1. Practical aspects of fluorescence microscopy

2. I want to improve …. What can I do? Colour separation Sensitivity
Resolution
What can I do?

3. How to avoid cross-talk between channels

4. How does cross-talk happen?
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

5. How can you test cross-talk?
Remove each primary antibody.
Corresponding signals should disappear.

6. How to choose antibodies
Primary antibodies
Use two antibodies made by different species.
(can label primary antibodies directly if you are desperate)
Secondary antibodies
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.
Donkey secondary anti-sheep Goat secondary anti-rabbit
eg.
Sheep primary antibody Rabbit primary antibody

7. How to choose filter sets and fluorescent dyes
A combination of
Excitation filters (lights)
avoid exciting other dyes
2. Emission filters
avoid passing emission lights from other dyes.

8. How to choose filter sets.
Excitation
Cy2
(=FITC)
Cy3
(=Rhodamin)
Emission
400
500
600nm

9. How to choose filter sets.
Cy3 is also excited
Excitation
Cy2
Cy3
Emission
400
500
600nm

10. How to choose filter sets.
Cy3 is also excited
Excitation
Cy2
Cy3
Cy3 emission is blocked
Emission
400
500
600nm

11. How to choose filter sets.
Excitation
Cy2
Cy3
Emission
Cy2 emission is not
blocked
400
500
600nm

12. How to choose filter sets.
Cy2 is not excited
Excitation
Cy2
Cy3
Emission
Cy2 emission is not
blocked
400
500
600nm

13. How to choose filter sets.
Excitation
Cy2
Cy3
Emission
400
500
600nm

14. 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
Do NOT trust a microscope rep!
Check if you use a new dye or fluorescent proteins.

15. How to tell the property of filters
Long pass (LP) filter
Band pass (BP) filter
Short pass (SP) filter
LP500
500nm
BP or BP515/30 nm
Multiband pass filter

16. Sensitivity
how to get brighter images

17. 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)

18. Live-imaging: sensitivity
objective lens
filters
camera
fluorecent molecules
contrast enhancement

19. Objective lens Brightness: propotional to (NA)4 / (magnification)2
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
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)

20. Filters If single channel,
consider a long-pass filter (broad-band pass)
NB, blocking auto-fluorescence may increase contrast
Emission spectra
You are loosing these light!
GFP
500
600

21. Camera – sensitivity High quantum efficiency Monochrome
Low noise camera
cooling the chip, or on-chip amplification (EMCCD)
Large pixel size
13 m is 4X brighter than 6.5m
(sacrifices resolution)
Binning
2X binning gives 4x brighter images
Gain increase noise as well

22. 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

23. Getting bright live images
objective lens
high NA, low mag, less correction, no phase
filters Long pass, or broader band pass
longer exposure time vs photo bleaching/speed
only expose when capturing
camera
binning, gain,
high quantum efficiency, monochrome, large pixel size
use bright/stable fluorecent molecules
"enhanced" FP (eGFP, …), tandem GFP
contrast enhancement after capture

24. Resolution
How to get finer images

25. 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
(=10m on chip for 100x lens)
2 dots
200nm
apart
under 'scope
Low NA
High NA
pixel
200nm
pixel
100nm

26. Resolution in reality Resolution is often limited by
the quality of your samples and optical system!
Out-of-focus light
Camera noise
Dirty lens
Bad illumination
Burnt out filters
High sample background
Bad fixation

27. Time resolution Facters affecting time resolution
Exposure time (vs sensitivity, resolution)
can reduce by increasing sensitivity (eg, binning)
Readout time from camera
can reduce by a subarray readout or binning
Computer (software) speed
Filter/laser switch time
filter cube (~1s), filter wheel (~100ms), laser (<1ms)
Focus moving time
reduce by a Piezo driven focus

28. Fancy microscopy
Choose a method according to your sample and purpose

29. Various fluorescence microscopy
Deconvolution : good for point signals
high sensitivity, slow to process
Confocal : good for diffused signals in thick samples
low 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 study
FRAP, FLIM, FLIP, iFRAP, FRET, FCS …..
Super-resolution microscopy (nanoscopy)
PALM, STED, SIM ….

30. END
Now go back to your lab
and improve your images !