Fluorescence

Microscopy
16 November 2007

By Chen Xiuli, Liu Yuchun, Ng Yen Shan, Cynthia Ong,

Adeline Sham & Eileen Yang
Overview
 Introduction
 Principleof Fluorescence Microscopy
 Fluorescence Microscope
 Fluorescence Dyes

 Experiment
 Objective
 Sample Preparation
 Experimental Procedures
 Results & Discussion

 Comparison with AFM/NSOM

 Conclusion
 Introduction

 Fluorescence Microscopy
•Increase in contrast
•Chemical specificity
•Dissect different functional
aspects of biological
systems

Source: White, N.S. & Errington, R.J. (2005). Fluorescence techniques for drug delivery
research: theory and practice. Advanced drug delivery reviews 57 (2005) 17-42
 Imaging of specific regions of
biological samples..

A cross section of cotton

stained with Rhodamine B.
Fluorescence double-labeling of mammalian cells.

Source:
http://nobelprize.org/educational_games/physics/microscopes/fluorescence/gallery/index.html
Pulmonary artery. Human cells.

Chinese hamster ovary cell. Rat tongue.

 Principle of Fluorescence
Microscopy
1. Introduction of fluorophore
into sample

Source: 1. http:/nobelprize.org/educational_games/physics/microscopes/fluorescence/gallery/12.html
 2. Excitation of Fluorophore
Principle

3. Stokes Shift

2. Shining Fluorescence Details. Basics of Light Microscopy and Imaging. Retrieved on Nov 10, 2007, from
http://www.microscopy.olympus.eu/microscopes/images/4_ShiningFluorescencedetails.pdf
Fluorescence Microscopy

 2 key aspects
Fluorescence Microscope
Fluorescence Dyes
Olympus Fluorescent Microscope BX41
 Olympus Fluorescent Microscope BX41

Internal Light Source

• Built-in transmitted
Koehler illumination of
6V 30W halogen bulb
• For bright field imaging

External Light Source

• Mercury Arc Lamp
• For fluorescence imaging
 Olympus Fluorescent Microscope BX41

Excitation & Emission Filters

• Band-pass filters
• Different filter sets are used for
different dyes
Assembly of
Fluorescence Imaging System
Fluorochromes

• Simultaneous/
multiple staining
possible

• All fluorochromes
show distinct spectral
properties
 Fluorochromes classification

1. Requires other molecules to bind to

specific targets
2. Contains fluorescent proteins
produced by organisms themselves
3. Have special properties
• Inherent binding capacities
• Fluorescent property developed by
enzymatic action
 Fluorochromes with inherent
binding capacity
Propidum iodide (PI)
• An indicator for surface membrane
integrity
• Binds to nuclei of dying or dead
cells
• Excitation λ= 520 nm,
Emission λ= 620 nm (red)
• Gives quantitative information
 Fluorochromes with inherent
binding capacity
4',6-Diamidino-2-phenylindole
(DAPI)
• cell permeable
• binds to the minor groove of double-
stranded DNA
• Excitation λ= 350 nm,
Emission λ= 400 nm (blue)
• often used as counter-stain
 Fluorochromes developed by
enzymatic action
Fluorescein diacetate (FDA)
hydrolase
FDA Fluorescein
(colourless) (green fluorescence)

• excitation λ = 480 nm, emission λ = 520 nm

• use to stain live cells
• quantitative information
Experiments
 Objectives

Main experiments
Using Pancreatic β-cells (β-TC-6) sample

To use fluorescence microscopy imaging technique to

study cell viability of β-TC-6 cells

Additional experiments
Using Human Umbilical Vein Endothelial Cells (HUVEC)
sample – FDA and PI dyes

To reconfirm viability of dead and live HUVEC samples

Sample Preparation Dead (floating)
BTC 6
Samples
 β-TC-6
 HUVEC HUVEC
Live (adhered
-> subcultured)
3 dyes for sample staining
PI and FDA to examine cell viability;
DAPI acting as a counter stain.

Left to right
PI (PBS)– dark red 2mg/ml
DAPI (DMF) – light yellowish 1mg/ml
FDA (acetone) – colourless 5mg/ml
 Experimental Procedures

1 2

Sample cells cultured beforehand Extraction of dye – FDA/PI/DAPI

3 Incubate
4
Adding dye to sample Wash with PBS
 5 6 7

Placed on microscope stage Adjusted to the correct filter Fluorescent light source
(bright field light source)

Focused, live previewed, imaged

Results,
Data Analysis &
Processing
 Results, Data Analysis and
Processing

Experiments Conducted:
1. Cell viability
2. Rate of fluorescein efflux
3. Cell fixation
 1. Cell Viability
To determine which cells in a given
sample are living and which are dead.
 Viability Staining

Bright Field FDA: Stains

Microscope living cells
Image

DAPI: Stains PI: Stains

all nuclei nuclei of
dead cells
 Red = Green = Blue =
Overlay dead cells living cells all cells
PI FDA DAPI

• To compare the relative distribution

of cells of different colors

Bright Field Overlay

Red Subset (Dead Cells) + Green Subset (Living Cells)

= Blue Set (All Cells)
 Overlay Red = Green =
dead cells living cells
PI FDA
• Generally, red and green do not overlap
• Patches of overlap due to multiple cell
layers

Bright Field Overlay

Overlapping
cells
 Experiment with HUVEC Cells

Sample A Sample B
Trivia:
PI PI
Which sample is
living?

FDA FDA
 2. Rate of Fluorescein Efflux
 Observation: Background
noise increased over time for
fluorescein labeled cells.
 Hypothesis: Fluorescein is
hydrolase
constantly being transported FDA Fluorescein

out of cells (active transport / Efflux

passive diffusion).
 Experimental Aim: To observe FDA Fluorescein
the rate of efflux.
 Fluorescein Efflux
1 min 4 min 7 min

0 1 4 7 Time
(min)
Note: Not the same field of view
Fluorescein Efflux

• We observed
7 min
fluorescein efflux
but not PI or
DAPI efflux.
• PI and DAPI are
bound to the DNA
in the nucleus.
7 min
• Cannot be moved
out of the cells.
 3. Cell Fixation
To determine the effect of fixation
(using pure methanol) on fluorescent
staining of cells.
 Cell Fixation
Steps: Why it works:

 After the usual  All the water in the sample (both

staining procedure, inside and outside the cells) should
immerse the slide in be replaced by methanol, which
cold methanol for 5 evaporates during the drying step.
min.
 The cells are hence kept in a fixed
 Take the slide out, let position.
dry.
 Also, without water as a medium,
fluorescein efflux would not be
observed.
 Cell Fixation
FDA PI Overlay

Green fluorescence is much weaker Green and red

than red fluorescence fluorescence
overlap
completely
 Cell Fixation
 Unexpected observation:
+
FDA PI

all the cells in the sample

were dead. Overlay
 Probably because we left the
cells at room temperature for
too long.
 Fluorescein efflux was
not observed even after
15 minutes.
 Ways to Improve Results

1. Avoid leaving cells outside the incubator for unnecessarily

long periods of time.
 Prevents unnecessary cell death.
2. Culture cells for at least 3 days on the slide, so they have
sufficient time to adhere.
 Alternatively, fix the cells on the slide.
3. Adjust exposure time according to objectives.
4. For more accurate experiments on viability, we suggest the
use of flow cytometry or other methods to count cells.
Comparison with
AFM/NSOM images
 AFM/NSOM vs.
Fluorescence Microscopy

a) AFM c) Bright Field

b) NSOM d) Fluorescence
 Figure :
Stained H9C2
individual cell

Reference : Ianoul, Anatoli. Melissa Street and Donna Grant. "Near-Field Scanning Fluorescence Microscopy
Study of Ion Channel." Biophysical Journal Volume 87 November 2004 3525–3535
Method Advantages Disadvantages
 Out-of-focus flares
 High sensitivity
Fluorescence  High specificity in targeting
 Presence of auto-fluorescence
Microscopy  Photobleaching effect of
 Easy and fast
fluorophores

 High lateral and depth resolution  Samples have to be very clean

Atomic Force  Biological samples can be  Sidewall angle induced artifacts
Microscopy studied in native state from inappropriate tip choice
 3D surface profiling  Long image acquisition time

Near-field  Subjected to artifacts e.g.

 High spatial resolution
Scanning  Minimal sample preparation topography
Optical  Low depth of imaging
 Operated in ambient environment
Microscopy  Long image acquisition time
Conclusion
 Principle of Fluorescence Microscopy
 Experiment
 Preparation & Procedures
 Fluorescence microscopy can be used to
quantify cell viability
 Limitations & Comparison with
AFM/NSOM
 Fast and efficient method for biological
samples
Light path on a fluorescence
microscope
Filter Nomenclature
 Applications – Cell Staining
Dyes used Cell component Color of stain
stained
DiOC6(3) Endoplasmic Orange
(3,3'-dihexyloxacarbocyanine reticulum
iodide)

Rhodamine 123 Mitochondria Green

Fura-2 Cytoplasm Depends on Ca

concentration

Alexa Fluor 633 phallodin Actin filament Magenta