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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 !
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