Live cell imaging Flowchart

Vanessa Eugenio
1002895307
Part A. Koehler Illumination
Procedures Explanation
1. Set phase ring to bridge field. Close the  We first need light to come in for focusing
field iris diaphragm using the knurled ring image
at the top of the light source.

2. Focus the edge of the field iris diaphragm  Giving a clear image
by raising the condenser unit.

3. Open the field iris diaphragm until the  We need to form a circle of light fills the
light is at the edge of the field of view field under the microscope to maximize its
resolution
4. Adjust the condenser iris diaphragm, close  Obtaining the best contrast and resolution
the diaphragm just until the point of image
begins to darken.

5. Switch to 40x objective lens. And increase  Amplify image by switching to a higher
the light intensity slightly. value objective lens. Increase light
intensity to give a clear image
6. Rotate the eyepiece until the lines of the  A parallel line is needed as ruler, to divided
reticle are parallel with those of the stage into equal sized arbitrary units, and the
micrometer and then align the lines at the calibrated stage micrometer is used to
left edges of both the reticle and the stage find the actual value of each eyepiece
by moving the stage micrometer with X reticle unit
and Y axis knob.

7. Locate the point as far to the extreme  Provide the greatest accuracy.
right as possible where two lines are
exactly superimposed upon each other.
Count the no. of divisions on the
micrometer between the zero line and
superimposed lines.

Part B. Observation of living cells (tomato stem trichomes)

Procedures Explanation
1. Place few drops of water on slides. Use a  Water dissolve the tomato and give a
razor blade to scrape off a small cluster of clearer image.
trichomes from the stem of tomato plant.

2. Using an inoculating needle to transfer the  Start making the specimen, and gently
trichomes from the razor blade to the cover it by coverslip to avoid bubbles
drop of distilled water. Gently lower a formed
coverslip onto the specimen.
3. Locate the trichomes under 10x objective  A phase-contrast microscopy must include
and switch to 40x after focusing the single filters, to block the light and only indirect
trichomes. The phase ring of condenser must refracted and diffracted light goes to
set at Ph2. specimen. So the phase ring needed to be
set
4.Locate the nucleis of a living cell, and look  The best cytoplasmic streaming usually
for the chloroplast and other organelles, its occur in cytoplasmic strands radiating out
usually around the nucleus. from the nucleus. The vibrating particles in
the cels are Brownian motion.
5.Add 5 L stock FDA to 495 L distilled water.  FDA is used to test cell viability and cell
membrane integrity. FDA will convert to
fluorescent, polar molecule, fluorescein
once inside a living cell.