BIO 206L: Exercise 3 Lab Analysis

Please answer the following questions during lab. Keep in mind that you need to work independently. Write in
complete sentences, using proper grammar and spelling when an explanation is required. Be complete yet
concise in your answers. Submit your report as a pdf file on Canvas before the beginning of the next lab.
1) Create a numbered list of the specimens (e.g., cheek cell, diatoms, etc.) you examined during Exercise 3.
For each specimen, describe the type(s) of microscopy you used to examine the specimen. Include images of
each specimen- indicate magnification and the type of microscopy used.
1. Cheek cells (brightfield microscopy unstained, 400x total magnification)

2. Cheek cells (brightfield microscopy stained with methylene blue, 100x total magnification)

3. Cheek cells (fluorescence microscopy stained with DAPI/Texas Red Phalloidin, 400x total
magnification)

4. Diatoms (darkfield microscopy, 400x total magnification)

5. Diatoms (phase contrast microscopy, 400x total magnification)

6. Algal cells (fluorescence microscopy, 100x total magnification)

7. BPAE (fluorescence microscopy, 100x total magnification)

2) Use the table below to describe the advantages and disadvantages of the following techniques.
Technique

Advantages

Disadvantages

Clear, bright image

Low contrast

Stain
adds
contrast
(especially for identifying
organelles)
Provides most contrast

The color of the specimen is not

what it actually is

Darkfield

Nice way to analyze the

exterior of a specimen

Fluorescence

Illuminates
different
functional
parts
of
a
specimen which are seen as
different colors

Specimen must be strongly

illuminated which can harm
specimen
Can result in bleaching and
blurring

Brightfield without Stain

Brightfield with Stain
Phase Contrast

Not good for thick specimen

3) During Procedure 3.2 (preparation of a wet mount of human cheek cells), you added FIX and PERM
solutions to your sample of cheek cells. What does each solution contain? What is the purpose of each
solution? What might happen if you forgot to add the PERM solution?
The FIX solution is used to fix the cells, mainly purposed to prevent decay. The PERM solution is to
permeabilize the cell by removing lipids to allow for antibodies to go thru the cell membrane. FIX
contains paraformaldehyde in a buffered solution. PERM contains a mild buffered detergent. If you
forgot to add the PERM, the dye might not full enter and stain the cell.
Cited: https://www.thermofisher.com/us/en/home/life-science/cell-analysis/cell-analysis-learningcenter/molecular-probes-school-of-fluorescence/sample-considerations/preparing-fixed-cells-imaging.html
4) You observed your cheek cells using fluorescent stains. Which two stains were used to prepare these cells?
Which wavelengths of light were used to excite each stain? Which wavelength of light were emitted by each
stain? What is the relationship between excitation and emission wavelengths?
DAPI and Texas Red were the two stains used. DAPI was excited with 365 nm light and Texas Red was
excited with 552 nm. DAPI emitted >420 nm light and Texas Red emitted 570 nm light. The emitted light
is of a higher wavelength, meaning lower energy the energy is lower because some of the energy is lost
as heat.
Cited: http://www.olympusmicro.com/primer/lightandcolor/fluoroexcitation.html
5) What cellular components were visible in your cheek cells? Include a composite image exhibiting both
stains simultaneously. Label the stained cellular structures. Why were other areas in the cell black?
From our images, the only components visible in our cheek cells were the nucleus, cell membrane, and
cytoplasm. The black areas are parts of the cell that were not bound to a dye.
cytoplasm
Nucleus
Cell membrane
6) You observed BPAE cells using fluorescent microscopy. What stains were used to prepare these cells?
Which wavelengths of light were used to excite each stain? Which wavelengths of light were emitted by each
stain? What is the purpose of the fluorescent filter cube turret?
The BPAE cells were stained with BODIPY FL, Texas Red, and DAPI. DAPI was excited with 365 nm
light, Texas Red was excited with 552 nm, and BODIPY FL was excited with 495 nm light. DAPI emitted
>420 nm light, Texas Red emitted 570 nm, and BODIPY FL emitted 525 nm light. The filters purpose is
to only allow light of a specified wavelength through while blocking others that are not useful to the
experimenter.

7) What cellular components were visible in the BPAE cells? Which fluorophore was used to identify the
location of each structure? Include a composite image exhibiting all three stains simultaneously. Label the
cellular components that were stained.
The cellular components visible in the BPAE cells were the actin microfilaments, the tubulin network
(microtubules), and the nucleus. DAPI was used to identify the nucleus (it binds to nucleic acids), Texas
Red was used to identify Actin, and BODIPY FL was used to identify microtubules (made of tubulin).

Nucleus (blue)
Actin microfilaments (red)
Microtubules (green)

8) Give an example from lab and then compare autofluorescence, direct fluorescence, and
immunofluorescence. How would you classify Rhodamine phalloidin?
Direct fluorescence was seen with the DAPI which directly bound to the A-T rich regions in the nucleus.
Autofluorescence was seen in the algal cells the chlorophyll abrsorbed blue/green light and emitted red
light as seen in our algal picture in #1 above. It is autoflourescing because the algae automatically
fluoresces without the need of an external fluorophore to bind to the cell. We saw immunofluorescence
with the BODIPY FL. The BODIPY FL dye was conjugated to an antibody which binds to tubulin. I
would classify Rhodamine phalloidin as an direct fluorescence fluorophore because the rhodamine is
conjugated to phalloidin which directly binds to the actin moreover, no antibodies are used so it is not
immunofluorescence. To relate the the three types of fluorescence: direct fluorescence makes use of
fluorophores that directly bind to cell parts that we are targeting, autofluorescence makes uses of
endogenous biomolecules that automatically fluoresce, and immunofluorescence
Cited: Dr. Maass powerpoint
9) Research HeLa cells. Explain their origin. Why are they unique? Why are they controversial? Give an
example of how these cells have been used in medical research. Be sure to cite your sources.
HeLa cells are descendants of a continually reproduced line of cervical cancer cells that came from
Henrietta Lacks in 1951. They are unique because unlike other cells that reproduce, and later die, these
cell stayed alive. They are controversial because the doctor who obtained the cells from Lack did not get
her consent. These cells were used by Jonas Salk to develop the polio vaccine.
Cited: Rahbari R, Sheahan T, Modes V, Collier P, Macfarlane C, Badge RM; Sheahan; Modes; Collier;
MacFarlane; Badge (2009). "A novel L1 retrotransposon marker for HeLa cell line identification".
BioTechniques. 46 (4): 27784. doi:10.2144/000113089 (inactive 2015-01-13)
Claiborne, Ron; Wright IV, Sydney (2010-01-31). "How One Woman's Cells Changed Medicine"
Batts DW (2010-05-10). "Cancer cells killed Henrietta Lacks then made her immortal". The VirginianPilot. pp. 1, 1214. Retrieved 2012-03-17.; Note: Some sources report her birthday as August 2, 1920, vice
August 1, 1920.

10) Find an image of HeLa cells that have been fluorescently labeled. Include the image in your assignment.
What types of fluorophores were used to stain the cells? What cellular structures were stained? Be sure to cite
your sources.
Fluorophores used: Phalloidin CruzFluor 488 conjugate, DAPI counterstain. The cellular structures
visible are the f-actin (green) and nucleic acids in the nucleus (blue).

Cited: https://www3.scbt.com/scbt/product/phalloidin-cruzfluor-488-conjugate