I. Excitation/emission bands commonly used in fluorescence microscopy and representative fluorescent dyes
DAPI、Hoechst 33342/33258、Pacific Blue、Cascade Blue
CFP, BFP, Alexa Fluor 430, Partial Calcium Indicator
GFP、FITC、Alexa Fluor 488、mNeonGreen、SYTO 9、Calcein AM
YFP、EYFP、Venus、Citrine、Alexa Fluor 514
TRITC、Cy3、Alexa Fluor 555/568、Rhodamine、DiI
mCherry、Texas Red、Alexa Fluor 594、PI、MitoTracker Red
Cy5、Alexa Fluor 647、APC、MitoTracker Deep Red
Cy7、Alexa Fluor 680、IRDye 680/700/800
Actual values may vary slightly depending on the instrument, filter, solvent, and conjugate. Reagent specifications and instrument filter parameters shall prevail.
The filter group is usually written as "excitation filter/dichroic mirror/emission filter".
EX 350–400 nm，EM 450–490 nm
DAPI, Hoechst, Nucleus
EX 450–490 nm，EM 500–550 nm
GFP、FITC、Alexa 488
EX 530–560 nm，EM 570–640 nm
TRITC、Cy3、Alexa 555/568
Texas Red/mCherry
EX 540–580 nm，EM 600–680 nm
mCherry、Texas Red、Alexa 594
EX 620–650 nm，EM 660–720 nm
Cy5、Alexa 647、APC
EX 650–690 nm，EM 700–760 nm
Cy7, Alexa 680, partial in vivo imaging
The writing of bandpass filters is also common, for example:
· 525/50: approx. 500–550 nm
· 600/50: approx. 575–625 nm
· 700/75: approx. 662–738 nm
DAPI、Hoechst 33342、PI、SYTOX
Hoechst 33342, Syto series
PI、SYTOX Green/Red、7-AAD
Annexin V-FITC、Annexin V-Alexa 647、TUNEL
Fluo-4、Fura-2、Rhod-2、GCaMP
MitoTracker Green/Red/Deep Red、MitoSOX
LysoTracker Green/Red/Deep Red
ER-Tracker Green/Red
Phalloidin-Alexa 488/555/647, antibody-labeled microtubules
DiI、DiO、DiD、FM 4-64、CellMask
DCFH-DA、MitoSOX、CellROX
BCECF、pHrodo、LysoSensor
Alexa Fluor 488/555/594/647、DyLight、Cy3、Cy5
1. The excitation light should fall in the excitation band of the dye. For example, the 488 nm laser is very suitable for GFP/FITC, but the 561 nm laser is more suitable for mCherry and TRITC.
2. The emission filter should be placed in the main area of the emission band, while avoiding excitation light. For example, GFP is detected at 500–550 nm, rather than 480–500 nm.
3. To avoid spectral overlap in multicolor experiments, GFP and FITC are usually not well distinguished; YFP and TRITC are also easy to cross-color. Combinations with large spectral intervals, such as DAPI/GFP/mCherry/Cy5, are preferred.
4. Far red and near infrared channels have a lower background In samples with strong tissue, live or autofluorescence, Alexa 647, Cy5, and Alexa 680 tend to be cleaner than green channels.
5. When it is necessary to do monochromatic control and spectral resolution multicolor fluorescence, spontaneous fluorescence or channel cross-color, a single label control should be done and spectral resolution/linear resolution should be considered.
In a fluorescence microscope, fluorescent molecules undergo a cycle:
Absorption excitation → electron transitions to the high energy level and → briefly stays → back to the low energy level and emits fluorescence
Excitation wavelength (λex) The wavelength at which fluorescent molecules are most easily absorbed and thus “lit up”. Practical experiments are usually determined by lasers, LEDs, or excitation filters. For example, 488 nm excitation is commonly used for GFP.
Emission wavelength (λem) The fluorescence wavelength emitted when the fluorescent molecule returns to the low energy level. The emitted light is generally longer in wavelength and lower in energy than the excitation light. For example, GFP is often detected at about 510 nm.
The difference between the two is called the Stokes shift. Because of the different wavelengths of excitation light and emission light, microscopes can use dichroic mirrors and emission filters to separate weak fluorescence from strong excitation light.
· Neither excitation nor emission is a “single point,” but a wide band.
· The term 488/509 in the literature generally refers to peak wavelengths.
· The peak position moves from a few nanometers to tens of nanometers with factors such as pH, solvent, protein conformation, and conjugated antibodies.
· Multiphoton microscopy is an exception: two-photon/three-photon excitation with 800–1000 nm near-infrared light, but the emission may still be in the visible region.
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