BE132P Instrumentation in Biological Engineering 1

Virtual Experiment 5:
Phase Contrast Microscopy and Fluorescence Microscopy

A. Phase Contrast Microscopy

Objective:
Familiarization of the working principles and instrumentation of phase contrast microscopy

Phase Plate/Ring Alignment

https://www.olympus-lifescience.com/en/microscope
%20resource/primer/java/phasecontrast/phasemicroscope/

1. Perform phase plate/ring alignment for 5 specimens at 40X:

a) Cheek cells
b) Chinese hamster ovary
c) Closterium
d) Mixed desmid
e) Rabbit muscle
2. Copy and paste the results (specimen images).
3. Answer experiment concept.

Positive and Negative Phase Contrast

https://www.microscopyu.com/tutorials/positive-and-negative-phase-contrast/

1. Compare images of 5 specimens using the three modes: brightfield, positive phase contrast and
negative phase contrast
a) Ctenoid Fish Scale
b) Frog Erythrocyte
c) Insect Wing Hairs
d) Mouse intestine
e) Paramecia
2. Copy and paste the results (specimen images).
3. Answer experiment concept.
BE132P Instrumentation in Biological Engineering 1

B. Fluorescence Microscopy
Objective:
Familiarization of the working principles instrumentation of fluorescence microscopy

Excitation and Barrier Filters

https://www.olympus-lifescience.com/en/microscope-resource/primer/java/fluorescence/opticalpaths/

1. With the given list of fluorophore dyes, adjust the settings for the fluorescence microscope.
• Select an appropriate filter cube
• Adjust the excitation filter
• Adjust the barrier filter

Fluorophores:
a) Alexa Fluor 488
b) Alexa Fluor 568
c) DAPI
d) Cy5
e) FITC

2. Copy and paste the results.

3. Answer experiment concept.

Excitation Filter Sets

https://www.microscopyu.com/techniques/fluorescence/nikon-fluorescence-filter-sets/blue-excitation-
filter-sets

1. Adjust the filters for the 5 specimens:

a) Endothelial cytokinesis
b) Fibroblast culture cell
c) Hela cell mitochondria
d) Metaphase centrosome

2. Take a snapshot or photo of the expected image of the specimen. Take note of the fluorophore dye
or set of fluorophore dyes used and filter set used.
3. Answer experiment concept.

References:
OLYMPUS Microscopy Resource Center. https://www.olympus-lifescience.com/
Microscopy U: The source for microscopy eduction, NikonR. https://www.microscopyu.com/
BE132P Instrumentation in Biological Engineering 1

Name: CUEVAS, Bernadette V. Date:

Student Number: 2015102318 Program/Year: BE/4

Virtual Experiment 5:
Phase Contrast Microscopy and Fluorescence Microscopy

A. Phase Contrast Microscopy

a. Results of Phase Plate/Ring Alignment

a.) Cheek cells b.) Chinese hamster ovary
BE132P Instrumentation in Biological Engineering 1

c.) Closterium d.) Mixed desmid

e.) Rabbit muscle

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b.) Results of Positive and Negative Phase Contrast

a.) Ctenoid Fish Scale

Negative Phase Contrast Brightfield

Positive Phase Contrast

BE132P Instrumentation in Biological Engineering 1

b.) Frog Erythrocyte

Negative Phase Contrast

Brightfield

Positive Phase Contrast

BE132P Instrumentation in Biological Engineering 1

c.) Insect Wing Hairs

Negative Phase Contrast

Brightfield

Positive Phase Contrast

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d.) Mouse intestine

Negative Phase Contrast

Brightfield

Positive Phase Contrast

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e.) Paramecia

Negative Phase Contrast

Brightfield

Positive Phase Contrast

Experiment Concepts:
BE132P Instrumentation in Biological Engineering 1

1. There are three various light waves generated in microscopy. Describe briefly these three waves
(i.) surround (S) wave; (ii.) diffracted spherical wavefront (D-wave); and (iii) resultant particle (P)
wave.
 Surround (S) wave- It is the undeviated or undiffracted planar wavefront that passes
through and around the specimen without interacting with it.
 Diffracted spherical wavefront (D-wave)- It is a deviated/diffracted spherical wavefront
that scatters over a wide arc, passing through the full aperture of the objective.
 Resultant Particle (P) wave- It is the resulting wave from the combination of S waves and
the D waves combined by interference that is focused at the intermediate image plane
upon entering the objective front lens elements which occurs after leaving the specimen
plane.

2. In brightfield microscope, why do transparent specimen lack contrast with its background?
 Transparent specimen lack contrast in the background using brightfield microscope due to
amplitudes of S waves and P waves are almost the same.

3. In order to have contrast between the specimen and its background in a phase contrast
microscope, what should be the characteristics of the S-wave and D-wave?
 Bright image of the condenser annulus are formed by the zeroth order/S-waves in the rear
aperture of the objective due to the objective rear focal plane is conjugated to the
condenser front aperture plane. This occurs under Kohler illumination conditions. On the
other hand, D waves distribution are dependent on the number, size, and refracted index
of the light-scattering objects in the specimen. Small portion of incident light waves are
diffracted while huge portion of light passes through undiffracted to illuminate the whole
image plane.

4. Briefly describe the difference between negative phase contrast and positive phase contrast.
Describe the magnitude of the S-wave, D-wave and P-wave for these two types of phase
contrast.
 In Positive Phase Contrast, because of the etched ring in the glass plate which reduces
physical path taken by the waves that is due to the high refractive index plate, surround
waves (S-waves) advances by a quarter wavelength. When interacting with the specimen,
D-waves are retarded by a quarter-wavelength. This results to one-half wavelength when
the optical path difference between the surround and diffracted waves emerges from the
phase plate. On the other hand, objects appear relatively darker than the background
because the amplitude of the P-wave is lower than the S-wave.
 In Negative Phase Contrast, in relation to the phase diffracted wave, the zeroth-order
surround wave retards by a quarter wavelength due to the objective phase plate
containing elevated ring. When passing through the specimen, the diffracted wave is
retarded by a quarter-wavelength. The P wave is higher in amplitude than the surround
(S) wave.

5. Draw the schematic diagram for the optical path in phase contrast microscope
BE132P Instrumentation in Biological Engineering 1

B. Fluorescence Microscopy
a. Results of Excitation Filters and Barrier Filters

 Alexa Fluor 488 Alexa Fluor 568

BE132P Instrumentation in Biological Engineering 1

 DAPI Cy5

 FITC
BE132P Instrumentation in Biological Engineering 1

b. Results of Excitation Filter Sets

 Endothelial Cytokinesis Fibroblast Cell Culture

BE132P Instrumentation in Biological Engineering 1

 Hela Cell Mitochondria Metaphase Centrosomes

Experiment Concepts:

1. Fill the table with the required information:

Fluorophore Excitation wavelength Emission wavelength

Alexa Fluor 488
495nm 519nm
579nm
Alexa Fluor 568 603nm
359nm
DAPI 457nm
651nm
Cy5 670nm
491nm
FITC 516nm

2. Fluorescence microscopy has three basic filters: an excitation filter (or exciter), a dichroic beam
splitter (or dichromatic mirror), and an emission filter (or barrier filter). Briefly describe and
differentiate one from the other.
BE132P Instrumentation in Biological Engineering 1

 Excitation of other sources of fluorescence are minimized and excitation light are blocked in
the fluorescence emission bond due to the presence of a bandpass filter called an Excitation
Filter (exciter) which allows wavelengths absorbed by the fluorophore to pass through.
 Light in the excitation band are reflected by the Dichroic beamsplitter which is a filter used at
an oblique angle of incidence.
 The type of filter in which wavelengths emitted by fluorophore passes through and blocks
unnecessary light of this bands, especially excitation light is the Emission Filter (barrier filter).

3. Briefly explain why it is necessary to determine fluorescence filter combination for a particular
fluorophore.
 It is necessary to determine fluorescence filter combination for a particular fluorophore
because fluorophores works by absorbing energy (Excitation range) and re-emitting
energy(Emission range) at specific wavelength region. At high frequency illumination,
fluorophore will be excited (absorb energy) while it emits energy at lower frequencies. Each
fluorophore consists of a wavelength where they absorb energy, maximally.

4. Describe briefly what detector is used in fluorescence microscopy and how it is able to create an
image of the specimen.
 The detector used is a conventional charge-coupled device. Images are formed by the effective
separation and detection of excitation and emission wavelengths. Emitted fluorescence
produced by the illuminated specimen is gathered by the objective then serves its usual
image-forming function. This then passes through the dichromatic mirror due to the emitted
light that consists of longer wavelengths and upward to the observation tubes or detectors.

Summary/ Conclusions:

A Phase Contrast Microscope is a device that is used for producing high-contrast images of
transparent specimens such as living cells. It is composed of the light source, the collector lens,
apertures, condenser annulus, condenser, objective lens, phase plate, and a digital camera system.

Surround (S) wave is the undeviated or undiffracted planar wavefront that passes through and
around the specimen without interacting with it. Diffracted spherical wavefront (D-wave) is a
deviated/diffracted spherical wavefront that scatters over a wide arc, passing through the full
aperture of the objective. Resultant Particle (P) wave is the resulting wave from the combination of S
waves and the D waves combined by interference that is focused at the intermediate image plane
upon entering the objective front lens elements which occurs after leaving the specimen plane.

In Positive Phase Contrast, because of the etched ring in the glass plate which reduces physical
path taken by the waves that is due to the high refractive index plate, surround waves (S-waves)
advances by a quarter wavelength. When interacting with the specimen, D-waves are retarded by a
quarter-wavelength. This results to one-half wavelength when the optical path difference between
the surround and diffracted waves emerges from the phase plate. On the other hand, objects appear
relatively darker than the background because the amplitude of the P-wave is lower than the S-wave.
On the other hand, Negative Phase Contrast, in relation to the phase diffracted wave, the zeroth-
order surround wave retards by a quarter wavelength due to the objective phase plate containing
BE132P Instrumentation in Biological Engineering 1

elevated ring. When passing through the specimen, the diffracted wave is retarded by a quarter-
wavelength. The P wave is higher in amplitude than the surround (S) wave.

Fluorescence Microscopy is a technique that uses fluorescence labeling (fluorophores) in order

to identify target molecules. Each fluorophore consists of a wavelength where they absorb energy,
maximally. Fluorophores works by absorbing energy (Excitation range) and re-emitting
energy(Emission range) at specific wavelength region. At high frequency illumination, fluorophore
will be excited (absorb energy) while it emits energy at lower frequencies.

Fluorescence microscope consists of three basic filters. Excitation of other sources of fluorescence are
minimized and excitation light are blocked in the fluorescence emission bond due to the presence of a
bandpass filter called an Excitation Filter (exciter) which allows wavelengths absorbed by the
fluorophore to pass through. Light in the excitation band are reflected by the Dichroic beamsplitter
which is a filter used at an oblique angle of incidence. The type of filter in which wavelengths emitted
by fluorophore passes through and blocks unnecessary light of this bands, especially excitation light is
the Emission Filter (barrier filter).

References:

“Fluorescence Filter Combinations.” Nikon's MicroscopyU,

www.microscopyu.com/techniques/fluorescence/nikon-fluorescence-filter-sets.

“Fluorescence Filter Spectral Transmission Profiles.” Fluorescence Filter Spectral Transmission Profiles
- Java Tutorial | Olympus Life Science, www.olympus-lifescience.com/en/microscope-
resource/primer/java/fluorescence/fluorocubes/.

“Fluorophores and Optical Filters for Fluorescence Microscopy.” Edmund Optics Worldwide,
www.edmundoptics.com/knowledge-center/application-notes/optics/fluorophores-and-optical-
filters-for-fluorescence-microscopy/.

“Introduction to Fluorescence Filters.” Semrock, www.semrock.com/introduction-to-fluorescence-

filters.aspx#:~:text=Most%20fluorescence%20instruments%2C%20including
%20fluorescence,filter%20(or%20barrier%20filter).

“Introduction to Fluorescence Microscopy.” Nikon's MicroscopyU,

www.microscopyu.com/techniques/fluorescence/introduction-to-fluorescence-microscopy.
BE132P Instrumentation in Biological Engineering 1

“Introduction to Phase Contrast Microscopy.” Nikon's MicroscopyU,

www.microscopyu.com/techniques/phase-contrast/introduction-to-phase-contrast-
microscopy#:~:text=Figure%203%20%2D%20Brightfield%20Microscopy%20Wave%20Phase
%20Relationships&text=Because%20the%20amplitudes%20of%20the,superimposed%20against
%20the%20bright%20background.

“Positive and Negative Phase Contrast.” Nikon's MicroscopyU, www.microscopyu.com/tutorials/positive-

and-negative-phase-contrast.

“Spectrum [Alexa Fluor 488]: AAT Bioquest.” Spectrum [Alexa Fluor 488] | AAT Bioquest,
www.aatbio.com/fluorescence-excitation-emission-spectrum-graph-
viewer/Alexa_Fluor_488#:~:text=Amino%20Acids-,Spectrum%20%5BAlexa%20Fluor
%20488%5D,emission%20peak%20at%20520%20nm.

“Spectrum [Alexa Fluor 568]: AAT Bioquest.” Spectrum [Alexa Fluor 568] | AAT Bioquest,
www.aatbio.com/fluorescence-excitation-emission-spectrum-graph-viewer/Alexa_Fluor_568.

“Spectrum [Cy5 (Cyanine-5)]: AAT Bioquest.” Spectrum [Cy5 (Cyanine-5)] | AAT Bioquest,
www.aatbio.com/fluorescence-excitation-emission-spectrum-graph-viewer/Cy5_cyanine_5.

“Spectrum [DAPI (4,6-Diamidino-2-Phenylindole)]: AAT Bioquest.” Spectrum [DAPI (4,6-Diamidino-2-

Phenylindole)] | AAT Bioquest, www.aatbio.com/fluorescence-excitation-emission-spectrum-
graph-viewer/DAPI_4_6_diamidino_2_phenylindole.

“Spectrum [FITC (Fluorescein-5-Isothiocyanate)]: AAT Bioquest.” Spectrum [FITC (Fluorescein-5-

Isothiocyanate)] | AAT Bioquest, www.aatbio.com/fluorescence-excitation-emission-spectrum-
graph-viewer/5_FITC_fluorescein_5_isothiocyanate.