16 November 2007

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

Adeline Sham & Eileen Yang

Fluorescence Microscopy

Overview Introduction

Principle of Fluorescence Microscopy Fluorescence Microscope Fluorescence Dyes

Experiment Objective Sample Preparation Experimental Procedures Results & Discussion

Comparison with AFM/NSOM Conclusion

Introduction

Fluorescence Microscopy

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

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

Imaging of specific regions of biological samples..

A cross section of cotton stained with Rhodamine B.

Source: http://nobelprize.org/educational_games/physics/microscopes/fluorescence/gallery/index.html

Fluorescence double-labeling of mammalian cells.

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

Principle 2. Excitation of Fluorophore

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 aspectsFluorescence MicroscopeFluorescence 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)

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

• use to stain live cells

• quantitative information

FDA Fluorescein hydrolase

(colourless) (green fluorescence)

Experiments

Main experimentsUsing Pancreatic β-cells (β-TC-6) sample

To use fluorescence microscopy imaging technique to study cell viability of β-TC-6 cells

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

To reconfirm viability of dead and live HUVEC samples

Objectives

Sample Preparation

Samples β-TC-6 HUVEC

3 dyes for sample stainingPI and FDA to examine cell viability;DAPI acting as a counter stain.

Left to rightPI (PBS)– dark red 2mg/mlDAPI (DMF) – light yellowish 1mg/mlFDA (acetone) – colourless 5mg/ml

BTC 6

HUVEC

Dead (floating)

Live (adhered -> subcultured)

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

Adding dye to sample

Incubate

Wash with PBS

1 2

3 4

Experimental Procedures

Placed on microscope stage (bright field light source)

Adjusted to the correct filter

6 7

Focused, live previewed, imaged

Fluorescent light source

8

5

Results, Data Analysis & Processing

Results, Data Analysis and Results, Data Analysis and ProcessingProcessing

1. Cell viability

2. Rate of fluorescein efflux

3. Cell fixation

Experiments Conducted:Experiments Conducted:

1. Cell Viability

To determine which cells in a given sample are living and which are dead.

Bright FieldMicroscope

Image

DAPI: Stains all nuclei

FDA: Stains living cells

PI: Stains nuclei of dead cells

Viability Staining

Overlay

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

= = Blue Set (All Cells)Blue Set (All Cells)

• To compare the relative distribution of cells of different colors

Red = dead cells

Green = living cells

Blue = all cells

Bright FieldBright Field OverlayOverlay

FDAFDAPIPI DAPIDAPI

Overlay

Overlapping Overlapping cellscells

Red = dead cells

Green = living cells

Bright FieldBright Field OverlayOverlay

• Generally, red and green do not overlap

• Patches of overlap due to multiple cell layers

FDAFDAPIPI

Experiment with HUVEC Cells

Sample ASample A

PIPI

FDAFDA

Sample BSample B

PIPI

FDAFDA

Trivia:Trivia:

Which sample is Which sample is living?living?

2. Rate of Fluorescein Efflux

Observation:Observation: Background Background

noise increased over time for noise increased over time for

fluorescein labeled cells.fluorescein labeled cells.

Hypothesis:Hypothesis: Fluorescein is Fluorescein is

constantly being transported constantly being transported

out of cells (active transport / out of cells (active transport /

passive diffusion).passive diffusion).

Experimental Aim:Experimental Aim: To observe To observe

the rate of efflux.the rate of efflux.

FDA Fluoresceinhydrolase

FDAFDA

Efflux

FluoresceinFluorescein

Fluorescein Efflux

00 11 44 77 TimeTime(min)(min)

1 min1 min 4 min4 min 7 min7 min

NoteNote: Not the same field of view: Not the same field of view

Fluorescein Efflux

7 min

7 min

• We observed fluorescein efflux but not PI or DAPI efflux.

• PI and DAPI are bound to the DNA in the nucleus.

• 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:Steps:

After the usual After the usual staining procedure, staining procedure, immerse the slide in immerse the slide in cold methanol for 5 cold methanol for 5 min.min.

Take the slide out, let Take the slide out, let dry.dry.

Why it works:Why it works:

All the water in the sample (both inside and outside the cells) should be replaced by methanol, which evaporates during the drying step.

The cells are hence kept in a fixed position.

Also, without water as a medium, fluorescein efflux would not be observed.

Cell Fixation

FDAFDA PIPI OverlayOverlay

Green fluorescence is much weakerGreen fluorescence is much weakerthan red fluorescencethan red fluorescence

Green and red Green and red fluorescence fluorescence

overlap overlap completelycompletely

Cell Fixation

Unexpected observation:

all the cells in the sample

were dead. Probably because we left the

cells at room temperature for

too long.

Fluorescein efflux was

not observed even after

15 minutes.

OverlayOverlay

++FDAFDA PIPI

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

b) NSOM

c) Bright Field

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

FluorescenceMicroscopy

High sensitivity High specificity in targeting Easy and fast

Out-of-focus flares Presence of auto-fluorescence Photobleaching effect of

fluorophores

Atomic ForceMicroscopy

High lateral and depth resolution Biological samples can be

studied in native state 3D surface profiling

Samples have to be very clean Sidewall angle induced artifacts

from inappropriate tip choice Long image acquisition time

Near-fieldScanningOpticalMicroscopy

High spatial resolution Minimal sample preparation Operated in ambient environment

Subjected to artifacts e.g. topography

Low depth of imaging 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 stained

Color of stain

DiOC6(3) (3,3'-dihexyloxacarbocyanine

iodide)

Endoplasmic reticulum

Orange

Rhodamine 123 Mitochondria Green

Fura-2 Cytoplasm Depends on Ca concentration

Alexa Fluor 633 phallodin Actin filament Magenta