Practical aspects of fluorescence microscopy

Colour separation

Sensitivity

Resolution

I want to improve ….

What can I do?

How to avoid cross-talk between channels

Antigen1

Primary antibody1

Secondary antibody1

Fluorescent dye1

Excitation light 1

Emission light 1

Antigen2

Primary antibody2

Secondary antibody2

Fluorescent dye2

Excitation light 2

Emission light 2

How does cross-talk happen?

How can you test cross-talk?

Remove each primary antibody.

Corresponding signals should disappear.

Primary antibodies

1. Use two antibodies made by different species. (can label primary antibodies directly if you are desperate)

Secondary antibodies 1. Secondary antibodies should not recognise immunoglobulin of host

species of other primary or secondary antibodies.

Use antibodies cross-absorbed with immunoglobulin of the other species(careful! goat~sheep, mouse~rat)

2. Use antibodies with fluorescent dyes matched with fliter sets.

How to choose antibodies

Donkey secondary anti-sheep Goat secondary anti-rabbit

Sheep primary antibody Rabbit primary antibody

eg.

How to choose filter sets and fluorescent dyes

1. Excitation filters (lights)avoid exciting other dyes

2. Emission filtersavoid passing emission lights from other dyes.

A combination of

Excitation

Emission

500 600nm400

How to choose filter sets.

Cy2(=FITC)

Cy3(=Rhodamin)

Excitation

Emission

500 600nm400

Cy2

Cy3

How to choose filter sets.

Cy3 is also excited

Excitation

Emission

500 600nm400

Cy2

Cy3

How to choose filter sets.

Cy3 is also excited

Cy3 emission is blocked

Excitation

Emission

500 600nm400

Cy2

Cy3

How to choose filter sets.

Cy2 emission is notblocked

Excitation

Emission

500 600nm400

Cy2

Cy3

How to choose filter sets.

Cy2 is not excited

Cy2 emission is notblocked

Excitation

Emission

500 600nm400

Cy2

Cy3

How to choose filter sets.

Do NOT trust a microscope rep!

Check if you use a new dye or fluorescent proteins.

Choosing filter sets is very important for

sensitivity and avoiding cross talk.

Filters are the cheapest component

but paid least attention

can make a huge difference

Long pass (LP) filter

Band pass (BP) filter

Short pass (SP) filter

BP500-530 or BP515/30

LP500

500nm

500 530nm

How to tell the property of filters

Multiband pass filter

Sensitivityhow to get brighter images

Sensitivity: how to get brighter images

For immunofluorescence

• use bright/stable dyes Cy or Alexa (not FITC, rhodamine, Texas Red)

• use a higher concentration of antibodies or dyes

• use a longer exposure time/ gain.

• use contrast enhancement (post-capture)

Live-imaging: sensitivity

objective lens

filters

camera

fluorecent molecules

contrast enhancement

Objective lens

Brightness: propotional to (NA)4 / (magnification)2

Use a lens with a high NA (and low magnification).

Consider a lens with less correction. (Corrected lens has more internal lenses which absorbe light)

Avoid phase contrast lenses (use DIC lenses if required)

X63 NA1.4 vs X63 NA1.2 (>1.8X brighter)

X63 NA1.4 vs X100 NA1.4 (>2.5X brighter)

"resolution": propotional to 1/NA

Filters

If single channel, consider a long-pass filter (broad-band pass)

NB, blocking auto-fluorescence may increase contrast

500 600

GFP

Emission spectra

You are loosing these light!

Camera – sensitivityHigh quantum efficiency

Monochrome

Low noise cameracooling the chip, or on-chip amplification (EMCCD)

Large pixel size 13 m is 4X brighter than 6.5m (sacrifices resolution)

Binning 2X binning gives 4x brighter images(sacrifices resolution)

Gain increase noise as well

Avoid photobleaching

Lower excitation light use a neutral density filter

Shorter time for excitation(only excite when capturing images)

Use stable/bright molecules"enhanced" FP (eGFP …), tandem GFP

Getting bright live imagesobjective lens

high NA, low mag, less correction, no phase

filters Long pass, or broader band pass

longer exposure time vs photo bleaching/speedonly expose when capturing

camerabinning, gain, high quantum efficiency, monochrome, large pixel size

use bright/stable fluorecent molecules "enhanced" FP (eGFP, …), tandem GFP

contrast enhancement after capture

Resolution

How to get finer images

Resolution

Theoritical limit ~200nm

Limited by NA, not magnification ~0.6 x (wave length)/NA

NA1.4 ~200nm

Camera pixel size need a half size of the optical resolution eg, To get 200nm resolution,

you need 100nm pixel size (=10m on chip for 100x lens)

2 dots200nmapart

under 'scope

pixel200nm

pixel100nm

200nm

Low NA

High NA

Resolution is often limited by

the quality of your samples and optical system!

Resolution in reality

Out-of-focus light

Camera noise

Dirty lens

Bad illumination

Burnt out filters

High sample background

Bad fixation

Time resolution

Exposure time (vs sensitivity, resolution)can reduce by increasing sensitivity (eg, binning)

Readout time from cameracan reduce by a subarray readout or binning

Computer (software) speed

Filter/laser switch timefilter cube (~1s), filter wheel (~100ms), laser (<1ms)

Focus moving timereduce by a Piezo driven focus

Facters affecting time resolution

Fancy microscopy

Choose a method according to your sample and purpose

Various fluorescence microscopyDeconvolution : good for point signals

high sensitivity, slow to process

Confocal : good for diffused signals in thick sampleslow sensitivity, slow capture

Spinning disc confocal : high speed, low bleaching(good for live imaging)

TIRF (total internal reflection fluorescence) microscopy: imaging only the surface (with low background)

Molecular dynamics studyFRAP, FLIM, FLIP, iFRAP, FRET, FCS …..

Super-resolution microscopy (nanoscopy)PALM, STED, SIM ….

END

Now go back to your lab and improve your images !