Principles of fluorescence microscopy

Andrew Vaughan – Light Microscopy Facility

Bright-field microscopy

• illumination usually from the opposite side to observation (trans-illumination)

• bright-field images can have low contrast if the specimen is thin
• contrast can be added back in with techniques like phase contrast and DIC
• histochemically staining specimens creates colour contrast

phase contrast histochemical stain

Dark-field microscopy

• true dark-field is a trans-illumination technique

• an annulus in the condenser blocks all but scattered light
• in fluorescence the specimen generates its own light
• fluorescent specimens are usually illuminated from above (epi-illumination)

dark field fluorescence

Basics of fluorescence

• light energy cannot be lost kinetically because the rings are constrained
• some is lost as vibration but most is re-emitted at a longer wavelength
• forbidden triplet state (D in the Jablonski diagram) important for dSTORM
• emission spectrum is shifted to the red and is a mirror image of excitation

fluorescein Jablonski diagram of energy excitation and emission

transitions in fluorescence spectra of fluorescein
Key properties of fluorophores

• excitation is shorter than emission wavelength (Stokes shift)

• emission usually skewed into the red (affects cross-talk)
• brightness is a function of the extinction coefficient and quantum yield
• modern dyes are optimised for photo-stability
• STED may need large Stokes shift, cross-section of stimulated emission
• STORM dyes should go into the dark state easily and have a high duty cycle

Emission spectra of the Alexa Fluor dyes

Fluorescent proteins

• GFP requires no cofactors and fluoresces at physiological temperatures

• fluorescent proteins of different colours from different organisms
• genetic modification has led to even more colours
• functional properties (pH sensors, calcium sensors)
• photoactivatable and photoswitchable proteins used for PALM

modified FPs from the sponge Discosoma The photochemistry of

photoconversion
Fluorescence microscope filters

• the Stokes shift of excitation and emission mean the two can be separated
• excitation and emission filters and a dichroic mirror are used
• these can be mounted in a cube or separately in filter wheels
• some confocal systems use a method where light is spectrally separated

Alexa 647 spectra filter and dichroic mirror anatomy of a filter arrangement of Leica’s
spectra for Alexa 647 cube confocal detector system
Anatomy of a fluorescence
microscope

upright stand inverted stand

The microscope objective lens

• Most important part of the microscope because it forms the image

• Nomenclature indicates things like field flatness and colour correction
• Resolution is defined by numerical aperture (NA)
• Magnification defines the field of view - and resolution if a camera is used
• Working distance generally is shorter for high resolution lenses
• It is important to match refractive indexes to avoid aberration
• Some techniques rely on refractive index mismatch (TIRF, PALM/STORM)
• SR needs high NA so it tends to use oil – not great for thick specimens
Wide-field microscopy

• whole field is illuminated at once

• imaging is quick and uses a camera
• useful for 2D imaging of thin specimens
• thick specimens are challenge because of blur and scatter (refraction)

phase is always thin fluorescent thick specimens can be a challenge

wide-field specimens are okay
Confocal microscopy

• confocal pinholes reduce blur by excluding out-of-focus light from the detector
• deconvolution computationally reassigns blur to the correct focal plane and
can be used on wide-field and confocal data