optics Microscopes
Confocal Microscopy
Facility Trivia
Specimen Preparation
Fluorescence
Potpourri
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Usually marked 10x or 20x, these parts of a
microscope allow the user to see the image
created by the objective.
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What are the eyepieces (oculars)?
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On one, the objectives are located above the
stage, on the other, the objectives are
underneath the stage.
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What is the main difference between an upright
and an inverted microscope?
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Differential Interference Contrast
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What does DIC stand for?
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This number, which can range from lt 0.1 to 1.6,
is inscribed on an objective and is a measure of
its light-gathering ability and resolving power.
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What is the numerical aperture (NA)?
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The bending of light rays as they pass from one
transparent medium to another (eg. from glass to
air or vice versa).
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What is refraction?
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The absorption of light of one wavelength and
re-emission (within 1 msec) of light of a longer
wavelength.
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What is fluorescence?
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Fluorescein, GFP, Alexa 488, Cy2
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What are some fluorescent molecules that can be
optimally excited by light of approx. 490 nm?
-OR What are some fluorescent compounds that
look green?
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The fluorescent protein whose DNA sequence is
most frequently appended to that of other
proteins, transfected into cells, and used to
determine the proteins location in a cell.
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What is GFP?
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Fluorescence originating from cellular components
that have not been labeled with a fluorescent
dye.
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What is autofluorescence?
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The Stokes shift.
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What term describes the wavelength change between
the excitation and emission photons in the
fluorescence process?
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The excitation light sources used on all of the
Microscopy Facility confocal systems.
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What are lasers?
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Z stack or Z series
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What term is used to describe a series of
confocal images acquired at multiple, sequential
focus (Z) positions?
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DAILY DOUBLE!!
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This aperture, located in front of the
fluorescence detector, makes a confocal system
confocal.
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What is the pinhole (or iris)?
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On the Zeiss confocal, its a Configuration on
the BioRad, its called a Method.
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What do you select when setting up the confocal
in order to have correct laser excitation and
fluorescence collection for the fluorescent
labels on the slide?
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You use it because it reduces out-of-focus
fluorescence and gives a clearer picture of
localization than does widefield fluorescence.
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What are the main reasons for doing confocal
imaging?
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http//hsc.unm.edu/crtc/microscopy/
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What is the URL/web address of the Microscopy
Facility? (Its our new address)
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Approximately 225
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How many people have been trained on Facility
microscopes since the Microscopy Facility started
in 2000?
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The name of the company that wrote the calendar
software used by the Facility.
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What is BosDev?
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The Meta (spectral) photodetector and a blue
laser diode (405 nm).
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What are the two main features of the Meta system
confocal that differ from the other confocals in
the Microscopy Facility?
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Kate
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What is Genevieves middle name?
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Paraformaldehyde, methanol, acetone
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What are compounds that are commonly used to fix
cells and tissues?
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What Becky says to people who try to image
poorly-prepared slides.
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What is You cant take good pictures of bad
slides?
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Labeling technique that uses antibody binding to
attach a fluorescent label to a specific protein.

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What is immunofluorescence?
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This specimen preparation step can be skipped if
the protein epitope being labeled is on the
outside of the cell.
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What is permeabilization?
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0.17 mm
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What is the coverslip thickness recommended when
making slides (the thickness for which most high
power objectives are designed)?
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tif, jpeg, bmp, pic, lsm
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What are some different types of image files
(image formats)?
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Substance placed between a high power objective
and the coverslip in order to maximize
resolution.
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What is immersion oil?
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Approximately 400 700 nm
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What is the wavelength range of visible light?
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The smallest distance at which two objects can be
distinguished from each other.
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What is the limit of resolution?
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Antony van Leeuwenhoek, Zacharias Janssen, Galileo
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Who were people involved in developing the first
microscopes?
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FINAL JEOPARDY
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Signal from one fluorescence label that gets into
a confocal channel or through a filter set
intended to collect fluorescence from a different
label.
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What is fluorescence bleedthrough?