School of Chemical, Biological and Materials Engineering and Sciences

BE14L Biological Techniques Laboratory

3rd Quarter SY 2019-2020

Fluorescence Microscopy
Apan, Jhon Jasper D 1

1 Student, BE14L/B1, School of Chemical, Biological and Materials Engineering and Sciences, Mapúa University

ABSTRACT

One alternative to bright field microscopy that enhances the resolution of microscope imaging is fluorescence
microscopy. The objectives of the students in this experiment are (1) to learn and be familiarized with the use of aa
fluorescence microscope; and (2) to identify characteristics of cells using fluorescence microscope. Cheek swabs were
obtained and suspended to 3 mL of sterilized water. Five hundred (500) µL of the cheek cell solution was mixed with one
drop of both NucBlue and NucGreen, and ten (10) µL of propidium iodide stain. Ten (10) µL of the resulting solution was
viewed under the microscope using bright field and fluorescent selection. Under the bright field microscope, the cells
were difficult to study since the intensity of the light source is not enough to excite the fluorochromes. Fluorescence
imaging of cheek cells using UV filter showed that their nuclei were stained bright blue as expected. Using the green
filter, the nuclei of some cells were observed to be on the range of yellow to green, indicating that dead cells were in the
sample. Using blue filter, the green emission was an unexpected outcome which might have been caused by the
phenomenon of photoconversion of the Hoechst stain. No concrete conclusions about the role of propidium iodide can
be provided since no red filter was used.

Keywords: fluorescence microscopy, fluorochrome, cheek cells, photoconversion

into the sample by the dichroic mirror [4]. The sample

INTRODUCTION
Brightfield microscopy relies on the difference in light
absorption of the substances within the sample to produce
contrast between the specimen and its environment that
allows the specimen to be seen under the microscope. Due
to this, the magnified image has limited resolution [1]. One
alternative to bright field microscopy that enhances the
resolution of the microscope imaging is fluorescence
microscopy.
As its name suggests, the underlying principle for
fluorescence microscopy is fluorescence. Fluorescence is
the capability of a substance to re-emit light which it has
initially absorbed [2].
A fluorescence microscope functions by exciting the
fluorescent species by its light source. Compared to visible
light (400-700 nm) used in bright field microscope, the light
source used in the fluorescence microscope has a much
higher intensity [3]. An exciter filter allows only selected
wavelengths (excitation wavelengths) to pass through it. The
excitation wavelengths are reflected through the objectives
absorbs the excitation light, and the fluorescent species re-
emits light which is called the fluorescent light or emission
light. The objectives gather the fluorescent light. Since the
fluorescent light has a longer wavelength compared to the
excitation light, it is able to pass the dichroic mirror [2].
Another filter called the barrier filters out the light to
selectively allow emission wavelengths to pass towards the
eye or other detectors [4].
However, in fluorescence microscopy, the specimen must be
naturally fluorescent or must be induced using dyes.
Fluorescent dyes are usually referred to as fluorochromes.
They bind with the substrates in the sample (e.g. DNA,
proteins) that allows for the substrate to be observed under
the microscope [5]. In this experiment, the dyes to be used
are propidium iodide, NucGreen nucleic acid stain, and
NucBlue, a derivative of Hoehchst 33342.
Propidium iodide (PI) is a red-fluorescent nuclear stain that
is not permeant to live cells whose excitation/emission
maxima is 493/636 nm. Once the dye is bound, the
excitation/emission maximum is shifted to 535/617 nm which
can be viewed using the rhodamine (red) filter [6][7].

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 School of Chemical, Biological and Materials Engineering and Sciences
BE14L Biological Techniques Laboratory
3rd Quarter SY 2019-2020

Similarly, the NucGreen stain is impermeant to live cells. Its ..

excitation/emission maximum is at 504/523 nm which can be
detected by standard green filter [8][9]. Meanwhile, NucBlue,
whose excitation/emission maximum is at 360/460 nm, is cell
permeant, allowing it to stain the nucleus of both dead and
live cells. It is detectable using blue/cyan or standard DAPI
filters [10].

The objectives of the students in this experiment are (1) to

learn and be familiarized with the use of aa fluorescence
microscope; and (2) to identify characteristics of cells using
fluorescence microscope.

MATERIALS AND METHODS

Figure 1. Bright field imaging of cheek cells
The first part of the experiment was dedicated for the
preparation of cheek cell sample. Prior to the experiment, the Figure 1 shows the cheek cells when viewed under the bright
pipette tips and the microcentrifuge tubes that would be used field microscope. From the resulting image, it can be noticed
in the experiment were autoclaved. that it is difficult to observe the cells when viewed under the
bright field microscope. No stain has been observed despite
The students gargled with distilled water twice to ensure that the addition of dyes on the solution. This is the case since
the sample would not contain any foreign materials. Then, the intensity of the light source is not enough to excite the
the sample of cheek cells was obtained by swabbing a fluorochromes.
popsicle stick inside their cheek and dipping it inside a test
tube with 3 mL of UV-sterilized distilled water. Five hundred
(500) µL of the cheek cell solution was placed in the micro
centrifuge tube along with one drop of both the NucGreen
and NucBlue stains and ten (10) µL of propidium iodide stain.

In the next part of the experiment, ten (10) µL of the prepared

sample was viewed using an inverted fluorescence
microscope at 10x magnification. The sample was viewed
first using bright field selection of the microscope. Then, the
sample was viewed using the fluorescence selection with
ultraviolet, blue, and green filter.

RESULTS AND DISCUSSION

Using microscopy, the students were able to observe the
cheek cells that are stained with NucBlue, NucGreen, and
propidium iodide . The following figures show the images of
the sample as viewed under the microscope using bright field
microscopy and fluorescence microscopy with the use of
different filters
Figure 2. Fluorescence imaging of cheek cells with ultraviolet
filter
In Figure 2, fluorescence imaging of cheek cells showed that
their nuclei were stained bright blue. This is as expected
since NucBlue, a derivative of Hoechst 33342, binds with
both dead and live cells. In UV-excitation, Hoechst 33342 is
visualized as bright blue [11].

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 School of Chemical, Biological and Materials Engineering and Sciences
Figure 3. Fluorescence imaging of cheek cells with green
filter
Figure 3 shows the imaging of cheek cells with the green
filter. The nuclei of some cells were observed to be on the
range of yellow to green. The presence of such emissions
shows that these stained cells are dead since the NucGreen
only reacts with the nucleic acids of cells with degraded
plasma membrane.
Figure 4. Fluorescence imaging of cheek cells with blue filter
In Figure 4, the resulting image using the blue filter is shown.
Unexpectedly, the emission was green instead of blue which
must be the color of the NucBlue stain when excited by
wavelength corresponding to blue.
This unexpected outcome might have been caused by the
phenomenon of photoconversion or a change in the spectral
property of the Hoechst stain. Prior to using the blue filter,
the sample was first subjected to UV excitation. Zurek-
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BE14L Biological Techniques Laboratory
3rd Quarter SY 2019-2020
Biesiada, Kedracka-Krok, and Dobrucki (2013) concluded in
their study that the exposure of Hoechst stain to UV causes
photoconversion where the dye becomes excited by blue
light and emits maximally in green [12].
No red filter was used in the experiment, hence, no concrete
conclusions about the role of propidium iodide, which could
be detected using the red filter, can be provided in this
experiment.
CONCLUSIONS AND RECOMMENDATIONS
The objectives of the students in this experiment are (1) to
learn and be familiarized with the use of a fluorescence
microscope; and (2) to identify characteristics of cells using
fluorescence microscope.
The students have been able to learn and be familiarized
with the use of fluorescence microscope in this experiment.
They were able to learn the underlying principle of the said
equipment. They were able to adjust the different parts of the
microscope to be able to perform the experiment.
The students have also identified the characteristics of cells
using fluorescence microscope with the aid of different
fluorescent dyes.
In future experiments, it is recommended that the sample first
be observed first using the green and blue filter instead of
the ultraviolet filter to since it is possible that the high energy
from UV-excitation caused a change in the spectral property
of the dye. it is recommended that a special enclosure be
used around the microscope to be able to fully prevent
natural light from affecting the results. Other dyes and
microscope filters can also be incorporated in the experiment
for comparison.
REFERENCES
[1] ThermoFisher Scientific, How Fluorescence
Microscopy Works, (n.d.).
https://www.thermofisher.com/ph/en/home/life-
science/cell-analysis/cell-analysis-learning-
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fluorescence/imaging-basics/fundamentals-of-
fluorescence-microscopy/how-fluorescence-
microscopy-works.html#magreso (accessed March
3, 2020).
 School of Chemical, Biological and Materials Engineering and Sciences
BE14L Biological Techniques Laboratory
3rd Quarter SY 2019-2020

[2] K. Spring, M. Davidson, Nikon Instruments Inc., hoechst-33342 (accessed March 3, 2020).
Introduction to Fluorescence Microscopy, (n.d.). [12] D. Zurek-Biesiada, S. Kedracka-Krok, J.W.
https://www.microscopyu.com/techniques/fluoresce Dobrucki, UV-activated conversion of Hoechst
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(accessed March 3, 2020). dyes into blue-excited, green-emitting protonated
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[9] ThermoFisher Scientific, ReadyProbesTM Cell
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[10] ThermoFisher Scientific, NucBlueTM Live
ReadyProbesTM Reagent (Hoechst 33342), (n.d.).
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March 3, 2020).
[11] Science Direct, Hoechst 33342, (n.d.).
https://www.sciencedirect.com/topics/neuroscience/

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