FLUORESCENCE MICROSCOPY
FLUORESCENCE MICROSCOPY
FLUORESCENCE MICROSCOPY
FLUORESCENCE MICROSCOPY
FLUORESCENCE MICROSCOPY
FLUORESCENCE MICROSCOPY
FLUORESCENCE MICROSCOPY
PRINCIPLE OF USE
Detector Fluorescence
Property of some atoms and molecules to
absorb light at a specific wavelength.
Image Subsequent emission of light of longer
wavelength after a brief interval.
Excitation Emission
filter
Basic Premise of Fluorescence
filter
Microscopy
Dichroic Components are stained with fluorescent dyes
mirror
(fluorophores or fluorochromes).
Light source
Molecules absorb excitation light at a given
Objective wavelength (usually UV).
Emit light at a longer wavelength after a short
Specimen
delay.
PRINCIPLE OF USE
Fluorescence Microscopy
Acridine orange
Fluorescent compounds exhibiting an affinity for particular cell
macromolecules can function as stains.
E.g Acridine orange - binds both DNA and RNA
Commonly employed to image specific features of small specimens such
as microbes
Visualize precise intricate physiological processes in terms of both time
and space
Gain valuable insights into the pathobiology of various disease that would
otherwise be very challenging to uncover.
PRINCIPLE OF USE
Illumination, Excitation and Emission
Detector
in Fluorescence Microscopy
High-intensity light illuminates and excites
fluorescence species in the sample.
Image
Fluorescent species emit light of a longer
wavelength.
Image based on emitted light (emission
Excitation Emission wavelength) rather than the excitatory light.
filter filter
Optical Path in Fluorescence
Dichroic Microscopy
mirror
Light source
Excitation wavelength focused on the specimen
through the objective lens.
Objective Fluorescence emitted by the specimen focused
on the detector by the objective.
Only reflected excitatory light and emitted light
Specimen reach the objective.
FLUORESCENCE MICROSCOPE
PARTS OF A FLUORESCENCE MICROSCOPE
Condenser
Tube Aperture
Lens Diaphragm Light
Field Source
Diaphragm
Eyepiece
Observation
Tubes
Fluorescence
Filter Cube
Objective
Lens
Stage Microscope
Frame/Arm
Stage Translation
Control Fine & Coarse
Adjustment
Knobs
Base
PARTS OF A FLUORESCENCE MICROSCOPE
SPECIALIZED PARTS & FUNCTIONS
Light Source
Transmits light with
ultraviolet, blue, and
green wavelengths
(340-465 nm)
Ex. Mercury Arc
Lamp, Xenon Arc
Lamp, Metal-Hallide
Lamp, and Light-
emitting diode (LED)
SPECIALIZED PARTS & FUNCTIONS
Fluorescence Filter Cube
Unique in fluorescent microscopy
Aligns the filters in the excitation and
emission light path
Contains an excitation filter, a
dichroic mirror, and a barrier/
emission filter
Directs light from the excitation
source to the specimen
Directs light emitted by specimen to
the detector
SPECIALIZED PARTS & FUNCTIONS
Excitation Filter
Ensures that only required
wavelengths are transmitted to the
specimen
Transmits wavelengths ranging
between 460 and 500 nanometers
Filters proper wavelength of light
for excitation of fluorophores such
as fluorescein
SPECIALIZED PARTS & FUNCTIONS
Dichroic Mirror
Positioned diagonally (45-degree
angle)
Primary optical element that
separates the excitation and
emission light
Shorter excitation wavelength is
reflected toward the specimen
Longer emitted wavelength is
transmitted is transmitted
SPECIALIZED PARTS & FUNCTIONS
Emission Filter
Further purifies emission
wavelengths passing through the
dichromatic mirror
Only allows light of wavelengths
between 515 and 565
nanometers to reach the
microscope eyepieces and/or
detectors
ADVANTAGES
High Specificity and Sensitivity.
Fluorescence microscopes can
detect weak signals or low
concentrations of fluorescent
molecules.
High specificity ensures selective
capturing of the structure of interest
while minimizing background signals.
ADVANTAGES
Selective Labeling with Multiple
Fluorophores.
Fluorophores will absorb and
emit light at different
wavelengths.
In a single sample, multiple
molecules can be distinguished
simultaneously.
ADVANTAGES
Real-time tracking of dynamic
cellular processes.
Fluorescence microscopy is a
common method used for live-cell
imaging. Without the need for sample
fixation, non-invasive fluorescent
tagging allows real-time observation
of dynamic processes in living tissues.
DISADVANTAGES
Phototoxicity
The intense light from the excitation
light can damage the samples and
lead to sample death. (e.g:
photooxidation)
DISADVANTAGES
Fading Specimen Fluorescence
The fluorescence intensity diminishes
during the imaging process over time
due to the fluorophores losing their
ability to emit light. It impacts the
accuracy and quality of the image.
DISADVANTAGES
Low Light Levels
The shortage of light used in imaging the specimen
that can be caused by physical constraints which
results to poor image contrast.
DISADVANTAGES
Expensive
THANK YOU!
Fluorescence Microscopy can be
costly, in the Philippines, the price
ranges from 100k and above
which makes it a barrier for many
research groups who don’t
necessarily have access to it.
REFERENCES
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THANK YOU!
orescence%20photomicrography.
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https://microbenotes.com/fluorescence-microscope-principle-instrumentation-applications-advantages-limitations/
Hickey, S. M., Ung, B. S., Bader, C. A., Brooks, R. D., Łaźniewska, J., Johnson, I. R., Sorvina, A., Logan, J. M., Martini, C., Moore, C.,
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Liver Pathophysiology. (n.d.). Free-radical damage. Free-Radical Damage - an overview | ScienceDirect Topics.
https://www.sciencedirect.com/topics/medicine-and-dentistry/free-radical-
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https://goldbio.com/articles/article/fluorescence-microscopy-a-basic-introduction
Sanderson MJ, Smith I, Parker I, Bootman MD. Fluorescence microscopy. Cold Spring Harb Protoc. 2014 Oct 1;2014(10):pdb.top071795. doi:
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What is a Fluorescence Microscope and What is It Used For. (n.d.). New York Microscope Company.
https://microscopeinternational.com/what-is-a-fluorescence-microscope-and-what-is-it-used-for/
Wu, Y., & Shroff, H. (2022). Multiscale fluorescence imaging of living samples. Histochemistry and Cell Biology, 158(4), 301–323.
https://doi.org/10.1007/s00418-022-02147-4
ZEISS Microscopy Online Campus | Microscopy Basics | Fluorescence Microscopy. (n.d.). https://zeiss-
campus.magnet.fsu.edu/articles/basics/fluorescence.html#:~:text=The%20excitation%20filter%20spectrum%20(Figure,green%20fluores
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