Fluorescence Microscopy Final

Fluorescence Microscopy 16 November 2007 By Chen Xiuli, Liu Yuchun, Ng Yen Shan, Cynthia Ong, Adeline Sham & Eileen Yang

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

•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.

Chinese hamster ovary cell.

Human cells.

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 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 Requires other molecules to bind to specific targets Contains fluorescent proteins produced by organisms themselves Have special properties

1.

2.

3. • •

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 doublestranded DNA Excitation λ= 350 nm, Emission λ= 400 nm (blue) often used as counter-stain

Fluorochromes developed by enzymatic action Fluorescein diacetate (FDA) FDA

hydrolase

(colourless)

Fluorescein

(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 Samples  β-TC-6  HUVEC

Dead (floating)

BTC 6

HUVEC

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

Live (adhered -> subcultured)

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

3

Extraction of dye – FDA/PI/DAPI

Incubate

Adding dye to sample

4 Wash with PBS

6

5 Placed on microscope stage (bright field light source)

Adjusted to the correct filter

8 Focused, live previewed, imaged

7 Fluorescent light source

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 Microscope Image

DAPI: Stains all nuclei

FDA: Stains living cells

PI: Stains nuclei of dead cells

Overlay

Red = dead cells PI

•

Green = living cells FDA

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)

Blue = all cells DAPI

Overlay

Red = Green = dead cells living cells PI

•

Generally, red and green do not overlap

•

Patches of overlap due to multiple cell layers

Bright Field

Overlay

Overlapping cells

FDA

Experiment with HUVEC Cells Sample A

Sample B

PI

PI

FDA

FDA

Trivia: Which sample is living?

2. Rate of Fluorescein Efflux 

Observation: Background noise increased over time for fluorescein labeled cells.



Hypothesis: Fluorescein is constantly being transported

FDA

out of cells (active transport / passive diffusion). 

Experimental Aim: To observe the rate of efflux.

hydrolase

Fluorescein Efflux

FDA

Fluorescein

Fluorescein Efflux 1 min

0

1

4 min

4 Note: Not the same field of view

7 min

7

Time (min)

Fluorescein Efflux 7 min

• We observed 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: 



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

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.

Take the slide out, let dry.

Cell Fixation FDA

PI

Green fluorescence is much weaker than red fluorescence

Overlay

Green and red fluorescence overlap completely

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.

FDA

Overlay

+

PI

Ways to Improve Results 1.

Avoid leaving cells outside the incubator for unnecessarily long periods of time. 

2.

Prevents unnecessary cell death.

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

Fluorescence Microscopy

 High sensitivity  High specificity in targeting  Easy and fast

Atomic Force Microscopy

 High lateral and depth resolution  Biological samples can be studied in native state  3D surface profiling

Near-field Scanning Optical Microscopy

  High spatial resolution  Minimal sample preparation   Operated in ambient environment 

  

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

 

Samples have to be very clean Sidewall angle induced artifacts from inappropriate tip choice Long image acquisition time



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

Endoplasmic reticulum

Orange

Rhodamine 123

Mitochondria

Green

Fura-2

Cytoplasm

Depends on Ca concentration

Alexa Fluor 633 phallodin

Actin filament

Magenta

DiOC6(3) (3,3'-dihexyloxacarbocyanine iodide)